Immunomodulators in Aquaculture

and Fish Health

This reference book provides updated information about different immunomodulators for managing fish health and
sustainable aquaculture. Immunomodulators are dietary additives that enhance innate defense mechanisms and increase
resistance against specific pathogens and diseases. The book covers the different types of immunostimulants, their modes
of action, and their efficacies. It also reviews safety concerns, ethical regulations, limitations, and outreach to farmers. It
discusses the application of herbal immunomodulators, antioxidants, and pre- and pro-biotics in disease management.

Features:

• Reviews the pressing topic of reduction of antibiotic use in aquaculture

• Discusses herbal immunomodulators, nutrients, antioxidants, and pre- and pro-biotics
• Covers the topic of progressive immunomodulation using nanotechnology
• Discusses fish health management in the ever-growing aquaculture industry
• Includes natural and synthetic immunomodulators

The book is meant for researchers and industry experts in aquaculture, fisheries science, and veterinary medicine.
Immunomodulators in Aquaculture
and Fish Health

Edited by
Preetham Elumalai
Mehdi Soltani
Sreeja Lakshmi
Design cover image: image courtesy Preetham Elumalai, Mehdi Soltani and Sreeja Lakshmi

First edition published 2024

by CRC Press
2385 Executive Center Drive, Suite 320, Boca Raton FL 33431

and by CRC Press

4 Park Square, Milton Park, Abingdon, Oxon, OX14 4RN

CRC Press is an imprint of Taylor & Francis Group, LLC

© 2024 selection and editorial matter, Preetham Elumalai, Mehdi Soltani and Sreeja Lakshmi; individual chapters, the contributors

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Library of Congress Cataloging-in-Publication Data

Names: Elumalai, Preetham, editor. | Soltani, Mehdi, editor. | Lakshmi, Sreeja, editor.
Title: Immunomodulators in aquaculture and fish health / edited by Preetham Elumalai, Mehdi Soltani, and Sreeja Lakshmi
Description: First edition | Boca Raton, FL : CRC Press, 2024 | Includes bibliographical references and index. | Summary: "This
reference book provides updated information about different immunomodulators for managing fish health and sustainable aquaculture.
Immunomodulators are dietary additives that enhance innate defense mechanisms and increase resistance against specific pathogens and
diseases. The book covers the different types of immunostimulants, their modes of action, and their efficacies. It also reviews safety
concerns, ethical regulations, limitations, and outreach to farmers. It discusses the application of herbal immunomodulators,
antioxidants, pre- and pro-biotics, in disease management.
Features: Reviews the pressing topic of reduction of antibiotic use in aquaculture Discusses herbal immunomodulators, nutrients,
antioxidants and pre- and pro-biotics Covers the topic of progressive immunomodulation using nanotechnology Discusses fish health
management in the ever-growing aquaculture industry Includes natural and synthetic immunomodulators The book is meant for
researchers and industry experts in aquaculture, fisheries science, and veterinary medicine"-- Provided by publisher.
Identifiers: LCCN 2023015962 (print) | LCCN 2023015963 (ebook) | ISBN 9781032407388 (hardback) | ISBN 9781032420905
(paperback) | ISBN 9781003361183 (ebook)
Subjects: LCSH: Fish culture. | Immunological adjuvants. | Biological response modifiers.
Classification: LCC SH151 .I46 2024 (print) | LCC SH151 (ebook) | DDC 639.3--dc23/eng/20230726
LC record available at [Link]
LC ebook record available at [Link]
ISBN: 978-1-032-40738-8 (hbk)
ISBN: 978-1-032-42090-5 (pbk)
ISBN: 978-1-003-36118-3 (ebk)
DOI: 10.1201/9781003361183

Typeset in Times
by MPS Limited, Dehradun
Contents

Foreword .......................................................................................................................................................................................... vii

Editors ............................................................................................................................................................................................. viii
Contributors .......................................................................................................................................................................................ix

Section I Immunomodulators: An Overview

1. Immunomodulators: An Introduction ...................................................................................................................................3
Falco F, Banaee M, Mauro M, Faggio C, Arathi Kollath, and Preetham Elumalai

2. Natural and Synthetic Immunomodulators: Inferences for Stress Responses in Aquaculture Fish...........................18
Shubhajit Saha, Azubuike V. Chukwuka, Nimai Chandra Saha, Caterina Faggio, and Hamed Mousavi Sabet

3. Immunomodulators: Mode of Action ..................................................................................................................................29

Subramaniam Sivakumar, C. Shanmuga Sundaram, Maderi Velayutham Dassprakash, and
Rantham Subramaniam Venkatesan

4. Immunomodulators and Stress Oxidative...........................................................................................................................43

Tamilselvan Gokul, Paulraj Balaji, Karthikeyan Venkatachalam, Subramanian Ramya, Ramaraj Jayakumararaj,
Chinnathambi Pothiraj, and Kamatchi Ramesh Kumar

Section II Immunomodulators and Sustainable Aquaculture Development

5. Immunomodulators to Prevent Diseases and Minimize Antimicrobial Use ...................................................................59
Akshay Thuruthiyil Rajesh, Sajna Beegum, Neha Omgy, Sreeja Lakshmi, Hethesh Chellapandian,
Sivakamavalli Jeyachandran, Einar Ringø, and Preetham Elumalai

6. Immunomodulation in Aquaculture Health Management: Opportunities and Obstacles............................................76

Ramchandran Ishwarya, Baskaralingam Vaseeharan, Rengarajan Jayakumar, Subramaniam Sivakumar, and
Preetham Elumalai

7. Disease Management and Prophylaxis by Immunostimulants .........................................................................................89

Chinnathambi Pothiraj, Divya Jyoti, Subramanian Ramya, Ramaraj Jayakumararaj, Aseem Grover,
Reshma Sinha, Palanichamy Ayyappan, Caterina Faggio, and Paulraj Balaji

8. Application of Immunostimulants for Aquaculture Health Management....................................................................103

Femi John Fawole, Shamna Nazeemashahul, Thongam Ibemcha Chanu, Arun Sharma,
Gbadamosi Oluyemi Kazeem, S. Ferosekhan, and Tejaswini Kinnera

Section III Immunomodulators in Aquaculture Health Management

9. Herbal Immunomodulators for Aquaculture ...................................................................................................................119
Shamna Nazeemashahul, Femi John Fawole, Babitha Rani A.M., Manish Jayant, Neha Qureshi,
Hussain Nottanalan, Ashutosh D. Deo, and Parimal Sardar

10. Prebiotics and Probiotics as Effective Immunomodulators in Aquaculture ................................................................136

Mehdi Soltani, Koushik Ghosh, Dipanjan Dutta, and Einar Ringø

v
vi Contents

11. Immunomodulation in Fish Through Nutrients, Antioxidants and Hormones............................................................169

Chiranjiv Pradhan, Nikhila Peter, and Sweta Das

12. Cytokines and Fish Health..................................................................................................................................................186

Aifa Fathima, Yaser Arafath, Saqib Hassan, George Seghal Kiran, and Joseph Selvin

13. Progressive Immunomodulation Through Nanotechnology............................................................................................200

Heba Mahboub, Hiam Elabd, Mian Adnan Kakakhel, Gehad E. Elshopakey, Maram H. Abduljabbar, and
Manal E. Alosaimi

Section IV Current Status of Immunomodulators in Aquaculture

14. Efficacy and Limitations of Immunomodulators .............................................................................................................213
Arathi Kollath, Lokesh Pawar, Ankeet Bhagat, Sunil Sharma, Owias Iqbal Dar, and Preetham Elumalai

15. Current Status and Recent Advancements with Immunostimulants in Aquaculture.................................................233

Parasuraman Aiya Subramani, S. Kalaivani Priyadarshini, Ramalakshmi Balasubramanian,
M. Divya Gnaneswari, Devasree Ganesh Kumar, Priyatharsini Rajendran, Catherine Alexander, and
R. Dinakaran Michael

Index.................................................................................................................................................................................... 263
Foreword

It is my great pleasure to write this The latest FAO State of the World Fisheries and Aquaculture
foreword for a very timely book, Report (2022) estimated global aquaculture production of aquatic
“Immunomodulators in Aquaculture animals at a record 87.5 million tonnes, with a value of USD
and Fish Health,” edited by my 264.8 billion; this equates to 49% of total aquatic animal supply
colleagues, Preetham Elumalai, Mehdi by volume and 65% by value. Approximately 2.5 million people
Soltani, and Sreeja Lakshmi. are directly employed in the aquaculture sector around the
As a believer in identifying opportuni- world, with most of these in Asia, followed by Africa and Latin
ties and linking scientific evidence, America. Women comprise 28% of these employment figures,
innovation with improved sustainable slightly more than the average of 25% for the agriculture sector as
aquatic production, together with my a whole.
professional commitment in the aquaculture industry of over Infectious diseases represent a major constraint to the
40 years, I have found this volume of work to be a very extensive continued growth of global aquaculture, with estimated annual
review. The wealth of information on the potential therapeutic losses of at least USD 6 billion. The industry is particularly prone
and preventative roles of immunomodulators, in combating to disease outbreaks because of high stocking densities which
diseases in farmed aquatic species is truly insightful. increase pathogen transmission rates and reduce water quality,
The importance of this book defines an era, where the and also because of low genetic diversity in many breeding
performance of aquatic foods has been greatly recognized and stocks, which may compromise the immune response to infection
concerted efforts have been initiated to enhance production and of cultured animals. Antimicrobials and antiparasitics are used
to bridge the ever-expanding supply-demand gap, for aquatic therapeutically and prophylactically, but these treatments are
blue foods worldwide. often expensive and there is concern over the potential adverse
The burden of disease is high in aquatic production and effects of their widespread use, particularly the promotion of
currently estimated as $10 billion USD annually. With the antibiotic resistance. Vaccination is another option for disease
decades of experience and lessons learned in aquatic animal control, but vaccines often have limited efficacy, particularly for
health management, it is convinced that prevention is better juvenile fish which do not have a fully developed immune
than cure for aquatics and investing in prevention is more cost- response. There is therefore increasing interest in alternative
effective than investing in therapy. approaches to disease control in aquaculture.
In this regard, I believe we should be aiming for more tools Immunomodulators are substances that affect the functioning
and procedures such as vaccines and vaccination and more of the immune system. A range of natural and synthetic products
research efforts should be supported, both at academic and have been used or proposed, with varying degrees of scientific
commercial levels. evaluation, to control infectious diseases in aquaculture. This
I congratulate the editors for this comprehensive volume and book provides a very comprehensive and timely exploration of
hope it will serve the purpose of increasing awareness of their efficacy and potential role in an aquatic animal health
immunomodulators towards the implementation within aquatic management system. The various sections of the book provide an
animal health management. overview of immunomodulators and their mode of action; the
potential of immunomodulators to provide a more sustainable
Dr Rohana Subasinghe approach to disease control; the current use of immunomodulator
Founder + Director, products in aquaculture; and finally, their efficacy, limitations,
FUTUREFISH Co. Ltd. and future prospects for the aquaculture industry. The book will
[Link] be of great benefit to researchers in aquatic animal health, aquatic
veterinarians, aquaculture managers, and all of us who wish to
promote an economically viable and environmentally sustainable
Aquaculture plays a vital role in global aquaculture industry.
food security and economic development. Professor Alan Lymbery
It is the fastest-growing food production Director, Centre for Sustainable Aquatic Ecosystems
sector in the world and has been for some Harry Butler Institute, Murdoch University
considerable time, with an average annual Australia
growth rate over the last 50 years of 8%.

vii
Editors

Preetham Elumalai, PhD, is an associate professor at the fisheries and veterinary science. He chaired government
Department of Marine Biology, Cochin University of Science advisory committees in fisheries and aquaculture and has also
and Technology, Kochi, Kerala, India. He earned a master’s worked closely with the aquaculture industry. He developed and
degree from the University of Madras and a PhD in biochemistry patented a number of fish vaccines, which are registered
and molecular immunology from the Institute for Immunology, throughout the Middle East. He also taught numerous
University of Regensburg, Germany. His research practice undergraduate courses and supervised many higher degree
includes bioassay-guided identification of novel marine students. His research interests include vaccine development
compounds, unveiling fish lectins in innate immune defense, for fish pathogens; immunopathogenesis of infectious agents in
aquatic vaccine development, evaluation of cost-effective feed fish/shellfish; and development of alternative therapies such as
additives and nutrigenomics, and effects of environmental immunostimulants, probiotics, and phytobiotics for disease
pollutants on marine ecosystems. He has been a partner in control in farmed fish and shellfish.
numerous EU-, Indian-, and UK-funded projects (e.g., IVVN,
BactiVac). He has written more than 70 peer-reviewed articles Sreeja Lakshmi, PhD, is a postdoctoral research scientist in
and has two patents in his name apart from editing five books collaboration with Moredun Research Institute (MRI), UK. She
(Springer, CRC Press) and has presented his work at more than graduated from Calicut University and earned a PhD in
60 national and international conferences. He has been awarded biochemistry and functional genomics from the Institute for
the prestigious INSA fellowship (2018); MASTS, Fellowship Molecular Biology, University of Regensburg, Germany. She has
(2019); IVVN award, UK (2020); FRSB award (2021); and published research articles in peer-reviewed international
BactiVac award, UK (2022). journals and authored books and book chapters. She has been
awarded prestigious research grants from the Bavarian Research
Mehdi Soltani, PhD, is a distinguished professor at the Foundation (Bayerische Forschungsstiftung), Government of
University of Tehran and an adjunct professor at Murdoch Bavaria, Germany; an HRD-Fellowship for Women Scientists
University, Australia. He earned a DVM from the University of from the Department of Health Research, Government of India;
Tehran and PhD in aquatic animal health from the University of and a MASTS (Marine Alliance Science and Technology,
Tasmania, Australia. Professor Soltani has an international Scotland) Award for Postdoctoral and Early Career Research
reputation for research on aquatic animal health, with 290 Exchanges (PECRE). She has visited the University of Aberdeen,
published scientific papers, collaborations with researchers Scotland, and received an IVVN Fellowship grant from the
throughout the world, and editorship of scientific journals in International Veterinary Vaccinology Network (IVVN), UK.

viii
Contributors

Maram H. Abduljabbar Faggio C

Department of Pharmacology and Toxicology, College of Department of Chemical, Biological, Pharmaceutical and
Pharmacy Environmental Sciences
Taif University University of Messina
Taif, Saudi Arabia Messina, Italy

Catherine Alexander Thongam Ibemcha Chanu

Jayaraj Annapackiam College for Women Aquaculture Division
Thamaraikulam, Periyakulam, Tamil Nadu ICAR – Central Institute of Fisheries Education
Mumbai, Maharashtra, India
Manal E. Alosaimi
Department of Basic Science, College of Medicine Hethesh Chellapandian
Princess Nourah Bint Abdulrahman University PG and Research Department of Biotechnology and Microbiology
Riyadh, Saudi Arabia National College (Autonomous)
Tiruchirappalli, Tamil Nadu, India
Babitha Rani A.M.
Aquaculture Division Azubuike V. Chukwuka
ICAR – Central Institute of Fisheries Education National Environmental Standards and Regulations
Mumbai, Maharashtra, India Enforcement Agency (NESREA)
Nigeria
Yaser Arafath
Department of Microbiology, School of Life Sciences Owias Iqbal Dar
Pondicherry University Aquatic Toxicology Lab, Department of Zoology
Puducherry, India Guru Nanak Dev University
Amritsar, Punjab, India
Palanichamy Ayyappan and
Department of Botany Department of Biosciences
Government Arts College Chandigarh University
Melur, Madurai, Tamil Nadu, India Gharuan, Punjab, India
Paulraj Balaji Sweta Das
PG and Research Centre in Biotechnology Department of Aquatic Animal Health Management
MGR College Kerala University of Fisheries and Ocean Studies
Hosur, Tamil Nadu, India Panangad, Kochi, Kerala
Ramalakshmi Balasubramanian Maderi Velayutham Dassprakash
Department of Zoology Department of Biochemistry
Lady Doak College Sri Sankara Arts and Science College
Tallakulam, Tamil Nadu Enathur, Kanchipuram, Tamilnadu, India
Sajna Beegum Ashutosh D. Deo
Department of Marine Biology, Microbiology and Biochemistry, Fish Nutrition, Biochemistry and Physiology Division
School of Marine Sciences ICAR – Central Institute of Fisheries Education
Cochin University of Science and Technology (CUSAT) Mumbai, Maharashtra, India
Lakeside Campus, Fine Arts Avenue, Cochin, Kerala, India
Dipanjan Dutta
Ankeet Bhagat Aquaculture Laboratory, Department of Zoology
Aquatic Toxicology Lab, Department of Zoology The University of Burdwan
Guru Nanak Dev University Golapbag, Burdwan, West Bengal, India
Amritsar, Punjab, India and
Post Graduate Department of Zoology
Hooghly Mohsin College
Chinsurah, Hooghly, West Bengal, India

ix
x Contributors

Hiam Elabd Aseem Grover

Aquatic Animals’ Medicine Department, Faculty of Veterinary Department of Chemistry, Biochemistry and Forensic
Medicine Sciences, Amity School of Applied Sciences
Benha University Amity University Haryana
Moshtohor, Toukh, Egypt Panchgaon, Haryana, India

Gehad E. Elshopakey Saqib Hassan

Department of Clinical Pathology, Faculty of Veterinary Department of Biotechnology, School of Bio and Chemical
Medicine Engineering
Mansoura University Sathyabhama Institute of Science and Technology
Mansoura, Dakahlia, Egypt Chennai, Tamilnadu, India
and
Preetham Elumalai FutureLeaders Mentoring Fellow
Department of Marine Biology, Microbiology and Biochemistry, American Society for Microbiology
School of Marine Sciences Washington, USA
Cochin University of Science and Technology (CUSAT)
Lakeside Campus Fine Arts Avenue, Cochin, India Ramchandran Ishwarya
Mandapam Regional Centre, ICAR – Central
Falco F Marine Fisheries Research Institute
Institute of Marine Biological Resources and Biotechnologies, Mandapam, Tamil Nadu, India
National Research Council (CNR)
Mazara Del Vallo, Italy Rengarajan Jayakumar
ICAR – Central Institute of Brackishwater Aquaculture
Caterina Faggio (CIBA)
Department of Chemical, Biological, Pharmaceutical and MRC Nagar, Chennai, Tamil Nadu, India
Environmental Sciences
University of Messina Ramaraj Jayakumararaj
Messina, Italy Department of Botany
Government Arts College
Aifa Fathima Melur, Madurai, Tamil Nadu, India
Department of Microbiology, School of Life Sciences
Pondicherry University Manish Jayant
Puducherry, India Fish Nutrition, Biochemistry and Physiology Division
ICAR – Central Institute of Fisheries Education
Femi John Fawole Mumbai, Maharashtra, India
Fish Nutrition and Biochemistry Unit, Department of Aquaculture
and Fisheries Sivakamavalli Jeyachandran
University of Ilorin PG and Research Department of Biotechnology and
Ilorin, Nigeria Microbiology
National College (Autonomous)
S. Ferosekhan Tiruchirappalli, Tamil Nadu, India
Department of Aquaculture
ICAR – Central Institute of Freshwater Aquaculture Divya Jyoti
Bhubaneswar, Odisha, India School of Biological and Environmental Sciences
Shoolini University
Koushik Ghosh Solan, India
Aquaculture Laboratory, Department of Zoology
The University of Burdwan Mian Adnan Kakakhel
Golapbag, Burdwan, West Bengal, India MOE Key Laboratory of Cell Activities and Stress
Adaptations, School of Life Sciences
M. Divya Gnaneswari Lanzhou University
Department of Zoology Lanzhou, P.R. China
Gargi College
New Delhi, India

Tamilselvan Gokul
PG and Research Department of Zoology
Vivekananda College
Tiruvedakam (West), Madurai, Tamil Nadu, India
Contributors xi

Gbadamosi Oluyemi Kazeem Shamna Nazeemashahul

Department of Fisheries and Aquaculture Technology Fish Nutrition, Biochemistry and Physiology Division
Federal University of Technology ICAR – Central Institute of Fisheries Education
Akure, Ondo State, Nigeria Mumbai, Maharashtra, India

Tejaswini Kinnera Hussain Nottanalan

Fish Nutrition, Biochemistry and Physiology Division Fish Nutrition, Biochemistry and Physiology Division
ICAR – Central Institute of Fisheries Education ICAR – Central Institute of Fisheries Education
Mumbai, Maharashtra, India Mumbai, Maharashtra, India

George Seghal Kiran Neha Omgy

Department of Food Science and Technology, School of Life Kerala University of Fisheries and Ocean Studies, Panangad
Sciences Kochi, India
Pondicherry University
Puducherry, India Lokesh Pawar
College of Fisheries (Central Agricultural University – Imphal)
Arathi Kollath Agartala, Tripura, India
Department of Marine Biology, Microbiology and Biochemistry,
School of Marine Sciences Nikhila Peter
Cochin University of Science and Technology (CUSAT) Fish Nutrition and Feed Technology Laboratory, Department
Lakeside Campus, Fine Arts Avenue, Cochin, India of Aquaculture
Kerala University of Fisheries and Ocean Studies
Devasree Ganesh Kumar Panangad, Kochi, Kerala
The Madura College (Autonomous)
Vidya Nagar, Madurai, Tamil Nadu Chinnathambi Pothiraj
Department of Botany
Kamatchi Ramesh Kumar Government Arts College
PG and Research Department of Zoology Melur, Madurai, Tamil Nadu, India
Vivekananda College
Tiruvedakam (West), Madurai, Tamil Nadu, India Chiranjiv Pradhan
Fish Nutrition and Feed Technology Laboratory, Department
Sreeja Lakshmi of Aquaculture
Kerala University of Fisheries and Ocean Studies, Panangad Kerala University of Fisheries and Ocean Studies
Kochi, India Panangad, Kochi, Kerala

Banaee M S. Kalaivani Priyadarshini

Aquaculture Department, Faculty of Natural Resources and the Department of Biotechnology
Environment Lady Doak College
Behbahan Khatam Alanbia University of Technology Hakim Ajmal Khan Rd, Tallakulam, Tamil Nadu
Behbahan, Khuzestan Province, Iran
Neha Qureshi
Mauro M Fisheries Economics, Extension and Statistics Division
Department of Biological, Chemical and Pharmaceutical ICAR‐CIFE, Mumbai, Maharashtra, India
Sciences and Technologies (STEBICEF)
University of Palermo Priyatharsini Rajendran
Palermo, Italy Department of Zoology
Lady Doak College
Heba Mahboub Tallakulam, Tamil Nadu, India
Fish Diseases and Management Department, Faculty of
Veterinary Medicine Akshay Thuruthiyil Rajesh
Zagazig University Department of Marine Biology, Microbiology and Biochemistry,
Zagazig, Sharkia, Egypt School of Marine Sciences
Cochin University of Science and Technology (CUSAT)
R. Dinakaran Michael Lakeside Campus, Cochin, Kerala, India
Centre for Fish Immunology
Vels Institute of Science, Technology, and Advanced Studies Subramanian Ramya
(VISTAS) Department of Zoology
Pallavaram, Chennai, Tamil Nadu Yadava College (Men)
Madurai, Tamil Nadu, India
xii Contributors

Einar Ringø Hamed Mousavi Sabet

Norwegian College of Fishery Science, Faculty of Biosciences, Department of Fisheries, Faculty of Natural Resources
Fisheries and Economics University of Guilan
UiT, The Arctic University of Norway Sowmeh Sara, Iran
Tromsø, Norway
Mehdi Soltani
Nimai Chandra Saha Centre for Sustainable Aquatic Ecosystems, Harry Butler
University of Burdwan Institute
Purba Barddhaman, India Murdoch University
Perth, Australia
Shubhajit Saha and
Fisheries and Ecotoxicology Research Laboratory Department of Aquatic Animal Health Faculty of Veterinary
Department of Zoology Medicine
The University of Burdwan, Burdwan University of Tehran
West Bengal, India Tehran, Iran
Parimal Sardar Parasuraman Aiya Subramani
Fish Nutrition, Biochemistry and Physiology Division Centre for Fish Immunology
ICAR – Central Institute of Fisheries Education Vels Institute of Science, Technology, and Advanced Studies
Mumbai, Maharashtra, India (VISTAS)
Joseph Selvin Pallavaram, Chennai, Tamil Nadu
Department of Microbiology, School of Life Sciences C. Shanmuga Sundaram
Pondicherry University Department of Microbiology
Puducherry, India Hindustan College of Arts and Science
Arun Sharma Kelambakkam, Chennai, Tamilnadu, India
Aquatic Animal Health Management Division Baskaralingam Vaseeharan
ICAR – Central Institute of Fisheries Education Biomaterials and Biotechnology in Animal Health Lab,
Mumbai, Maharashtra, India Department of Animal Health and Management
Sunil Sharma Alagappa University
Aquatic Toxicology Lab, Department of Zoology Karaikudi, Tamil Nadu, India
Guru Nanak Dev University Karthikeyan Venkatachalam
Amritsar, Punjab, India Faculty of Innovative Agriculture and Fishery Establishment
Reshma Sinha Project, Prince of Songkla
Department of Animal Sciences University Surat Thani Campus
Central University of Himachal Pradesh Makham Tia, Mueang, Surat Thani, Thailand
Kangra, India Rantham Subramaniam Venkatesan
Subramaniam Sivakumar Lakshmi Bangaru Arts and Science College
Department of Biochemistry Melmaruvathur, Chengalpet, Tamilnadu, India
Sri Sankara Arts and Science College
Enathur, Kanchipuram, Tamilnadu, India
 Section I

Immunomodulators: An overview
1
Immunomodulators: An Introduction

Falco F1, Banaee M2, Mauro M3, Faggio C4, Arathi Kollath5, and Preetham Elumalai5
1
Institute of Marine Biological Resources and
Biotechnologies, National Research Council (CNR),
Mazara Del Vallo, Italy
2
Aquaculture Department, Faculty of Natural Resources
and the Environment, Behbahan Khatam Alanbia
University of Technology, Behbahan, Khuzestan
Province, Iran
3
Department of Biological, Chemical and
Pharmaceutical Sciences and Technologies
(STEBICEF), University of Palermo, Palermo, Italy
4
Department of Chemical, Biological, Pharmaceutical
and Environmental Sciences, University of Messina,
Messina, Italy
5
Department of Marine Biology, Microbiology and
Biochemistry, School of Marine Sciences, Cochin
University of Science and Technology (CUSAT),
Lakeside Campus, Fine Arts Avenue, Cochin, India

autoimmune diseases in fish. We prefer to divide the immune

1.1 Introduction system into two broad categories based on function, namely the
innate immune system (non-specific immune system) as the first
Before going into detail about immunomodulators, it’s important line of defence against pathogens (Carbone & Faggio 2016)
to understand how immune systems work and how different and the adaptive immune system (specific or acquired immune
variables can stimulate the immune system in different ways. system) (Marshall et al. 2018).
Many substances are known to exist in our environment, and they
are always capable of affecting the immune systems of living
beings. For example, it has been shown that the immune system 1.2 How Do the Immunostimulants Work in
plays an important role in aquatic organisms subjected to various
physical stress conditions (Mauro et al. 2021), noise pollution Fish?
(Mauro et al. 2020), drug pollution (Mauro et al. 2022), or bac­ Since their embryonic life stage, fish have relied on their innate
terial activity (Vizzini et al. 2021). Several biomarkers are also immune system, and their survival depends on it. The skin is the
used to evaluate the health status of important animals in aqua­ principal non-specific defence in fish and plays a key role in
culture (Mauro et al. 2022). In addition, aquatic organisms are protecting and preventing the entry of pathogens into the epi­
also an excellent source of molecules with antimicrobial and thelium through the secretion of a mucus layer involved in the
antitumor activity (Mauro et al. 2022). In this chapter, we briefly immunity system (Salinas et al. 2011). The cells and mediators
introduce the function and role of immunomodulators, especially involved will differ depending on the time, the trigger, the
in the fish immune system. Fish have an immune system that is anatomical location (inflammation can affect any tissue), and the
similar to that of higher vertebrates. As a result, every living entity severity of the inflammation (Calder et al. 2013). Teleosts have a
must preserve its integrity and health status when challenged and cellular defence system that includes macrophage-like phago­
must be able to recognise and distinguish between “self” (its cytic cells, neutrophils, and natural killer (NK) cells, as well as T
molecules, cells, and tissues) and “non-self” (all other organisms and B lymphocytes, as well as various humoral defence com­
or substances). The purpose of the immune system is to recognise ponents like complement (classical and alternative pathways),
the millions of non-self organisms that are potentially harmful to lysozyme, natural hemolysin, transferring factor, and C-reactive
the self and to eliminate them or reduce their impact so that no protein (Watts et al. 2001). Furthermore, teleosts and elasmo­
damage occurs to the self (Takx-Köhlen 1992). This chapter does branchs are the most primitive groups that possess the major
not explain self-immunity or self-tolerance phenomena that lead histocompatibility complex (MHC) and T-cell receptors, which
to suppression of the immune system and the spread of many are the primary components of the immune response against

DOI: 10.1201/9781003361183-2 3
4 Immunomodulators in Aquaculture and Fish Health

FIGURE 1.1 The immune system assumption in teleostfish.

The above figure represents types of immune system in fish. Innate immunity provides primary defence against pathogens by activating phago­
cytosis and antimicrobial, complement activation. On the other hand, adaptive immune system functions by stimulating specific lymphocytes.

pathogenic organisms (Zou & Secombes 2016). Sakai et al. When an agent depresses the immune system, also known as
(2021) investigated the role and function of cytokines in fish an immunosuppressant, they can cause a negative response.
(interferon, interleukin 2, and macrophage activating factors), Immunosuppression is described by Dohms and Saif (1984)
whereas among the lymphoid organs found in fish to mediate the as “a state of transient or persistent dysfunction of the
responses have been the thymus, spleen, and kidney (Zapata immune response resulting from insults to the immune
1996). The collaboration of fish innate and adaptive (memory) system and increasing susceptibility to disease,” and the
immune systems to eliminate intruders or activate defensive qualifier “and frequently a suboptimal antibody response.”
mechanisms is now well recognised; these two systems are Otherwise, if an immunomodulator can increase or promote
classified into cell-mediated defence and humoral components activity, it is called an immunostimulant. Finally, another
(soluble substances). Figure 1.1 describes their main functions. category is immune adjuvants, which hold the promise of
The innate system is made up of three parts: the tegument being the actual modulators (De Paula Barbosa 2014) of the
(skin and mucus), cellular components (granulocytes, mono­ immune response, especially to enhance the vaccine’s effi­
cytes, macrophages, and natural killer cells), and humoral cacy. An example in this regard is Freund’s complete adju­
components (granulocytes, monocytes, macrophages, and nat­ vant, which is being used to enhance the potency of poor
ural killer cells) (the complement system, antimicrobial enzyme immunogenic substances (Tengjaroenkul & Yowarach 2011).
system, and non-specific mediators such as interferon and Very often, the same agent can have both immunostimulant and
interleukin). Furthermore, inflammation is thought to be an immunosuppressive effects on an organism. Studies showed
innate immune response driven by complex interactions between that the physiological response of fish to different doses of
cellular and humoral components. Granulocytes are the first cells immune system stimulants could be different (Petit 2019).
to arrive at the site of inflammation and are responsible for In other words, in some cases, excessive use of several im­
killing infections. Innate and adaptive immune systems normally munostimulants simultaneously may cause immunosuppression
work in concert, with innate responses serving as the host’s first in fish (Raa 1996).
line of defence and enabling adaptive responses by antigen-
specific T and B cells to produce antibodies in the presence of
specific humoral components called histocompatibility mole­
1.4 Immune Suppressors
cules and glycoprotein receptors encoded by genes in the major
histocompatibility complex (MHC). Immunosuppressive compounds are chemical, biochemical, and
physical agents that suppress, decrease, or disrupt the immune
system functions in fish. Studies show that all immuno­
suppressant compounds carry a severe risk of infection. Various
1.3 Immunomodulators
types of immunosuppressive materials may change the immune
The immunomodulators are all antigens (chemical or phys­ response in fish when challenged by pathogens (Hidasi 2017).
ical) that manage to vary the immune system’s activities. Most immunosuppressants may weaken the immune system by
Immunomodulators 5

altering the gene expression of immune parameters. However, immune system in fish exposed to agrochemicals can increase
some immunosuppressive materials may decrease the absorption the susceptibility and vulnerability of fish to various pathogens
of vitamins and dietary supplements. Furthermore, immuno­ (Banaee et al. 2019; Farag et al. 2021). Changes in intrinsic
suppressive compounds reduce the ability of fish’s innate and and specific immunological indices are perhaps the most
specific immune systems to respond to foreign objects. The important reason for suppressing the immune system of fish
interaction of immune suppressants with drugs may also reduce exposed to agrochemicals (Hassan et al. 2022). This section
their effectiveness. The following part explains some immune explains the reasons for the decay of the fish immune system
suppression effects on the fish immune system. after exposure to agrochemicals.
Due to their lipophilic nature, most pesticides easily cross
biological barriers and enter the aquatic body. Pesticides can
1.4.1 Environmental Stressors
suppress the immune system by interacting with immune
The functions of the immune system of fish depend on nutrition, agents or causing oxidative damage in tissues involved in the
environmental conditions, health, gender, and ontogeny. immune system (Farag et al. 2021). Previous studies have
Temperature, dissolved oxygen, salinity, pH, and hardness shown that fish exposure to pesticides can cause changes in
ranges may also differ between fish species. Therefore, en­ haematological parameters, including a decrease in leucocytes
vironmental fluctuations could alter the fish’s physiological and an alteration in the differential count of white blood cells
status and immunological response. For example, hyperosmotic (Banaee et al. 2008). In fish treated with pesticides, decreased total
stress could decrease the immune system response of immunoglobulin, C3, and C4 complement activities have also
Scatophagusargus to bacterial infection (Lu et al. 2022). In been reported (Hatami et al. 2019). A significant decrease was
hostile biological conditions, fish may not be able to feed reported in lysozyme activity, respiratory burst activity, and total
properly. As a result, they will not have enough energy to immunoglobulin levels in Nile tilapia (Oreochromis niloticus)
allocate to the immune system (Estensoro et al. 2012). exposed to cypermethrin (Abdel-Tawwab et al. 2020). Exposure
Moreover, breeder malnutrition may lead to epigenetic changes to pesticides can also lead to changes in the gene expression of
in the offspring’s immune and metabolic genes. Improper fish inflammatory cytokines such as TNF-, IL-1, and IL-6 (Acar et al.
nutrition under stressful conditions can also lead to impaired 2021; Wang et al. 2020).One study found that glyphosate ex­
immune priming by dendritic cells and monocytes and impair posure altered the levels of interferon- (IFN-) and IL-1 in the
the function of effective memory T-cells. Acute and chronic hematopoietic tissues of common carp (Cyprinus carpio).
stress are critical agents in suppressing the immune functions of
fish (Guo et al. 2021).
[Link] Heavy Metals
Environmental stress can suppress the immune response in
fish. As a result, fish that live in stressful environments may have Exposure to heavy metals at levels higher than the accepted
a weaker immune system than fish that live in normal conditions. dosages could suppress the immune response in fish. Also, the
Guo et al. (2022) discovered that exposing Wuchang Bream transmission of heavy metal contamination through the food
(Megalobramaamblycephala) to ammonia nitrogen reduced chain can affect fish immune systems (Mohiseni et al. 2017). A
immunoglobulin M (IgM), interleukin 1 (IL-1), and tumour significant decrease in immune functions of Vardar chub
necrosis factor (TNF-) levels while decreasing TLR mRNA (Squalius vardarensis, Karaman) may be due to histopatholog­
expression. Following exposure to ammonia, there was a con­ ical damage to the kidney and spleen (Jordanova et al. 2017).
siderable drop in IgM and component C3 levels and lysozyme Bernier et al. (1995) demonstrated that bioaccumulation of
activity in the spleen and head kidney of Pelteobagrusvachellii heavy metals could cause suppression of the immune system,
(Qi et al. 2017). Moreover, those latter authors showed that high autoimmune diseases, increased susceptibility to pathogens, and
ammonia concentrations in the environment could disrupt the inflammation reactions in fish. Banaee et al. (2019) demon­
expression of immune-related genes in crucian carp (Carassius strated that alterations in humoral immune parameters in fish
auratus) (Mazini et al. 2022). It was observed that the immune exposed to heavy metals indicated quenching of innate immune
system’s response would be decreased when fish were exposed responses. Manganese reduced lysozyme activity and IgM levels
to the stress of transporting between different farms. They while increasing the expression of tlr3, tnf-, il-1, and il-6 in
discovered that an increase in corticosteroids was linked to juvenile yunlong groupers (Epinephelus maureus and E. lan­
immunological suppression in fish. Corticosteroids may affect ceolatus) (Wang et al. 2022). Heavy metals were exposed to
the effectiveness of the immune system by altering mRNA ex­ Centrarcus [Link]. changes in bactericidal activity in the skin
pressions implicated in immunological parameters. Therefore, to mucosa of gilthead seabream (Sparus aurata) after heavy metal
better understand the impact of environmental stressors, each of exposure have also been reported (Guardiola et al. 2015). A
the stressors has been discussed separately. significant decrease in HK-B cell proliferation, IgM level, and
serum bactericide potential was observed in catfish exposed to
[Link] Agrochemicals arsenic (Ghosh et al. 2007). Heavy metal exposure in fish
may affect leucocyte counts. Furthermore, changes in the
Agrochemicals include fertilisers, phytohormones, and pesti­ granulocyte/granulocyte ratio suggest that heavy metals
cides (e.g., insecticides, pesticides, herbicides, and fungicides) trigger the fish immune system. A significant increase in
used in the agriculture industry. Agrochemicals also include monocyte and neutrophil numbers and a significant decrease
medications, disinfectants, hormones, and growth stimulants in lymphocyte numbers were reported in goldfish (C. auratus)
used in cattle, poultry, and aquaculture. Suppression of the after exposure to manganese (Aliko et al. 2018).
6 Immunomodulators in Aquaculture and Fish Health

Genotoxicity may be linked to oxidative stress, DNA 1.4.5 Diseases

damage, and changes in mRNA expression. Indeed, Ghazy
Increased inflammatory cytokines in chronic diseases may be a
et al. (2017) found that the gene expression in the immune
reason to suppress the immune system. Furthermore, toxins
system changed in Nile tilapia that lived in waters contami­
and enzymes secreted by the primary pathogen can reduce the
nated with heavy metals. Heavy metals altered the expression
immunity of fish against secondary pathogens (Ilgová et al.
of IL1, TNF-, IFN, Mx, Lyz, C3B, and CXCL-Clc in zebrafish
2021). Simultaneous fish infection with two or more pathogens
(D. rerio) embryos (Cobbina et al. 2015). Moreover, signifi­
can weaken the immune system (Shameena et al. 2021). Ilgová
cant changes in the mRNA transcription of immune-related
et al. (2021) found that chronic pathogen infections could
genes were observed in the leucocytes of European sea bass
significantly delay the immune system response of fish. They
exposed to cadmium, lead, and mercury (Morcillo et al. 2015).
showed that infection with monogeneans could increase the
susceptibility of fish to secondary pathogens. Ilgová et al.
1.4.2 Other Xenobiotics (2017, 2020) demonstrated that infection with Eudiplozoon
nipponicum reduced TNF-gene expression in common carp
Segner et al. (2021) showed that xenobiotics could bind to the
macrophages in vitro.
aryl hydrocarbon receptor (AhR) as a vital transcription factor.
Then, complex AHR and xenobiotics were set on sequence
response elements and changed immune-related gene expres­ 1.4.6 Sex Hormones
sion. Suppression of the immune system in aquatic animals
Hormone manipulation and changes in the steroid hormones
exposed to xenobiotics may be due to energy shifts for detoxi­
may affect gene expression in a fish’s immune response.
fication. Decreased energy allocated to the immune system may
Therefore, a decrease in the immune system capacity of adult
reduce its performance.
fish may be related to changes in sex hormones. Szwejser et al.
(2017) found that fish leucocytes have receptors and cyto­
1.4.3 Natural Toxins chrome P450 aromatase. As a result, oestrogen levels in the
blood might affect the fish’s immune system. Cabas et al. (2018)
Qiao et al. (2013) investigated the immunotoxicity of cyano­
found a link between oestrogen levels and autoimmune illness
bacteria in Crucian carp. The immunological fish response
and chronic inflammation in fish and discovered that fluctuations
might be boosted by blood cyanobacteria, according to authors
in sex steroids affected the immune systems of spotted snake­
Rymuszka and Adaszek (2013), who assessed the effects of
heads (Channa punctatus). They found that increased sex ste­
microcystin derived from cyanobacteria on carp leucocyte
roids could mitigate innate and cellular immune responses.
proliferation under in vitro conditions. They showed micro­
Dietrich et al. (2021) showed that hormone therapy in common
cystin could increase apoptosis rates in white blood cells.
carp could change mRNA expression in hematopoietic tissues.
Rymuszka and Sieroslawska (2018) showed that carp leuco­
cytes’ half-life decreased after exposure to nodularin, a cya­
nobacteria toxin. Contamination of food with aflatoxin toxins 1.4.7 Drugs and Antibiotics
can reduce the potency of non-specific immune systems in fish
Antibiotics can suppress the fish immune system by disrupting
(Bitsayah et al. 2018). In common carp fed with aflatoxins-
the regulation of NF-B signalling and immunotoxic pathways
contaminated feed, Bitsayah et al. (2018) found a substantial
(Qiu et al. 2020). Yang et al. 2020 indicated that antibiotics are
shift in complement C3, C4, and CH50, lysozyme activity, and
toxic to fish at high dosages. They also showed that treatment
total immunoglobulin content.
with a high dose of antibiotics would suppress the fish’s immune
system. Increased mortality and an inflammatory response were
1.4.4 Anti-nutritional Agents observed in zebrafish treated with 260 ng of L-1 sulfameth­
Some antinutritional compounds in the diet can suppress the oxazole (Zhou et al. 2016). Common carp exposed to sulfa­
immune system in aquatic animals. However, the im­ methoxazole has reportedly seen a similar outcome (Iftikhar
munodepressive effects of anti-nutritional compounds on the et al. 2022). Liu et al. (2020) found that exposure of zebrafish
immune system depend on their bioavailability and their dosages larvae to high doses of sulfamethoxazole could change the
in feed. By generating cytotoxicity, anti-nutritional substances mRNA expression of cytokines such as IL-1, IFN-, IL-11, and
may have an impact on immune function. Furthermore, by TNF-. The genotoxicity effects of antibiotics on leucocytes
producing inflammation or modifying the quantity of inflam­ could mitigate the efficiency of cellular immunity in fish
matory markers in the blood, these substances may impair (Grondel et al. 1985).
immunological function. Inhibiting the absorption of micro­
nutrients in the intestine by anti-nutritional compounds can also
play a role in suppressing the aquatic immune system (Abdel-
1.5 Immunostimulants
Tawwab et al. 2018). The immune system may be harmed by
phytotoxins such as cyanogenic glycosides (Cho et al. 2013). Immunostimulants have a crucial role in activating the non-
Glycinin is a dietary allergen in soy, which can harm an animal’s specific defence mechanism in fish protection against patho­
immune system (Sun et al. 2008). Gossypol, an anti-nutritional gens, and they are valuable for controlling fish diseases.
compound found in cottonseed, can cause apoptosis by acti­ Previous studies have shown that numerous immunostimulants
vating caspase-3 (Sadahira et al. 2014). may be useful to fish cultures in aquafeed. Particularly for
Immunomodulators 7

enhancing and improving the immunity and disease resistance have additional effects on growth performance and the survival
in fish, increasing non-specific defence mechanisms immune rates of the fish under stress (Heo et al. 2001) (Figure 1.2).
stimulants may also be an effective strategy to increase fish
performance. Although the use of immunostimulants in the
1.5.1 Chemical Agents
aquaculture industry has been successful in some cases,
research in this field is still ongoing (Jadhav et al. 2006). Since immune system stimulants contain many chemical
Chemical agents, bacterial components such as probiotics compounds that may affect the function of the aquatic immune
(Abdel-Latif et al. 2022), polysaccharides (e.g., from plants) system, they can be administered alone or in combination with
(Faggio et al. 2016, 2015), animal or vegetable extracts (plant- vaccination. Some immunostimulants have been shown in studies
based) (Rashidian et al. 2021), feed additives and herbal ex­ to improve vaccination efficiency. Shahbazi and Bolhassani
tract (Elumalai, as pointed out above, immunostimulants) (2016) found that some biochemical compounds such as
might mainly facilitate the function of phagocytic cells and vitamin C and E, lactoferrin, interferon, growth hormone, pro­
increase their bactericidal activities (Abarike et al. 2019). lactin, and recombinant cytokines can act as immunostimulants.
Moreover, natural killer cells, complement, lysozyme, and The chemical agents could disrupt immunological structures and
other antibody responses may be stimulated by different types functions, making animals more susceptible to both infections
of immunostimulants. Immunological function and activation and non-infectious agents. A wide range of substances target the
are associated with improved protection against infectious immune system and prolonged exposure to these compounds
diseases in aquaculture due to their ability to serve as an can result in immunological dysfunction (Koller 2001). For ex­
alternative and supplement to vaccination. Moreover, they also ample, levamisole is the most useful chemical agent used and is a

FIGURE 1.2 Different categories of immunostimulants for fish.

Various natural and synthetic agents that enhance immunological activity in fish are depicted in the figure. Bacterial agents, specifically pre­
biotics and probiotics are of high potential, along with immensely used phytochemicals, yeast derivatives, chemical agents and metals.
8 Immunomodulators in Aquaculture and Fish Health

levo-isomer of tetramisole (Findlay & Munday 2000). It is a 1.5.3 Lipopolysaccharide

polysaccharide that can increase macrophage activity and provide
Lipopolysaccharide (LPS) is the main component of the outer
resistance to specific harmful microorganisms. On the other hand,
membrane of gram-negative bacteria such as Salmonella ty­
chitosan is a de-N-acetylated version of chitin. Both chitin and
phimurium and Escherichia coli (Miura & Mizushima 1968),
chitosan have the potential to be significant components in
and its preparations include O-antigens and endotoxins. The
aquaculture. Chitosan treatment by injection or immersion was
biological activity of LPS is a consequence of both hydro­
found to enhance brook trout (Salvelinus fontinalis) resistance to
phobic domains known as lipid A (or endotoxin), a “core”
an A. salmonicida infection. Chitosan treatments had a substantial
oligosaccharide, and a distal polysaccharide (or O-antigen)
influence on the non-specific immunity and immunological
(Neidhardt 1996). Moreover, LPS has been used as a potential
response of both healthy and cortisol-treated Labeo rohita
immunostimulant. Toll-like receptor (TLR)-4 is mainly
(Barman et al. 2013).
involved in the activation of the immune system by LPS
through the specific recognition of its endotoxin (lipid A)
1.5.2 Biological Substances and Bacterial Derivates moiety. LPS studies were made on fish both in vitro and in
vivo, and they reported that LPS influences the growth and
The most common biological substances are bacterial deriva­
health status of fish. (Guttvik et al. 2002) showed that Atlantic
tives, also known as killed pathogens, and their products. The
salmon fry fed with LPS-coated feed (0.1% LPS) for 63 days
use of immunostimulants such as probiotics and prebiotics has
had a reduced survival rate when challenged with a virulent
always been considered (Bachère 2003). Probiotics are a col­
strain of A. salmonicida. Furthermore, Paulsen et al. (2003)
lection of non-pathogenic microorganisms often found in
discovered that LPS stimulates plasma lysozyme activity
aquatic animal digestive systems. Oral administration of pro­
originating from macrophages in various organs (e.g., blood
biotics can change bacterial flora, boost the immune system,
polymorphonuclear and cells isolated from the head, kidney,
and stimulate growth performance (Villamil et al. 2002).
and intestine) in their experiment on Salmo salar. In an in vitro
Previous studies show that the administration of probiotics can
experiment, Paulsen et al. (2001) in Salmo salar found that in
significantly increase antibody production and non-specific
head kidney macrophages grown in the presence of LPS, there
immune parameters in fish (Abareethan & Amsath 2015).
was an increase in lysozyme production in the culture super­
Therefore, it is essential to identify and isolate different strains
natants, which coincided with an accumulation of lysozyme
of microorganisms to produce probiotics. Among the different
gene transcript in stimulated cells.
probiotic strains commonly used in fish are Bacillus, e.g., B.
subtilis and B. licheniforms; Lactobacillus sp. such as L. del­
brueckii subsp., L. bulgaricus, and L. acidophilus; and 1.5.4 Hormones and Cytokines
Bifidobacterium sp. Numerous reports on the use of probiotics
Hormones and cytokines are part of the neuroendocrine
in aquatic environments have been published (Van Doan et al.
system. Their role as immunomodulators in the immune
2020; Jahangiri & Esteban 2018; Chauhan & Singh 2019), and
system has been studied in recent years. Acute stress may often
for more details, readers are referred to Chapter 10.
be associated with fish life stages and have an impact on fish
immunity and health (Figure 1.4). An example could be stress
(resulting in potential advantages), thus involving short-term
Improve the challenges resulting in immune activation or enhancing pro­
feed value or cesses. Hormones generally can directly affect macrophages,
enhance its lymphocytes, NK cells, and mitotic activity. Cortisol, growth
Non- nutritional Improve the
pathogenic hormone (GH), prolactin (PRL), reproductive hormones,
value quality of its
and non-toxic ambient
melanin-concentrating hormone (MCH), and pro-opioid mel­
with less to no environment/ anocortin (POMC)-derived peptides have all been shown to
side effects Beneficial microflora affect immune function in many fish species (Harris & Bird
effects of 2000). The growth hormone (GH, or somatropin) is a hormone
probiotics Promote from the family of prolactin and somatostatin; the main role of
Help in GH and insulin-like growth factor-I (IGF-I) is in the regulation
immune
digestion
response and of body size in growing animals. Previous studies showed that
process
Reduce growth the administration of exogenous growth hormone (GH) im­
disease and proves many aspects of immune function, for example, cyto­
stress
toxic (Sakai et al. 1996), phagocytic (Yada et al. 2006),
haemolytic (Kim et al. 2013), and lysozyme activities (Harris
et al. 2000) as non-specific defences, and immunoglobulin
FIGURE 1.3 Benefits of probiotics. production as specific defences Yada (2007) observed that the
activation of immune function during seawater adaptation was
Figure 1.3 illustrates the functions of probiotics as an
immunostimulant in aquaculture. Probiotics promote closely associated with increased plasma GH levels in some
immunity, growth, digestion, reduce stress and improve euryhaline fishes. Moreover, a previous study (Yada et al.
the feed value and quality of microflora in fish. 2006) showed that ghrelin (an important regulator of GH
Immunomodulators 9

FIGURE 1.4 Interconnections between the general immune system and the endocrine system about environmental processes and fish life stages.

secretion) stimulates superoxide production associated with 1.5.5 Phytogenic Immunostimulants

phagocytosis in trout leucocytes. While GH increased the
(Plant extracts, herbals, garlic, ginger, triterpenic acid, poly­
mRNA levels of superoxide dismutase, which catalyses the
phenols, olive oil, seaweeds)
dismutation of superoxide into oxygen and hydrogen peroxide,
Phytogenics are plant-derived natural products characterised
Also, Pontigo & Vargas-Chacoff (2021) found that GH may
by their richness in biologically active compounds that are
modulate the immune response in the SHK-1 cell line and
mainly incorporated into the feed to enhance the innate immu­
leucocyte cultures of the head kidney in Atlantic salmon.
nity, health status, and growth performance of the animals. In
Therefore, their work points out the independent action of GH
farmed fish, phytogenics have been reported to contribute as
on the immune system and the GH/IGF axis.
antimicrobials, antioxidants, anti-inflammatory agents, im­
Cytokines are low-molecular-weight glycoproteins involved
munostimulants, and sedatives. They work as promoters of
in regulating the immune system. These molecules are mainly
growth and appetite stimulators, and they could influence the
secreted by cells of the innate and adaptive immune systems,
bile secretion and several enzymes associated with digestion
but they play an important role in the innate immune response
(Chakraborty et al. 2011, Firmino et al. 2021, and Caipang et al.
in fish. Kono et al. (1996) reported that IL-1 plays an essential
2021). Botanicals, including herbs and spices, contain aromatic
role in fish immunity by activating lymphocytes and phago­
compounds and essential oils (extracted from parts of plants
cytic cells and increasing resistance to A. hydrophila infection.
such as leaves, flowers, roots, and fruits), and many other
Type I IFNs (homologs to human IFN- and IFN-) also have
medicinal plants come under phytogenics (Caipang et al. 2021).
antiviral activity. Secombes & Belmonte (2016) discovered
Plant extracts are active substances with desirable properties that
that type II IFN (IFN-) had bactericidal activity against intra­
are extracted from plant tissue for specific purposes, such as
cellular parasitic bacteria. Studies to elucidate cytokines’
immunostimulant use. They have been known to have increased
functions in fish have recently begun. Still, more work is
lysozyme activity, complement activity, phagocytic activity, an
needed to select their appropriate functions, such as im­
antibody response, elevated respiratory burst activity, and higher
munostimulants and vaccine adjuvants, to prevent infection in
plasma protein (albumin and globulin) (Reverter et al. 2014;
farmed fish. Sakai et al. (2021) developed a multiplex reverse
Harikrishnan R et al. 2011a). Saponin compounds, herbs, ginger,
transcription-polymerase chain reaction assay to investigate
triterpenes from fungi and plants and seaweeds, etc. are some of
the immune response of fish when activated by an im­
the compounds and products which have an immunostimulatory
munostimulant. Moreover, cytokines are also defined as bio­
effects on fish health and welfare and are discussed in detail in
logical response modifiers because of their ability to enable
this book.
communication between different cell populations, in agree­
ment with what was reported by Wilson et al. (2002).
Conforming to changes in environmental factors such as 1.5.6 Nutritional and Dietary Factors
water quality, salinity and diseased conditions, the T-cell
proliferation and cytokine expression also range, which is (Dietary amino acids, vitamin C, vitamin E, dietary nucleotides,
mediated through hormonal regulations. Hormones expressed organic acids, polysaccharides, probiotics, and food waste)
during sexual maturation and larval growth enhance the ex­ Nutrition plays a crucial role in maintaining the body’s func­
pression of these immunological mediators. tioning and health. It provides all the essentials required by the
10 Immunomodulators in Aquaculture and Fish Health

Vitamins are organic compounds necessary for animal growth

Lysozymal and development. They are required in small quantities and must
antibacterial be provided with food, as they cannot be synthesised in the body.
activity in the At present, vitamins C and E have gained popularity as im­
presence of
complement Phagocytosis munostimulants (Kono et al. 1996). Vit E (tocopherols) are
Resistance process is one of bioactive phenolic compounds, and a proper dose of this can (i)
against parasitic the main promote the differentiation and proliferation of lymphocytes and
and viral mediators of cytokines, (ii) enhance the production of antibodies and enhance
infections innate immunity
to pathogens
complement activity when encountered with an antigen, and (iii)
Phytogenic as it can also improve phagocytosis activity and cytotoxicity. In
immunostimulants channel catfish and turbot, VE enhances macrophage phagocy­
induce tosis (Barman et al. 2013). Vitamin C (ascorbic acid) is involved
Antibody- as a cofactor in many bioactive processes, like neuromodulation,
Respiratory burst
mediate immune to limit the collagen synthesis, and cellular activities related to hormones,
response to growth of fish and the immune system.
encounter the pathogens Dietary nucleotides, chitin, and organic waste do also have
infections
Plasma protein, compounds with immunostimulatory effects and are discussed
albumin, globulin
in subsequent chapters. Bacterial and yeast cell walls mostly
and cause strong
innate response consist of glucans. When glucan was given to feed, it activated
in fishes phagocytic cells in fish, and they also demonstrated an increase
in the development rate of Litopenaeus vannamei juveniles,
enhancing phagocytosis and the capacity of the cells to elim­
FIGURE 1.5 Activities of plant extract as an immunostimulant. inate harmful pathogens. Additionally, they increase comple­
Figure 1.5 represents the role of plant extracts in enhancing ment and lysozyme activity (Kono et al. 1996). Additionally,
immunity in fishes. Plant extract stimulate immunity by different glucans improve the non-specific defence mechanisms of fish
cellular mechanism like phagocytosis, respiratory burst, antibody and shellfish and offer defence against bacterial infections
mediated responses, lysosymal antibacterial activity, and stimulate (Barman et al. 2013). Prebiotic compounds called fungal
immunogenic plasma proteins. polysaccharides are commonly regarded as a dietary compo­
nent for controlling growth and health issues. Higher fungi are
excellent providers of a variety of crucial natural compounds
body to maintain life, like metabolic energy and elements and (Mohan et al. 2019). More details are discussed in subsequent
compounds that act as co-factors for various physiological pro­ chapters (Figure 1.7).
cesses. When we talk about immunostimulants, dietary nutri­
tional components include dietary amino acids (AA), vitamin C,
1.5.7 Trace Elements and Metals
vitamin E, dietary nucleotides, organic acids, polysaccharides,
probiotics, food waste, etc. Dietary amino acids are essential Minerals that are less abundantly present in living tissues are
acids that can improve the haemocyte count, phagocytic activity, referred to as trace elements (or trace metals). They are con­
respiratory burst in haemolymph, and lysozyme activity in cell- sidered to be nutritionally essential, although if consumed at
free haemolymph (CFH). Moreover, they can remarkably sufficiently high levels, they may prove toxic. Copper, chro­
downregulate the malondialdehyde content (Luo et al. 2021). In mium, iron, iodine, fluoride, manganese, molybdenum, sele­
addition to these, AAs are necessary for endogenous synthesis of nium, and zinc are considered as essential trace elements.
protein and act as important energy substrates. These can also General functions of minerals include structural constituents of
modulate the necessary metabolic pathways (Dawood et al. tissues, formation of the exoskeleton, osmotic pressure balance,
2021) (Figure 1.6). muscle contractions, and nerve impulse transmission. They are

Channel catfish Increased disease resistance to the

Arginine bacteria Edwardsiella ictaluria
(Ictalurus punctatus)

Gilthead seabream Enhanced innate immune

Trypthophan response
(Sparus aurata)

Channel catfish (I. Improve immune response

Glutamine
punctatus)

FIGURE 1.6 Effects of different amino acids on fish health.

Essential amino acids such as arginine and tryptophan and non essential aminoacid like glutamine have increased effect on fish immunity.
Arginine, in channel catfish improve the resistance in fish against Edwardsiella ictaluria. Tryptophan in Gilthead seabream has a transient
immune enhancement activity in the fish. Similarly, glutamine improve the immune response in channel catfish.
Immunomodulators 11

1.5.9 Yeast Hydrolysate

Antimicrobi Yeast is known for keeping the digestive system of an animal’s
al activity body healthy and in balance; hence, it is widely used as a sup­
plement in aquatic feeds. Yeast has a high protein and energy
Immunostim
Promote
ulantory content, as well as high micronutrient content. Aside from being
growth
effects high in amino acids and proteins, yeast products also have im­
munomodulatory compounds like mannan oligosaccharides
(MOS), chitin, glucans, and nucleic acids. Mannan oligo­
saccharides are well known to enhance the growth performance
Prebiotics of rainbow trout (Oncorhynchus mykiss), Aespeciallyes cerevi­
Enhance
innate Hypoglyce siae is a well-known yeast from which glucan is extracted and
immune mic activity purified, and an intraperitoneal injection of this improves both
responses specific and non-specific immune responses in carp (Cyprinus
carpio) to the bacterial challenge posed by A. hydrophila. Yeast
Gut health hydrolysate is a hydrolysate of yeast cells obtained through
maintanence various methods like acids, enzymes, or other hydrolysis. The
Disease (improving
resistance the growth extract may be obtained through autolysis, where the enzymes
of intestinal found in yeast itself are used to break down the protein, or
microbiota)
through hydrolysis, where enzymes are added from external
sources as depicted in Figure 1.8 (Gong et al. 2019).
FIGURE 1.7 Functional properties of prebiotics.
Prebiotics have an enhancing effect on fish growth, antimicrobial
activity, and disease resistance. They improve favourable intestinal 1.6 How to Administer the Immunomodulators
microbiota and stimulate immune system in teleost.
to Fish?
Overall, the immunostimulants could be administered through
also prime components for co-factors in metabolism, catalysts, different routes. Although injection methods are the best strategy
enzymes, enzyme activators, hormones, pigments, and vitamins. to enhance non-specific immune system responses, this method is
costly and time-consuming. Furthermore, injection methods are
1.5.8 Synbiotics only performed by experts. Therefore, it is applied in experiments
where the fish are intended as brook stock in genetic studies.
Synbiotics are a combination of both prebiotics and probiotics Another method is immersion, but its efficiency is less than that
and work as growth and immunity promoters. These have been of injection; however, it requires crowding and an increase in the
used in aquaculture for over a decade, but the functional handling of fish stocks. Immune system stimulants may be pre­
mechanism is still not very clear. Prebiotics, which are parts of scribed as oral supplements. However, the immunostimulants‘
synbiotics when hydrolysed to simpler mono- or disaccharides, concentration in the diet depends on the size, ontogeny stages,
show an exceptional increase in biomass and colonisation of and initial weight of the fish. This method consists of oral
probiotic bacteria on the surface of intestinal epithelial cells in ingestion, produces a suitable non-specific immune response, and
the host. By releasing extracellular bacterial enzymes and can be the most cost-effective method of administration. Top
bioactive substances from their metabolic activities, they also dressing can help you achieve these. The surface of the food is
contribute to the growth of aquatic animals. These enzymes treated with the pure immunostimulant in this case. This is
also improve the nutrient absorption capacity, which in turn comparable to employing a layer of fish oil to top-dress antibiotic
help in effective utilisation of feed. Synbiotics stimulate the granules. This technique produces variable results depending on
immune system’s synthesis of nitric oxide, phagocytosis, and how well the immunostimulant adheres to the feed. At last, one
respiratory burst activity in fish. method that is much more advanced is bio-encapsulation.

Additional use Finally, the

Collected yeast of the nuclease, yeast
Saccharomyces hydrolysate
(Saccharomyces peptidase and
cerevisiae used as procured after
cerevisiae) began protease to Purification
strain
to autolyze hydrolyze and (condensing
catalyze and drying)

FIGURE 1.8 Extraction of hydrolysate.

Demonstrate the pathway for the extraction of hydrolysate from yeast to use as an immunostimulant for fishes. The yeast (Saccharomyces
cerevisiae) are collected, autolyzed yeast is subjected to nuclease, peptidase, and protease to get the hydrolysed product, which is subsequently
purified using condensing and drying.
12 Immunomodulators in Aquaculture and Fish Health

most effective against a range of pathogens. Vaccination

1.7 Limitations of Immunostimulants seems to be impractical for small fish. Immersion is
commonly used in intensive culture systems, despite the
• Though showing extraordinary growth in the field of
fact that it is less expensive than injection, produces a less
aquaculture, one of the important disadvantages of some
non-specific immune response, and stresses the fish
immunostimulants is their high cost.
during handling. It is most effective during the acclima­
• The administration of the drug is important to consider tion of juveniles to ponds in field conditions. Oral
as it depends on its efficacy. Immunostimulants show ingestion is good for extensive aquaculture systems. It is
limited efficiency upon parental administration. cost-effective and enhances non-specific immune
• Immunostimulants are not completely effective against responses (Barman et al. 2013).
all diseases.
• Overdoses of immunostimulants in feeds may cause
immunosuppression.
1.9 Evaluating the Efficacy of
• In some cases, aquatic animals may also fail to provide
enhanced protection or an increase in immunity.
Immunostimulants
• Immunostimulants are successfully used in aquaculture In vivo and in vitro methods can be used to assess the efficacy
against various infections and pathogens; however, the of an immunostimulant. The in-vivo method employs fish
ability to improve innate resistance against many dis­ pathogens to assess the efficacy of immunostimulants, whereas
eases (e.g., columnaris disease) has not been studied. the in-vitro method examines cellular and humoral immune
mechanisms. In-vivo and in-vitro methods should be per­
formed together to check the basic mechanisms for providing
protection. In preliminary studies, in vitro methods are pre­
1.8 Factors Affecting the Efficiency of ferred. In vitro evaluation is based on lymphocyte prolifera­
Immunostimulants tion, complement activation, total erythrocyte and leucocyte
The effectiveness of an immunostimulant can depend on var­ counts, chemokinesis, chemotaxis, lysozyme activity, and
ious factors, as follows: RBA phagocytosis. Other parameters include monitoring nat­
ural cytotoxic activity, macrophage-activating factor (MAF)
• Solubility: Laminaran is an algal extract that can boost levels, and C-reactive protein levels. However, these tests are
respiratory burst activity in leucocytes of the anterior too expensive to be conducted to check the efficacy of im­
kidney and activate macrophages in Atlantic salmon munostimulants. A deep level of research is required to check
because it is more soluble than the fungal and yeast the efficacy of various compounds for aquaculture species and
glucans. It has also been demonstrated to be a potential their pathogens and to ultimately decrease the cost of the
chemical for diet usage due to its greater solubility, so immunostimulants.
the solubility is considered an important aspect for
immunostimulants.
• Duration of dose: Salmon that received M-glucan 1.10 Timing of Administration
injections only took 2 days to produce their peak leu­
It is very important to use immunostimulants at the correct
cocyte responses. After 4–7 days of therapy with yeast
time and in the right concentration to boost the immune
beta-glucan, the respiratory burst activity increased. This
system. Anderson proposed in 1992 that it is best to use im­
demonstrated how immunostimulants might improve
munostimulants prior to the possibility of disease outbreaks in
non-specific immunity with very brief dosages.
order to minimise disease-related loss. Furthermore, the
• Dosage: A high dose or overdose certainly does not seem effective dose and timing of exposure have a significant impact
to have an enhancing effect and can, in turn, inhibit the and are complicated by the culture system and feeding sce­
immune responses. At concentrations of 0.1–1 g/mL, the nario. Studies in Atlantic salmon showed that the maximum
respiratory burst activity of glucans-treated macro­ non-specific disease resistance is attained only after the third
phages increased. Whereas glucan had no effect at a week of injecting glucan at 10 mg/100 g, whereas the effects
concentration of 10 g/mL, it was inhibitory at a con­ of low dosing at 1 mg/100 g last only for 1 week. Similarly, in
centration of 50 g/mL. Very high vitamin E levels in African catfish, administration of glucan led to a maximum
feed, such as those of a >1,000–5,000 IE/kg diet, have increase in the phagocytic cells at 7 days but not after 14 days.
an immunosuppressive effect. So, the dose per unit So, it is preferable to use them well in advance and at regular
weight has a significant effect on efficacy. intervals (Barman et al. 2013).
• Time of administration: applying immunostimulants at
the right time is very important in aquaculture. Mostly,
application is needed before the outbreak of disease so
that losses due to disease can be reduced. 1.11 Detection of Immunostimulants
• Method of Administration: The administration of im­ Detection of immunostimulants in fish body is detected by
munostimulants through injection has been reported to be using ‘omic’ technologies.
Immunomodulators 13

Methods of detection of immunostimulation are as follows

and it is discussed in further chapters in detail:

• In vitro measurement
• In vivo measurement
• Phagocytic activity

1.12 Attributes of Immunostimulants

The most important attribute of an immunostimulant is that it
directly influences the animal’s health. It is biodegradable and
biocompatible, and therefore safe for the environment. It en­ FIGURE 1.9 Vaccination via injection.
hances the immune system of animals, promoting good health, Figure 1.9 represents vaccine administration through injection via
and is non-toxic to both fish and shellfish with no side effects intramuscular or intraperitoneal method to increase resistance against
observed. In aquaculture, it provides disease resistance to an­ pathogens by stimulating immune system.
imals against a broad spectrum of pathogens and reduces
mortality caused by opportunistic pathogens. It can also keep
the host safe by providing enhanced immune stimulation to Fish immunisation started in 1942 when David C. B. Duff
fight viral diseases. It increases the effectiveness of many an­ successfully immunised trout orally against the bacteria
timicrobial substances, vaccines, and antibiotics. Moreover, it Aeromonas salmonicida (the first fish vaccine). He is known
is cheap, easily available, and most importantly, an eco- “Father of Fish Vaccination”. The first commercially approved
friendly method for immune stimulation (Barman et al. 2013). fish vaccination was a dead Yersinia ruckeri vaccine against
enteric redmouth disease that was administered by immersion
in 1976.

1.13 Vaccine
A vaccination is a biological treatment that increases immunity
1.14 Immunostimulants vs Vaccine
to a specific disease. Vaccine often comprises an agent that re­
sembles a microorganism that causes a disease and is frequently Table 1.1 is the comparison of immunostimulants and vaccines
created from weaker or dead versions of the pathogen. In order (Dawood et al. 2021).
for the immune system to more quickly identify and eliminate
any further interactions with this disease-causing micro­
organism, The agent prompts the body’s immune system to
identify the agent as foreign, eliminate it, and “remember” it so 1.15 New Paradigm
that the immune system will be better able to identify and
1.15.1 Nutrigenomics
eliminate any of these microorganisms that it comes into contact
with in the future. “Prevention is better than cure” is the core Nutrigenomics is a branch of science that integrates bio­
tenet of a vaccination. The name “vaccine” originated from informatics, nutrition, genomics, molecular biology, and epi­
Edward Jenner’s usage of the phrase “cow pox” in 1796 (Latin demiology. It links the relationship between nutrients and
“variolvaccin,” which was borrowed from the Latin “vaccn-us,” cellular processes and shows how the dietary components alter
from “vacca” cow). He was a pioneer in the use of cowpox the genetic makeup. Although the relationship between nutrition
vaccinations to stop the spread of smallpox. and the immune system is generally known, it is still unclear

TABLE 1.1
The Comparison of Immunostimulant with Vaccines
Immunostimulants (IS) Vaccine
• More treatments are required as the prophylactic effect is short term • One or two treatments are enough as prophylactic effect is
• Efficacy of immunostimulants is good long
• IS possess wide spectrum of activity • Efficacy of vaccine is excellent
• Nontoxic with less side effects • Vaccine possess limited spectrum of activity
• No toxic residue accumulation • Nontoxic with less side effects
• Positive/no environmental impact • No toxic residues accumulation
• Mainly enhance non-specific immune system before specific immune system • No environmental impact
matures. • Enhance specific and nonspecific immune response
• Can be used at any stage of life cycle • Cannot be used at any stage of life cycle
• Easy to supply to larvae of fish and shrimp • Difficult to supply to larvae of fish and shrimp
• Cost-effective • Costly
14 Immunomodulators in Aquaculture and Fish Health

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nologies like transcriptomics (microarray and RNA-seq) and Nile tilapia (Oreochromis niloticus) exposed to glyphosate-
proteomics. Modules of genes can reveal changes in both local based herbicide. Comparative Biochemistry and Physiology
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fasting, feed additives, and protein replacement on gene ex­ Antioxidant defense system, immune response and erythron
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tively new approach in aquaculture, but the scope it provides to acute manganese treatment. Fish & Shellfish Immunology,
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2
Natural and Synthetic Immunomodulators:
Inferences for Stress Responses in
Aquaculture Fish

Shubhajit Saha1, Azubuike V. Chukwuka2, Nimai Chandra Saha3, Caterina Faggio4, and Hamed Mousavi Sabet5
1
Fisheries and Ecotoxicology Research Laboratory,
Department of Zoology, The University of Burdwan,
Burdwan, West Bengal, India
2
National Environmental Standards and Regulations
Enforcement Agency (NESREA), Nigeria
3
University of Burdwan, Purba Barddhaman, India
4
Department of Chemical, Biological, Pharmaceutical
and Environmental Sciences, University of Messina,
Messina, Italy
5
Department of Fisheries, Faculty of Natural Resources,
University of Guilan, Sowmeh Sara, Iran

the preparation and presentation of phagocytosed antigens,

2.1 Introduction which have an impact on cell-mediated reactions.
Antibiotics are typically used in feed or water to manage
Fish are frequently stressed because they are exposed to several bacterial diseases, boost production, and reduce mortalities in
pathogenic agents, including bacteria, fungi, viruses, and para­ fish farms since immune systems may not have the full potential
sites transferred on their farms [1]. Disease-related issues have to effectively prevent and eliminate such infections [4].
been one of the industry’s biggest roadblocks over the past However, the emergence of antibiotic resistance makes its hap­
20 years. The rising prevalence of sickness has caused the spread hazard use potentially harmful to the ecosystem [5]. As a result,
of infectious organisms between nations. As a result, the shrimp different aquaculture systems have explored and adopted other
business in various climes has experienced enormous financial approaches to enhance or modify the immunological activity to
losses. Immunostimulants have emerged as the preferred improve fish responses to xenobiotics and pathogenic agents [6].
strategy for controlling fish and shrimp infections because there Immunostimulants are substances that directly interact and
are no effective treatments for these viral diseases [2]. activate immune system cells to improve innate or non-specific
Fish that successfully combat pathogen infection either live or immune responses when administered to an organism. They
die depending on how well they can stop the spread of the infec­ are primarily divided into two categories: synthetic (chemical)
tion. The potency of immune responses of an organism to attenuate agents and natural agents, such as those derived from
the onset and proliferation of pathogens determines outcomes of cytokines, polysaccharides, animal or plant extracts, or bacterial
eventual survival or mortality. Innate immunity and acquired preparations [7]. Immunostimulants have been used to suc­
immunity (specific) are two categories of the immune system of cessfully control a variety of pathogens in fish and shrimp,
fish (non-specific). Both rely on cellular, humoral, and physical including bacteria like Aeromonas hydrophila, A. salmonicida,
defenses against pathogenic microorganisms. The particular Edwardsiella tarda, E. ictaluri, Vibrio anguillarum, V. vulni­
immune systems offer a defense against a particular infection by ficus, V. salmonicida, Yersinia ruckeri, and Streptococcus spp.
identifying particular antigens on that organism. Innate immune Viral pathogens that have been overcome by immunostimulation
systems offer a collection of defense responses that are on standby of fisheries include acute hematopoietic necrosis infections,
to put a wide range of infections under check [3]. yellow head disease, Egtved disease, and the Ich parasitic ciliate.
The adaptive immune system is highly dependent on the An immunostimulant is a substance that enhances either the
innate immune system as its first line of defense against anti­ immune response or the non-specific defensive mechanism [8].
genic assault. Macrophages are thought to be a crucial com­ Aquaculture uses immunostimulants to combat the immuno­
ponent of the cellular immune system in fish and work to suppressive effects of stressors [9] as a preventative measure
defend the host by phagocytizing foreign substances, including against anticipated seasonal breakouts of known endemic ill­
pathogens. Additionally, macrophages actively participate in nesses [10] or as a suppressive measure against latent or sub-

18 DOI: 10.1201/9781003361183-3
Natural and Synthetic Immunomodulators 19

TABLE 2.1
Immunostimulants Evaluated in Fish and Shrimp (Adapted from Barman, Nen [ 2])
Immunostimulants Effects of Immunostimulants References
Synthetic chemicals Levamisole, fK.565 (Lactoylretrapeptide from Streptomyces olvaccogriseus). Quaternary
ammonium compounds (QACs)
Natural substances
1. Bacterial derivatives MOP (Muramyl dipeptide from Mycobocterlum species). Lipopolysaccharides (LPS), Freund’s [ 11]
complete adjuvant (FCA), EF203 (fermented egg product), Peptidoglucan (from
Brevibacrerium lactofermentum and Vibrio sp.), Clostridium butyricum cells, Achromobacter
stenohalis cells, Vibrio anguillarum cells (Vibrio vaccine)
2. Yeast derivatives b-1, 3 glucan, b- 1, 6 glucan
3. Nutritional factors Vitamin C and E, n-3 fatty acid
4. Hormones Growth hormone, Prolactin, triiodothyronine
5. Cytokines Interferon, interleukins
6. Polysaccharides Chitosan (brook trout), Chitin, Lentinan, Schizophyllan, Oligosaccharide [ 12]
7. animal extracts Fermented products of chicken egg (EF203) on rainbow trout [ 11] [ 11]
8. plant extracts Ocimum sanctum (Tulsi), Phyllanthus emblica (Amla), Azadirachta indica (Neem), Solanum [ 12]
trilobatum (Purple Fruited Pea Eggplant), Eclipta alba (Bhringraj), Zingiber officinale
(Ginger), Echinacea (Purple coneflowers), Allium sativum (Garlic), Camellia sinensis (Green
tea), Cynodon dactylon (Bermuda Grass), Achyranthes aspera (Prickly Chaff Flower,
Nyctanthes arbortristis (Night-flowering Jasmine)
9. Fermented vegetable The phagocytic activities and superoxide generation of peritoneal induced leukocytes were [ 13]
product (FVP) significantly higher in fish Japanese flounder (Paralichthys olivaceus) fed the FVP
supplemented diet
10. Mucor circinelloides (Fungi) Gilthead seabream growth was slightly higher in specimens fed M. circinelloides-supplemented [ 14]
diets
11. Spirulina platensis Spirulina enhanced responses of bactericidal, phagocytic activity and lysozyme activity in [ 15]
tilapia, Oreochromis niloticus
12. Others Lactoferrin, QuiI A, Achyranthes aspera (Herb)

lethal infections. Additionally, immunostimulants can aid in the However, the indiscriminate use of antibiotics has led to the
recovery from stress-induced immunosuppression states [7]. emergence of bacterial strains that are resistant to them. In both
Table 2.1 provides a summary of the various immunostimulants treatments for parasites, trichlorfon or praziquantel, for
tested on fish and shrimp. instance, have drawbacks such as the emergence of resistance,
Chemicals called immunostimulants cause leukocytes to potential risks to animal health, and environmental pollution
become active [16]. One of the first immunostimulants to be [5]. Additionally, a single vaccination only protects against a
used in animals to increase a particular immune response was specific kind of infection, and immunizing young fish is time-
adjuvant (FCA), which recorded desirable results in combined consuming and expensive. Sequel to the above-mentioned
administration with fish bacterins injection [8]. Glucose mole­ challenges, the use of natural immunostimulants, i.e., sub­
cule variants i.e., the glucans polymer extracted from plant cell stances of natural origin have been a major alternative
walls, and microbes (fungi, and bacteria), have been docu­ approach to ensure sustainable aquaculture (Table 2.1).
mented with a greater level of potency particularly through oral Particular plant extracts have been found to activate the
administration for aquaculture fish and shrimp [17]. It is prac­ immune system in fish cultures, increase growth, increase appe­
tical to use these various immunostimulants to improve the tite, and have anti-parasite and antibacterial characteristics.
immunological capacity and disease resistance of fish and Popular examples of herbal immunostimulants include saponins,
shellfish. Numerous drugs are now being used in aquaculture, phenolics, glycosides, and essential oils. Others include alka­
and research on immune stimulants is ongoing. loids, flavonoids, terpenoids, tannins, and steroids [6]. The an­
The two broad classes of immunomodulators, their peculiar tistress, antipathogen, appetite-stimulating, growth-promoting,
dimensions of contributing to fish health and aspects of their tonicity-enhancing, and immunostimulatory actions of plants and
limitations are discussed in the following section. their extracts have been demonstrated in aquacultures [18–20].
Over the past ten years, various aquaculture systems have
used medicinal plants and their derivatives extensively as im­
munostimulants [21]. In both freshwater and marine fish species,
2.2 Natural Immunomodulators
herbal medicine has been shown to enhance the immune
As already mentioned, fish rely on innate immunity, but im­ response and hematological parameters as well as reduce losses
munostimulants significantly improve innate immune from bacteria, viruses, and parasites [22].
responses. To control disease in aquaculture, chemother­ The health and physiological status of the fish can be mon­
apeutics, vaccinations, and antibiotics have been employed. itored using hematological and non-specific immunological
20 Immunomodulators in Aquaculture and Fish Health

markers [23]. Pourmoghim and Haghighi [24] documented plant to improve health, boost the body’s defenses against infection,
extracts that enhanced the plasma levels of total protein, glob­ and treat and prevent a wide range of diseases [25]. The innate
ulin, and albumin compared with the control. These serum immune system can be modulated by herbal immunostimulants,
proteins function as humoral components and detected increases which are less expensive or less effective than the medications,
suggested an improvement in the fishes’ non-specific immune including synthetic antibiotic drugs currently administered to
reactions. In many nations, immune-boosting herbs are utilized diseased fish and shellfish [26] (Table 2.2).

TABLE 2.2
Effects of Various Compounds on Immune System of Fish
S. No Name of Compounds Immunological Responses References
1 Polyphenols • Proliferation of the immunocompetent cells. Including T helper 1 (Th1), [ 27]
natural killer cells, dendritic cells (DCs), and macrophages in secondary
lymphoid organs.
• Increasing splenic T and 8 cells populations.
Flavanols Suppress on of inducible nitric oxide synthase (INOS), cyclooxygenase-2 [ 28]
(COX-2) as well as cytokines TNF-a, IL-1ß, and IL-6 from macrophage.
a Epigalocatechin-3-gallate • Foxp3 and IL-I0 gene expression increases in Jurkat T cells. [ 29]
• Increases regulatory T cells proliferation in spleen, pancreatic lymph
nodes, and mesenteric lymph nodes. Decreases the Th1 and Th 17
populations and lower the expression of T-bet and ROR-yt in animal
models of experimental autoimmune encephalomyelitis.
• Decreases the Th9 cells proliferation along with decreased expression of
the PU-I.
• Immunosuppressive and decreases IFN-y production in vitro.
b Baicalin Under TGF- ß stimulus increase the Foxp3 gene expression and also [ 30]
induction of functional Treg from splenic CD4+CD25-T cells in HEK 293
T cells.
c Procyanidin CI Decreases the secretion of TNF-a, IL-I-ß, and IL-6. [ 31]
d Fisecin Inhibits the expression of IL-6 and TNF-a inTHP-1 monocytes cells exposed [ 32]
to high osmolarity.
e Theaflavin Decreases the expression of IL-6, Monocyte chemoattractant protein-I [ 33]
(MCP- 1) and Intracellular adhesion molecule-I (ICAM-1).
f Curcumin analog 3,5-Bls (2- Inhibits secretion and express on of TNF-a, L-1-, and IL-6. [ 34]
pyridinylmethy lidene)-4-
piperidone (EF31)
g Propolis It can act as vaccine adjuvant. [ 35]
h Resveratrol • Reducing the levels of pro-inflammatory cytokines TNF-a, L-I-. and IL-6 [ 36]
and inflammation.
• Increases proliferation CDl9+ cells and immunoglobulin synthesis in
8lymphocytes.
i Resveratrol analog RVSA-40 Increases anti-inflammatory cytokine IL-I-ß together with down- regulation [ 37]
of TNF-a and IL-6.
j 7-0-Methylnaringenin Down regulate TNF-a. L-6 and IL-1-f expression in a dose-dependent [ 38]
manner.
2 Pentachlorophenols • Enhances B and T-lymphocyte blastogenesis. [ 39]
• Decreases antibody titers and delayed type hypersensitivity response and
increases induced peritoneal macrophage count.
3 Hydroquinone • Inhibits IFN-y production and increases Th2 response mediated allergic [ 40]
diseases. Enhances IL-4 production In CD4+ T cells and also increases
age levels in antigen-primed mice.
• Blocks the IL-12 production via suppression of NF-Kß activity.
• Inhibition of the production of IL-1-ß, IL-2, and NO.
• Suppresses the production of TNF-a, IL-1-ß, and IL-6.
• Inhibits production of NO and ROS phagocytic uptake of FITC- labeled
dextran.
• Induces production of costimulatory molecules.
1 Organophosphate
1.a Chloropyrifos • Increases CD26 expression on cytotoxic T cells. [ 41]
• Reduces mitogenesis immune cells in response to concanavillin and
phytohemagglutinin.
• Amplifies autoantibodies production.
1.b Diisopropyl methyl phosphonate • Inhibits activity of human and murine NK cells and murine cytotoxic T [ 42]
cells.
Natural and Synthetic Immunomodulators 21

TABLE 2.2 (Continued)

Effects of Various Compounds on Immune System of Fish
S. No Name of Compounds Immunological Responses References
1.c Malathion • Suppresses of NO production and LPS-induced TNF-alpha generation. [ 43]
• Increases antibody production following immunization with a T-
lymphocyte dependent antigen and macrophage function and led to mast
cell degranulation.
2 Carbamate
2.a. Aldicarb • Increases TB lymphocytes population and decreases T4: TB cell ratio. [ 44]
• Increases lymphocytes proliferation and alters macrophage functions by
decreasing with IL-I production.
2.b. Dimethoate Decreases total serum lgG and lgM and T cells in the thymus, forming [ 45]
autologous rosettes.
2.c. Sodium methyl dithiocarbamate Inhibits expression of IL- Iα, IL- Iβ, IL- 1B, IL- 12, IFN-y, p35, p40 m-RNA [ 46]
level and macrophage migration inhibitory factor (MIF), whereas increases
IL- I 0 m-RNA level.
2.d. Mancozeb • NO production decreases with the in vitro exposure. [ 47]
• Suppresses TNF-y secretion in vitro whereas enhance release detected in
ex vivo experiment.
2.e. Carbendazim Decreases B lymphocytes proliferation and serum lgG, lgM, and lgA levels. [ 48]
3 Organochlorine
3.a. Endosulfan Decreases lgA and lgG production. [ 49]
3.b. Methoxychlor Decreases in lgM splenic plaque-forming cell responses, splenic T-cell [ 50]
(CD3+) populations and germinal center (GC) B-cell (CDl9+PNA+)
populations.
3.c. Hexachlorocyclohexane Increases population of CD3 (+) CD4 (+) T-lymphocytes and expression of [ 51]
CD45RO (+) on CD4 (+) and COB (+) T-lymphocytes.
Decreases CD4(+) CD25(+) T-lymphocytes and level of I L-2 and IFN -y in
SLE patients.
3.d. o, p‘-Dichlorodiphenyl- Increases the percentages of CD3(+) CD4(+) T-lymphocytes and IL-10 level. [ 51]
trichloroethane (DDT)
S. No. Metals Immunological Responses References
1 Cadmium • Increases production of NO. [ 52]
• Inhibits synthesis of lgE by B cells or PBMCs upon IL-4/aCD40
stimulation and decreases proliferation of B cells or PBMCs.
• Increase production of chemoattractant Leukotriene B4 from neutrophils
and monocytes.
2 Lead (Pb) • Decreases NO production in cytokine-induced cell lines. [ 53]
• Induces production of TNF, IL-6, and IL-12 and decreases IL- I 0
production.
3 Mercury(Inorganic) • Increases IL-12, IL- 17, IFN-y, and TNF-a production. [ 54]
• Overexpression of CD86 and HLA-DR and production of TNF and IL-8
in vitro.
4 Arsenic • Decreases IL-2, I L-4, IL-5, I L-10, TNF-a, and IFN-y secretion from T [ 55]
cells.
• Increases lgG, lgA, and lgE levels.
S. No. PAHs Immunological Responses References
1 2,3,7,8-Tetrachloro-dibenzo-p-dioxin • Increases the expression of genes involve in antigen processing and [ 56]
(TCDD) presentation.
• It also increases expression of genes of dendritic cell maturation
pathways.
2 Polychlorinated Biphenyls • Immunosuppressive. [ 57]
2,2’,4,4‘,5,5’-hexachlorobiphenyl • Deceases CCL22 gene expression.
3 Polybrominated diphenyl ethers • Decreases peripheral blood monocytes, NK cell activity and splenic CD4 [ 58]
(+) CDS (+) proliferation.
• Increases T and B cell proliferation on mitogen stimulation.
• Decreases proinflammatory cytokine production (TNF-y and IL-6).
22 Immunomodulators in Aquaculture and Fish Health

The use of natural immunostimulants to boost innate Levamisole has been approved by the U.S. Food and Drug
immunity may be able to reduce disease outbreaks in com­ Administration to treat helminth infections in ruminants, which
mercial fisheries [59], however, this method is currently dis­ has sparked interest in its immunostimulatory potential in fish
regarded by the scientific community because it is not in the USA and abroad. Levamisole, which is the active laevo-
standardized [60]. Herbal remedies are a more affordable isomer of tetramisole has also been discovered to have the
source of medicines, are more accurate than chemotherapeutic potential as a regulator of carp and rainbow trout immune
agents, and provide a workable solution to every issue that responses [72]. Both species were observed with up-regulated
aquaculture is currently facing. Numerous plant compounds innate immune responses with demonstrated tolerance to
with antibacterial, anti-stress, immunostimulant, and growth- pathogenic microorganisms under experimental conditions
promoting properties have a big impact on fish or shrimp larval [73]. Greater phagocyte activity, macrophage cytokine pres­
culture [61]. Proteoglycans, flavonoids, polysaccharides, and ence, lymphocyte infiltration following the introduction of
phenolics from plants all have a significant impact on the mitogens, and generation of antibodies may all be associated
prevention or management of pathogenic microorganisms [62]. with the enhanced protection.
Herbal substances have the power to scavenge free radicals and
prevent the formation of oxygen anions. Shrimps have suc­
cessfully employed Picrorhiza kurroa as an anti-stress chem­
2.4 Immunostimulant Administration Methods
ical [62] and Ocimum sanctum significantly improved
Oreochromis mossambicus’ immune stimulatory effects, and Immune stimulants boost the host’s natural capacity for immune
antibody response, and disease resistance against Aeromonas responses, enhancing its ability to fight off infections. The nat­
hydrophila infection [63]. The serum bactericidal activity and ural killer cells, complement, lysozyme, and antibody responses
phagocytosis in rohu Labeo rohita were likewise boosted by an of fish are also stimulated by several immunostimulants [74].
enriched meal that included Curcuma longa and allium sa­ Immunostimulants can be administered primarily in three ways:
tivum. In fingerling rohu, L. rohita fed with diets enriched with via injection, submersion, and oral absorption. Immunostimulant
Achyranthus at various doses, superoxide anions were pro­ injections can result in potent non-specific responses, but they
duced more frequently, serum lysozyme levels were higher, are expensive, labor-consuming, and only suited to large fish
and serum bactericidal levels were also higher [64]. The with a body weight of more than 10–15 g in an intense aqua­
phagocytic activity in certain fish is greatly increased when culture environment. According to reports, injections offer broad
immunostimulants, such as plant extracts, are applied [65]. protection against a variety of infections, as demonstrated by the
intra-peritoneal injection of glucan into channel catfish, which
reduced fish mortality when exposed to Edwardsiella ictaluri
2.3 Synthetic Immunomodulators [75]. It is impossible to vaccinate little fish. Although less ex­
pensive than the injection, immersion causes more stress to the
With varying degrees of efficacy, substances of non-biological fish when handled, making it useful in systems of intensive
origin (chemically developed antibiotics) have been adopted as culture. When introducing sub-adult life-staged fish into field
interventions of choice in situations of disease and infections in ponds, the prior dipping method is particularly effective.
aquaculture fish and shrimp [66]. Depending on the condition, Levamisole-immersed fish, showed increased leukocytes circu­
vaccines against various infections have been produced recently lation, phagocyte activity, and resistance against P. damselae
with varying degrees of efficacy. The administration of different sub sp. piscicida [76].
substances to enhance or potentiate the innate immune response Oral administration has also been shown to be a cost-
of farmed fish and shrimp has been a different strategy. These effective enhanced innate immune capacity. It is suitable for
substances, also known as immunostimulants, are thought to be large-scale aquacultural setup and running. Powdered feed is
an alluring and effective tool for preventing illnesses in fish and often fortified with immunostimulant substances are mixed
shellfish. Immunostimulants are now widely used for disease with and coated with fish oil. Bioencapsulation is now being
management in aquaculture techniques due to their proven utilized to immunize fish larvae with live-feeding animals
positive benefits in a variety of living systems [67]. during their early larval stages.
Levamisole is an anthelmintic drug used to treat nematode
infections in both humans and animals (Table 2.1). In vitro, it
can activate the immune system. Levamisole increased
2.5 Timing of Administration
phagocytic activity, the NBT reaction, and the number of cells
that produce antibodies. Levamisole increased lysozyme Immunostimulants must be used properly and at the appro­
activity in serum, leukocyte count, accelerated NBT decrease, priate time. [8] suggested that to prevent illness-related losses,
and phagocytic index of phagocytic cells when given orally immunostimulants should be used before disease outbreaks.
[68]. Although Levamisole was used in rainbow trout, changes Based on various culture systems with varied feeding regi­
in hematocrit, leucocrit, or immunoglobulin values were not mens, effective dose, and exposure duration will become even
significant [69]. Findlay et al. [70] have recommended the more complicated [77]. Injections of high doses of glucans
application of levamisole as an immunostimulant in fish. (100 mg/kg) in Atlantic salmon resulted in a week without
Rainbow trout treated with a bath treatment containing 5, 10, protection, although the full advantages are only felt after
or 25 g/ml of levamisole over two hours showed resistance to 3–4 weeks. Additionally, protecting for only one week at
Y. ruckeri [71]. modest injection doses of 2–10 mg/kg [59]. Similar to this, it
Natural and Synthetic Immunomodulators 23

has been observed that over 30 days but not over 45 days, By changing epigenetic processes including DNA methylation,
Clarias spp. fed with glucan or oligosaccharides exhibit an histone protein modification, and post-translational modifica­
increase in the number of NBT-positive cells [78]. tions, polyphenols influence the immunological response (acti­
vation and differentiation of immune cells) [82].
Fish meal and oil, which are feed items made from smaller,
2.6 Mode of Action wild pelagic fish such as herring and sardines, are POPs and
mercury sources for farmed fish. Both POPs and mercury enter
Immunostimulants work by stimulating the immune systems of the ocean as a result of air emissions, mostly from the com­
living things to boost defenses against pathogen invasion. The bustion of fossil fuels for POPs and agricultural pesticide that
method is quite varied or may not be well known, and it is also is used for mercury [83]. Farmed salmon tended to have lower
affected by the type or variety of immune stimulants, the dosage levels of POPs and mercury when fish-based feed is obtained
administered, the route of uptake, and exposure duration. from regions of the world. These small pelagic fish when
Several of the mechanisms of action are as follows: sourced from South America contain fewer contaminants,
compared to smaller pelagic fish from Northern Europe, where
• T-lymphocyte stimulators: Levamisole, Freund’s Complete higher levels of contaminants have been recorded [84]. Some
Adjuvant (FCA), Glucans, Muramyl dipeptide, FK-565 fish processors use active carbon filters to eliminate POPs from
(Lactoyl tetrapeptide from Streptomyces olivaceogriseus). fish meal and oil to reduce pollution in farmed salmon [85].
• Bacterial endotoxins and lipopolysaccharides stimulate Reducing the use of fish meal and oil in aquaculture is another
B-cells. method for lowering POPs in farmed fish. In addition to
• Glucans, Chitin, and Chitosan are macrophage activators. nutritional sources, exposure to alterations in ambient en­
• Inflammatory agents, such as chemo toxins vironmental elements inside aquaculture installations may
cause immunological modulation in fish. In addition to inten­
• Detergents and sodium dodecyl sulfate, quaternary
sifying farming practices, global warming reduces the amount
ammonium compounds (QAC), and saponins are cell
of oxygen on the water’s surface [23]. Limited quantities of
membrane modifiers.
dissolved oxygen in the culture water were revealed to be the
• Nutritional considerations include vitamins C and E, as cause of immune suppression in tilapia at higher temperatures
well as omega-3 fatty acids. [22]. According to these research results, hypoxia in fish
• Cytokines include leukotriene and interferon. modulates the immune system.
• Metal – e.g., Cd According to several studies, exposure to metals alters several
• Mitogens extracted from non-plant organisms immune system characteristics in the host, making them more
susceptible to allergies and infections [86]. Additionally,
Immune stimulants often increase the capacity of macro­ Arunkumar and Rajasekaran [87] hypothesized that exposure to
phages, complements, lymphocytes, and non-specific cytotoxic metals triggers immunological responses in fish, either directly
cells to phagocytose and kill germs, providing resistance and by binding the tertiary structures of physiologically active mo­
protection against a variety of illnesses and invasive microbes. lecules or inadvertently by acting as stressors that change cor­
ticosteroid concentrations. An essential immunological reaction
is a phagocytosis, in which harmful particles are taken up by
intracellular vacuoles and eliminated. Pillet and Lesage [88]
2.7 Environmental Immunomodulators
noted significant increases in phagocytic activity in Zn-exposed
Environmental immunomodulators, also known as xenobiotics, harbor seals (Phoca vitulina) and gray seals (Halichoerus
are manmade or natural substances that frequently have no grypus). It has been established that exposure to aquatic toxins
metabolic function and behave as foreign molecules to an can increase the production of lysozyme, a crucial element of
organism [79]. The impact of pesticides, synthetic chemicals, the innate immune response in fish [89]. Observable increases
metals, polycyclic aromatic hydrocarbons, mycotoxins, and in the serum lysozyme were also noted in rainbow trout,
other substances on the immune system has been demonstrated Oncorhynchus mykiss, subjected to Zn, cadmium, mercury, and
by epidemiological and animal investigations [80]. These im­ metal combinations, according to Sanchez‐Dardon, Voccia [90].
munomodulators fall into an intriguing category because their When exposed to Zn, Mozambique tilapia, Oreochromis mos­
existence or activity may unintentionally create physiological sambicus, showed an increase in both phagocytic and lysozyme
changes that undermine ongoing immunostimulant treatments activity, according to Çelik, Kaya [91]. Given that lysozyme is a
or interventions in aquaculture species. trustworthy indicator for assessing how changes in the en­
In addition to immune modulators purposely added to fish to vironment affect fish’s innate immunity [89]. The ectothermic
increase disease resistance, contaminated fish food can also un­ characteristic of fish, which directly affects their physiology and
intentionally introduce immune modulators to fish. For instance, immune function, has been attributed to this interaction between
polyphenols, which are micronutrients found in typical diets and environmental events and fish immunomodulation [92].
divided into non-flavonoids (phenolic amine) and flavonoids The blue mussel, Mytilus edulis, showed a considerable
(phenolic acids). Due to their antioxidant qualities and ability to increase in phagocytosis at a higher temperature, according to
prevent diseases associated with oxidative stress, such as neu­ Parry and Pipe [93]. Furthermore, nickel-exposed Pacific
rodegenerative diseases [81] polyphenols are frequently utilized abalone, Haliotis discus hannai, showed enhanced lysozyme
in the food sector for applications (color and flavor enrichment). activity in high water temperatures (Figure 2.1).
24 Immunomodulators in Aquaculture and Fish Health

FIGURE 2.1 Pathways of xenobiotic-induced immunomodulation (adapted from Sajid and Cameotra [ 79]).

formation. When the immune system is suppressed after being

2.8 Inferences for Stress Responses in exposed to xenobiotics, it makes people more susceptible to
Aquaculture Fish bacterial, viral, and parasite illnesses as well as more likely to
develop cancer. On the other side, the stimulation of immune
Regarding innate immunity, fish are a particularly significant responses might start autoimmune processes or cause overall
animal group because fish take longer to build an adaptive or tissue damage (Figure 2.2).
acquired immune response than mammals, which may make fish Different molecular event(s) in immune cells are triggered
suffer more severely from innate immunity impairment. In as a result of the diversity in physio-chemical characteristics
fish, the idea of species-specific immunological competence has of xenobiotics. The main mechanism behind changes in
been proven. This suggests that fish can adjust their immuno­ immunological response is the activation of oxidative stress
competence to the seasonal variations in environmental condi­ by the creation of ROS. Adduct formation and ROS genera­
tions or can respond favorably to specific immunomodulatory tion are two aspects of the polycyclic aromatic hydrocarbons’
substances like other fish species, but responses are often rela­ carcinogenic process. These pathways also impair immune
tive [94]. Numerous innate immunity genes have also been cells’ abilities and cause the breakdown of their genetic
identified in teleost fish, and it has been found that while some of integrity. Dietary polyphenols (secondary metabolites) func­
their signaling pathways are preserved from fish to mammals, tion as antioxidants and reduce the generation of ROS in
others have unique characteristics and exhibit significantly more immune cells, but they also influence epigenetic pathways that
species variation [95]. Monitoring fish health can also identify control immunological response. Pesticides used on a global
the state of the aquatic system or medium in which fish are scale have a variety of immunomodulatory effects. ROS
reared [96]. As a consequence, fish innate immunity can serve as production, caspase-3 activation, mitochondrial malfunction,
a warning system for potential dangers to fish populations [97]. and changes in the transcription factor activity all play a role
Furthermore, it is economically significant to comprehend how in the mechanism of pesticide-induced immunotoxicity. Aryl
toxicants affect fish’s innate immunity. The well-being of other hydrocarbon receptor-bound xenobiotics like dioxins, biphe­
fish species, such as forage species, which may be crucial to the nyls, and dibenzofurans influence immunological response
survival of a fishery, is as important as the target species’ health (AhR). Th17 cells produce more xenobiotic metabolizing
is important. Financial success in aquaculture may be hampered enzymes and IL22 when they are bound to the AhR receptor.
by a weakened innate immune system in farm populations. Even Protein-reactive xenobiotic compounds are produced by the
though the immune system is a defense mechanism against action of xenobiotic metabolizing enzymes. Some of these
pathogens and cancer, xenobiotic metabolism produces acti­ xenobiotic compound’s function as neoantigens and en­
vated metabolites that cause severe pathophysiological condi­ courage T-cell activation. Increased IL-22 production and
tions such as immune disruptions, neurological disruptions, T-cell activation worsen autoimmune disease symptoms.
cardiovascular, nephrological, and ultimately disturbances in the Additionally, exposure to heavy metals in the environment
body’s homeostasis. Xenobiotics can disrupt the immune sys­ and at work increases the likelihood of inflammation and
tem’s normal function, and these immunocompromised condi­ pathogen contact by significantly disrupting immunological
tions increase the likelihood of pathogen encounter and tumor responses.
Natural and Synthetic Immunomodulators 25

FIGURE 2.2 Toxicants and stress via immunomodulation.

health and for the environment. Environmental Microbiology,

2.9 Conclusion 2006. 8(7): p. 1137–1144.
6. Chakraborty, S.B. and C. Hancz, Application of phyto­
For the development of novel intervention techniques that
chemicals as immunostimulant, antipathogenic and antis­
increase aquaculture production, it is crucial to sufficiently tress agents in finfish culture. Reviews in Aquaculture,
grasp the dynamics of fish immune systems. This includes 2011. 3(3): p. 103–119.
understanding the immune system’s plasticity and how it helps 7. Sakai, M., Current research status of fish immunostimulants.
to better regulate and optimize rearing settings for improved Aquaculture, 1999. 172(1-2): p. 63–92.
fish welfare and performance. Furthermore, managers must 8. Anderson, D.P., Immunostimulants, adjuvants, and vaccine
exercise comprehensive management control and comprehend carriers in fish: applications to aquaculture. Annual
how environmental immunomodulator sources can confound Review of Fish Diseases, 1992. 2: p. 281–307.
efforts to maintain healthy aquaculture fisheries. 9. Jeney, G. and D.P. Anderson, Glucan injection or bath
exposure given alone or in combination with a bacterin
enhance the non-specific defence mechanisms in rainbow
Acknowledgment trout (Oncorhynchus mykiss). Aquaculture, 1993. 116(4):
p. 315–329.
The authors express immense gratitude to Dr. Tarun Mandal, 10. Nikl, L., T.T. Evelyn and L. Albright, Trials with an orally
Principal of Sundarban Hazi Desarat College, South 24 Par- and immersion-administered β-1, 3 glucan as an im­
ganas, for providing permission to write this chapter on im­ munoprophylactic against Aeromonas salmonicida in
munomodulators. juvenile chinook salmon Oncorhynchus tshawytscha.
Diseases of Aquatic Organisms, 1993. 17(3): p. 191–196.
Disclosure statement: The authors declare that they have no 11. Wang, S.-H. and J.-C. Chen, The protective effect of chitin
conflicts of interest. and chitosan against Vibrio alginolyticus in white shrimp
Litopenaeus vannamei. Fish & Shellfish Immunology,
2005. 19(3): p. 191–204.
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resistance in Atlantic salmon, Salmo salar L., by a glucan 76. Chen, D. and A. Ainsworth, Glucan administration
from Saccharomyces cerevisiae cell walls. Journal of Fish potentiates immune defence mechanisms of channel catfish,
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60. Ponni, A., Ethanotherapeutics. An approach to study the 1992. 15(4): p. 295–304.
antimicrobial evaluation of some siddha and ayurvedic 77. Galeotti, M., D. Volpatti and G. Jeney. The nature of
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Sc. Dissertation, MS University, Tamil Nadu, India. labrax) to Pasteurella piscicida following bath exposure
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78. GH, K. and S. YL, Vibriosis resistance induced by glucan of zinc on phagocytic activity. Marine Pollution Bulletin,
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3
Immunomodulators: Mode of Action

Subramaniam Sivakumar1, C. Shanmuga Sundaram2, Maderi Velayutham Dassprakash1, and

Rantham Subramaniam Venkatesan3
1
Department of Biochemistry, Sri Sankara Arts and
Science College, Enathur, Kanchipuram, Tamilnadu,
India
2
Department of Microbiology, Hindustan College of Arts
and Science, Kelambakkam, Chennai, Tamilnadu, India
3
Lakshmi Bangaru Arts and Science College,
Melmaruvathur, Chengalpet, Tamilnadu, India

of cancerous cells, or by promoting the ability of the host to

3.1 Introduction tolerate damage by cytotoxic pathways.(5)
The following agents are considered as biological response
Aquaculture is blooming as one of the most promising and modifiers (BRM)(3)
viable industries for keeping pace with the indispensable
requirement for animal protein, providing nutritional and • Immunoaugmenting agents
healthy food to humans, particularly those living in regions • Immunosuppressive agents
where livestock availability is relatively scarce. With every
• Interferons and interferon inducers
move toward intensification of increasing production in aqua­
culture methods, there is a rise of the stress level in the en­ • Lymphokines and monokines
vironment as well as the fish. Hence, disease outbreak is being • Growth factors
increasingly identified as one of the most important risks to the • Thymic factors
aquaculture production in most of the countries. The disease • Tumor antigens
control in aquaculture is mostly achieved by the use of
• Antitumor antibodies
chemical compounds and antibiotics. Due to side effects and
resistance development, the development of non-antibiotic • Antitumor effector cells
and environment friendly agents is the need of the present • Maturation factors
scenario in the field of aquaculture.(1) At this juncture, the • Recombinant genes rendering normal cells less suscep­
current studies are directed towards another promising area tible to cytotoxic effects of chemotherapeutic agents
i.e., the applications of immunomodulators to increase the • T cell immunoregulators
immune response and protection against the diseases affecting
aquaculture industry.(2) Probably due to the restricted knowledge of the fish’s immune
Immunomodulators are those intrinsic or extrinsic substances response, non-specific immunomodulators have been used in
which regulate or alter the competency, duration, type or scope limited numbers and the method of their application in aqua­
of the immune response.(3) The initial immune response to an­ culture. Along with the rapid growth of aquaculture sector over
tigenic stimulation induces specific antibody synthesis and the last 30 years, an associated increase in disease problems
production of active effector cells. The nature, magnitude, and also occurs as result of rapid expansion and amongst other
specific form is determined by an array of modulatory processes factors like high stocking densities. Immunomodulators have
which orchestrate this response. Multiple pathways of regulation been used as dietary additives to improve feed efficiency, weight
are used by the competent immune system, depending on the gain, and disease resistance in order to maintain fish health and
appropriate response to the organism’s need.(4) to improve fish growth performance of immunomodulators in
Biological response modifiers (BRMs) modify the animal aquaculture industry.(6)
responses by aiding or retrieving effector mechanisms or
agents of host defense, reducing the dangerous components of
the host’s reaction, exchange the defective effector or mediator
3.2 Lymphoid Organs
mechanisms with natural compounds or synthetic derivatives,
aiding the host response to vaccines and chemotherapeutic All the major primary and secondary lymphoid organs present in
agents, reducing transformation and increasing differentiation fish as in all vertebrates, except lymphatic nodules and bone

DOI: 10.1201/9781003361183-4 29
30 Immunomodulators in Aquaculture and Fish Health

marrow.(7) The head, kidney, or pronephros has hematopoietic same pathogen. Once the T-cells recognize antigens through
functions, and unlike in higher vertebrates, it is the immune TCR by the antigen presenting cells (APCs), they are activated.
organ involved in phagocytosis, antigen processing, production Activated T cells differentiate into either helper T (TH) lym­
of IgM, and immune memory through melanomacrophage phocytes or cytotoxic T (TC) lymphocytes. Helper T cells are
centers. The thymus, situated near the opercular cavity in tele­ capable of activating other immune cells such as B cells and
osts, produces T cells involved in allograft rejection, stimulation macrophages through cytokines, while cytotoxic T cells upon
of phagocytosis, and antibody production by B cells.(8) The activation are able to lyse the infected or self-altered cells.(12,17)
spleen, another lymphoid organ, plays a vital role in trapping and
processing of antigens.(9) Melanomacrophage centers are
present for clearance of ingested material and can be surrounded 3.4 Mechanisms of Immunomodulators
by immunoglobulin-positive cells, especially after immuniza­
tion. Proliferation of granular cells has also been observed in There has been a growing interest in the last decade in the use of
association with ellipsoids and melanomacrophage centers after plant, herb, and algae extracts as immunomodulators in dietary
the immunization of fish.(10–12) supplements for fish.(18,19) Several studies in mammals have
shown that plants extract modulate TLRs as evidenced by the
use of natural extracts for centuries by many civilizations.(20)
Some algae and plants derivatives can down-regulate gene ex­
3.3 Immune Response pression (21,22) and induction (23,24) of TLRs, regulating the TLR/
The immune system is essential for the living organisms to NF-kB signalling pathway.(25) Some other extracts promote the
protect them from diseases. Immunity can be divided into two proinflammatory response(26) and enhance TLR9 and IRF7
types, namely the innate immune response and the adaptive activation and in turn inducing Interferon-β (IFN-β) protein
immune response. The innate immune response is the non- expression.(27) They also elicit the M1 and M2 macrophage
specific type of defense against infection, which are provided by responses through the TLR/NF-kB signaling pathway.(28)
physical barriers and cellular response. The adaptive immune To date, lipopolysaccharides (LPS), peptidoglycan (PGN),
response is the specific type of defense against pathogens. and beta-glucans are the most commonly used PAMPs in fish
immunomodulator diets. In mammals, TLR4 recognized LPS,
a component of the outer membrane of gram-negative bacteria,
3.3.1 Innate Immune Response and forms a complex with co-receptors lymphocyte antigen 96
Through specific receptors like toll receptors (TLRs) of the (MD2), and CD14 on the cell surface that leads to the acti­
innate immune system, it identifies conserved molecular patterns vation of multiple signaling components and the subsequent
common to pathogens such as DNA, RNA, lipopolysaccharides, production of proinflammatory cytokines.(29)
polysaccharides, and peptidoglycans. This type of recognition The mechanism of action of many immunomodulators
may lead to the removal of pathogens, for example by phago­ depends upon their interaction with TLRs, a class of proteins
cytosis, or may induce adaptive immune responses.(13) The that plays a key role in the innate immune system as shown in
innate immune system possesses different types cells with a Figure 3.1.(30) Their mode of action is a very specialized
diverse array of functions. The phagocytic cells remove patho­ pathway that can identify endogenous microorganisms and
gens by engulfing and degrading them. Other cells can stimulate microbial products as well as induce the specific immune
the migration of immune cells to the site of infection by pro­ response for each antigen.(31) In fish, at least 11 types of TLRs
ducing and secreting the cytokines and chemokines and which have been discovered, each with different functions and lig­
further directs the immune response to clear the pathogens.(14) ands.(18) The TLR-2, TLR-5, and TLR-9 receptors were highly
expressed in zebrafish (Danio rerio) in addition to normal
TLRs.(32) The eleven types of TLR homologs were also
3.3.2 Adaptive Immune Response identified in Japanese flounder (Paralichthys olivaceus).(33)
The adaptive immune system recognizes foreign bodies by From the growing use of innovative technologies for knowl­
means of two receptors of B and T lymphocytes, the B cell edge and evaluation of the immune system, such as proteomics
receptor (BCR), and the T cell receptor (TCR), respectively. analyses and transcriptomics to evaluate the modulation of the
Adaptive immunity is attained by variety of mechanisms. It acts immune system, studies on TLRs have opened a new research
by the antigen exposure and elicit immunological memory. The area with high potential in the field of fish immunology.(34)
immunological memory property is the basis of vaccine devel­ TLRs are localized on the cell surface and in the endosome for
opment.(15,16) The effector phase of adaptive response generally ligand recognition. A member of the TLR1 family, TLR14,
starts three to five days after infection and is capable of recog­ dimerizes with other TLR1 family molecule to recognize lipo­
nizing specific antigens, which increases the immune response proteins. Tiger puffer TLR3 and TLR22 recognize dsRNA.
level in both speed and magnitude for the future exposures.(17) Teleost TLR4 genes have only been reported in Cypriniformes.
The vital players of the adaptive immune response are the B and However, the homologs of the TLR4 co-receptors, CD14 and
T lymphocytes. Once the B-cells recognize antigens through MD2, are lacking in teleosts. Soluble TLR5 (TLRS5) evolved in
BCR, they are activated. After activation, they are capable of the teleost lineage after it separated from the mammalian
differentiating into plasma cells and memory cells. Plasma cells lineage. Rainbow trout TLRS5 acts synergistically with
(effector B-cells) can secrete antibodies whereas memory B- membrane-bound TLR5 to activate NF-κB activation upon
cells will play important role for the successive infection by the recognition of bacterial flagellin. Teleost TLR7 and TLR8
Immunomodulators 31

FIGURE 3.1 A putative model of the fish TLR system( 18, 30, 35).

Abbreviations used are IKK - IκB kinase, IL – Interleukin, IRAK - IL-1R-associated kinase, IRF - Interferon regulatory factor, MAPKs -
Mitogen-activated protein kinases, MD-2 - Myeloid differentiation factor 2, MyD88 - Myeloid differentiation primary response protein 88,
NF-κB - Nuclear factor kappa B, RIP1 - Receptor-interacting protein1, TAK1 - TGF- β-activated kinase 1, TANK - TRAF family-member-
associated NF-κB activator, TBK1 TANK-binding kinase 1, TGF – Tumour growth factor, TIR – Toll-interleukin-1 receptor domain, TRIF,
TICAM1 – TIR - domain-containing adaptor inducing IFN- γ,TIRAP or Mal TIR domain-containing adaptor protein, TNFR – Tumor necrosis
factor receptor, TRAF – TNFR-associated factor.

recognizes sRNA and its analogues, such as the imidazoquino­ However, other IRAK homologs (IRAK1 and IRAK4) are
lines. Japanese flounder TLR9 recognizes the CpG DNA ana­ conserved in the teleost lineage. These IRAK homologs may
logue, CpG ODN. Chicken TLR21 was also reported to play a pivotal role in signaling via the MyD88-dependent
recognize CpG DNA, suggesting a functional similarity with pathway. Immune kinases, including TAK1, IKK, and
teleost TLR21 as a CpG DNA receptor. Among the teleost- MAPKs have been identified in teleosts. These kinases activate
specific TLRs, both TLR19 and TLR20 have been found in transcription factors to produce inflammatory cytokines.(18)
zebrafish, whereas only TLR20 was found in channel catfish.
TLR19 and TLR20 have a shorter leucine-rich repeats (LRR) 3.4.1 Immune Response Followed by TLR-PAMPs
containing domain than the other TLR family members. TLR23 Pathway
is unique to the puffer fish lineage. The function of these teleost-
specific TLRs needs to be assessed further to identify novel TLR Once the pathogenic microbes and their products are recog­
functions. Regarding the TIR domain-containing adaptors, the nized by the APCs through the TLR-PAMPs recognition
universal adaptor, MyD88, is evident in many teleost species. process, the pathogenic agents are removed or neutralized by
TICAM-1 seems to be essential for the signalling of teleost any one of the following immune responses, namely elim­
TLR3 and TLR22. Zebrafish TICAM-1 associates with RIP1 to inating intracellular pathogens, targeting extracellular parasitic
activate the NFkB cascade. The direct interaction of zebrafish worms, and intoxicating extracellular microbes. The mecha­
TICAM-1 and TBK1 is reported, however, IRF3/IRF7 activa­ nisms were explained through pictorial representations in
tion by the TICAM-1-dependent pathway is not clear in teleosts. Figures 3.2, 3.3, and 3.4.(36,37)
TIRAP association with TLR2 and TLR4 seen in mammals is
also seen in teleosts. However, TICAM-2, which is involved in
3.4.2 Role of Fish Cytokines and Other Factors in
the MyD88- independent pathway in mammals, has also not
Elimination of Intracellular Pathogens
been found in any teleost species. In addition, zebrafish TLR4
negatively regulates NF-κB, unlike mammalian TLR4. This Interleukin-12 is secreted in large amounts by antigen presenting
suggests an impairment of LPS recognition by TLR4 in teleosts. cells (APCs) after their activation by PAMPs and damage/
IFN production induced via stimulation of salmonid TLR7 and danger-associated molecular patterns (DAMPs). IL-12 induces
TLR8 has been reported. It may indicate the conservation of the IFN-γ and IFN-γrel expression by NK cells and by developing
MyD88- dependent pathway among teleost TLR7 family T-cells.(38,39) When TH naive cells receive strong TCR signals
members. Among the IRAK homologs identified in teleosts, in the presence of Interleukin-12 and IFN-γ, T-box expressed in
IRAK2 is absent from the genome of tiger puffer and zebrafish. T cells (T-bet) or T-box transcription factor (TBX21) expression
32 Immunomodulators in Aquaculture and Fish Health

FIGURE 3.2 Potential fish cytokines and other factors involved in elimination of intracellular pathogens.

FIGURE 3.3 Potential fish cytokines and other factors involved in the removal of extracellular parasitic worms.

FIGURE 3.4 Potential fish cytokines and other factors involved in the removal of extracellular microbes.

is induced by IFN-γ/signal transducer and activator of transcrip­ 3.4.3 Role of Fish Cytokines and Other Factors in
tion (STAT)1 and IL-12/STAT4 signaling.(40) T-bet induces IFN- Removal of Extracellular Parasitic Worms
γ and IFN-γrel production, thereby upregulating T-bet expression
Cytokines such as thymic stromal lymphopoietein (TSLP) and
and fortify the Th1 phenotype in a positive feedback loop, and
IL-17C-like cytokine from non-lymphoid cellular sources are
increase the expression of IL-12R.(41) In addition, IL-2 and IL2L,
essential for Th2 cell differentiation.(44,45) IL-2 and IL2L pro­
produced by T cells after TCR activation, regulates Th1 differ­
duced by TCR signaling activate STAT5 and induce IL-4/13 A,
entiation via STAT-5-dependent upregulation of IL-12R
IL-4/13B and IL-4R expression. Th2 cells require an IL-4/13 A
expression.(42) By secretion of IFN-γ, IFN-γrel, tumour necrosis
or IL-4/13B/STAT6 signal for development.(46,47) The initial
factor-α(TNF-α), IL2L, and IL-2, Th1 cells help eliminate
sources of IL- 4/13 A and IL-4/13B can be from basophils, mast
intracellular pathogens.(37,43)
Immunomodulators 33

cells, NK cells, follicular Th cells and TH naive cells. IL-4/13 A regulating cytokine levels.(61) Cytokines play a vital role in
or IL-4/13B binds to IL-4R and activates STAT6 to control the both the innate and adaptive immune responses. All major
expression of IL-4 target genes including the Th2 master regu­ cytokine families have been shown to existing fish and appear
lator GATA-binding protein-3 (GATA-3), which is responsible to have similar roles to those observed in mammals. However,
for the expression of Th2 effector cytokines.(40) Th2 cells, by multiple forms exist in several fish species which may result in
releasing the cytokines IL-4/13 A, IL-4/13B, IL-5, IL-9, IL-10, variations in the function that cytokines play compared to their
and IL-17C, are specialized to elicit immune reactions against counterparts in other species.(62) The type II cytokines of fish
extracellular parasitic worms.(37,44,48–50) listed along with their immune functions in Table 3.1.(63) The
innate immune system stimulated by immunomodulators
derived from rice, human milk, and soybean tryptic hydroly­
3.4.4 Role of Fish Cytokines and Other Factors in sates. Chalamaiah et al. (2018) have reviewed about im­
Removal of Extracellular Microbes munomodulators from food protein.(64) Subhan et al. (2017)
IL-6, IL-21, IL-23, and TGF-β1 are the major signaling cytokines proved that peptides could downregulate the expression of
involved in Th 17 cell differentiation, and RORγ is the master cytokines from fish scale collagen in vitro.(65) Thus, peptides
regulator.(51–54) TGF-β1 and IL-6 in combination with a strong isolated from fish scale collagen had a useful effect as an anti-
TCR signal cause naive TH cells to express the IL-23R and to inflammatory agent.(66) Figure 3.5 depicts the mechanism of
induce retinoid- related orphan receptors γ (RORγ), as well as to action of cytokines as immunomodulators.(67) Details of fish
produce IL-17A/F1-3 and IL-21.(55,56) IL-6, IL-21 and IL-23 cytokines are given in Chapter 10.
activate STAT3 and induce the expression of RORγ and effector
cytokines.(57) IL-2 or IL2L activate STAT5, which compete with [Link] Chemokines
STAT3, down-regulate IL-23R expression and block differentia­
tion to the Th17 lineage.(58,59) Th17 cells, by the production of IL- Chemokines are a superfamily of approximately 40 different
17A/F1- 3, IL-21, and IL-22, induce the expression of numerous cytokines which promote the recruitment of immune cells to
chemokines and antimicrobial peptides in epithelial cells and fi­ the infection region. Chemokines exhibit their activity by
broblasts, which are important for the neutrophil-mediated binding to the G-protein-linked receptors. The chemokine
immune reactions against extracellular microbes.(37,54,56,60) superfamily consists of four distinct subgroups and are des­
ignated as CXC (or a), CC (or b), CX3C (or d), and C (or g),
which are defined by the arrangement of the first two cysteine
amino acid residues within their peptide sequences.(68)
3.5 Actions of Different Immunomodulators
The best-known immunomodulators are cytokines, glucans,
[Link] Transforming Growth Factor Beta
hormones, vitamins, minerals, lipopolysaccharides (LPS),
(TGF-beta)
peptidoglycans, synthetic compounds (levamisole; FK-565),
polysaccharides (chitin, chitosan), and plant extracts that may In various leukocyte lineages including lymphocytes, granu­
increase the immune responses of fish.(35) Only few of them locytes, macrophages, NK cells, and dendritic cells; TGF-beta
are discussed in this chapter. regulates cell development, proliferation, differentiation,
migration, and survival since it is a pleiotropic cytokine. TGF-
3.5.1 Cytokines beta1 is to suppress autoimmunity and maintain immune tol­
erance in the mammalian immune system. TGF-beta1 sup­
Immunomodulators can stimulate immune response by presses Th1 and Th2 cell proliferation, whereas it promotes T
increasing lymphocyte proliferation, antibody production and regulatory cell generation by inducing Foxp3 expression, thus

TABLE 3.1
Biological Activity of Fish Type II Cytokines( 63)
Sl. No. Cytokine Human Receptors Immune Functions
Homologue
1. IL-10 IL-10 IL-10R1/CRFB12, IL10R2/CRFB4 Suppress immune responses, inhibit
inflammation, promote T cell proliferation,
memory B-cells and IgM production
2. IL-22 IL-22 Not characterized Activate antimicrobial peptide genes and
antibacterial immunity
3. Group I IFN (IFN-a, d, e) Type I IFN IFNAR1/CRFB5, IFNAR2/CRFB1 Induce expression of antiviral effectors; promote
apoptosis, regulate inflammation
4. Group I IFN (IFN-b, c, f) Type I IFN IFNAR1/CRFB5, IFNAR2/CRFB2 Induce expression of antiviral effectors; promote
apoptosis
5. IFN-γ Type II IFN IFN-γR1/CRFB13, IFN-γR2/CRFB6 Activate macrophages, enhance antigen
presentation, promote Th1 cytokine expression
6. IFN-γrel Type II IFN CRFB17, IFN-γR2/CRFB6 Regulate antibacterial and antiviral immunity
34 Immunomodulators in Aquaculture and Fish Health

FIGURE 3.5 Mechanism of action of cytokines as immunomodulators( 67).

Abbreviations used are COX2 – Cyclooxygenase2, ERK – Extracellular signal-regulated kinase, EIK – Ets-like molecule 1, ETS – E26
transformation-specific protein, JNK – c-Jun N-terminal kinases, cJUN – Proto-oncogene of jun (ju-nana, the Japanese word for 17) gene AP-1
Transcription Factor Subunit.

TGF-beta1 as a potent immunosuppressive agent. On the other cells are the target cells of fish TNF alpha and involved in the
hand, TGF-beta induces immune responses by promoting the recruitment of immune cells to the inflammatory region rather
Th17 cells generation. Therefore, in mammals, the regulatory than their activation. The differential expression of fish TNF
roles of TGF-beta as a positive or negative control agent in alpha has been observed in rainbow trout leucocytes and have
immunity are widely recognized. In teleost, some research shown an increased response to various pro-inflammatory
works have revealed that TGF-beta1 also exerts powerful stimuli, such as human recombinant TNF alpha, zymosan, li­
immune-suppressing effects on activated leukocytes. For ex­ popolysaccharides and muramyl dipeptide as a peptidoglycan
ample, TGF-beta1 significantly blocks TNF alpha-induced component of bacteria. However, infectious pancreatic necrosis
activation of macrophage in common carp and goldfish, but virus (IPNV)-mediated up-regulation of TNF alpha controls
promotes the proliferation of the fibroblast cell line CCL71 of both the RIP1 (receptor-interacting protein-1)/ROS-mediated
goldfish. In grass carp, TGF-beta1 suppresses LPS/PHA- secondary necrosis pathway and the Bad/Bid-mediated apo­
stimulated proliferation of peripheral blood lymphocyte in ptotic pathway.(12,51)
contrast with the stimulatory effect of TGF-beta1 alone in the
same cells.(69) Similar phenomenon was also detected during
leukocyte migration under TGF-beta1 treatment, with or [Link] Interferons (IFNs)
without LPS challenges in red sea bream.(70) These studies not Interferons are involved in modulating innate and adaptive
only define TGF-beta1 as an immune modulator in teleost, but immune systems and are also responsible for cellular resistance
also proved that during the evolution of vertebrates, TGF-beta1 against viral pathogens. The IFNs are mainly classified into
may have retained similar functions in animal immunity.(12,53) two groups namely type I and type II.(71) Type I IFN includes
the classical IFN alpha/beta, which is induced by most of the
[Link] Tumor Necrosis Factor Alpha animal cells, whereas type II IFN is only composed of a single
gene called IFN gamma and is produced by natural killer (NK)
Tumor necrosis factor alpha (TNF alpha) plays an important role cells and T lymphocytes in response to interleukin-12 (IL-12),
in various host responses, including cell proliferation, differen­ IL-18, mitogens, and antigens. Structurally, both interferons
tiation, apoptosis, and necrosis. TNF alpha can induce either types belong to the class II alpha-helical cytokine family, but
apoptosis or NF-kB-mediated survival, depending on the cel­ have different three-dimensional structures and bind to dif­
lular status. TNF alpha induces powerful anti-microbial ferent receptors.(12)
responses, including killing infected cells, inducing apoptosis, From Atlantic salmon, two IFNs (IFN alpha 1 and IFN alpha 2)
up-regulating diverse host response genes and inhibiting intra­ have been cloned and characterized with respect to promoter,
cellular pathogen replication. In several bony fish, including gene structure, sequence, antiviral activity and induction of
carp, gilthead seabream, Japanese flounder, turbot, catfish, ISGs. Salmon IFN alpha 1, similar to mammalian IFN alpha/
tilapia, rainbow trout, and goldfish, TNF alpha has been iden­ beta and IFN lambda, induces both Mx and ISG15 proteins in
tified, cloned, and characterized. The occurrence of multiple TO cells. Furthermore, salmon IFN alpha1 induces potent
isoforms of TNF alpha in some teleost species has been iden­ antiviral activity against the IPNV under in vitro conditions,
tified. The high constitutive expression of TNF alpha gene in but this effect has not been observed in the in vivo studies, i.e.,
different tissues of healthy fish and its relatively poor up- Atlantic salmon challenged intraperitoneally with IPNV.(12)
regulation by immune challenge in vitro and in vivo are also Three types of type I IFNs have been discovered in rainbow
reported. The in vitro effects of TNF alpha on phagocyte acti­ trout namely rtIFN1, rtIFN2, and rtIFN3. The three rtIFNs can
vation in rainbow trout, gilthead seabream, turbot, and goldfish show a differential expression in cells and tissues. The IFN1
were also demonstrated. The intraperitoneal injection of (rtIFN1) and IFN2 (rtIFN2) display high sequence similarity to
recombinant TNF alpha in gilthead seabream promotes the Atlantic salmon IFN alpha 1 and IFN alpha2, which contain
recruitment of phagocytes to the injection site and further two cysteine residues. However, rtIFN3 contains four cysteine
increases the respiratory burst phenomenon. The endothelial residues, which further confirms the relationship between fish
Immunomodulators 35

IFNs and mammalian IFN alpha. Both recombinant rtIFN1 and immunomodulatory effects of corticosteroids and stress are well
rtIFN2 have been shown to increase the expression of Mx and reported and similar effects have been observed in fish also.(78)
inhibit VHSV replication in RTG-2 cells, whereas recombinant For example, a number of studies have shown that stress or
rtIFN3 was a poor inducer of Mx and antiviral activity.(72) The cortisol administration can decrease fish resistance to bacterial
three trout IFNs have different functions in fish immune and fungal pathogens.(79) Most studies in fish have focused on
system, which paved the way for further research.(73) The IFN the effects of cortisol administration on the immune system, both
gamma has been identified in several fish species, including in vitro and in vivo conditions.(80)
Atlantic salmon and rainbow trout. Fish and mammalian IFN Cortisol administration can reduce the number of circulating
gamma are similar in exon-intron structure and display gene T- and B-like lymphocytes, while the number of circulating
synteny. However, a second IFN gamma subtype named IFN phagocytes (neutrophils and macrophages) may increase. The
gamma rel was identified in some fish species, which is dif­ fate of lymphocytes following stress is unclear, although re-
ferent from the classical IFN gamma. Carp and rainbow trout trafficking of cells to lymphoid tissues may be involved. Thus,
IFN gamma have several common functions with mammalian acute stress or cortisol treatment in juvenile coho salmon
IFN gamma, including phagocytosis of bacteria in macro­ (Oncorhynchus kisutch) lowers the number of circulating leu­
phages, nitric oxide production and the ability to enhance kocytes, but increases the number of these cells in the thymus
respiratory burst activity. However, fish IFN gamma induces and head kidney. Cortisol, however, has also shown to induce an
antiviral activity against both IPNV and the salmon alpha virus apoptosis in B cells and this could be responsible for their
(SAV) in salmon cell lines.(12) clearance from the blood following stress conditions. Cortisol
has depressive effects on a number of fish immune responses,
including phagocytosis and lymphocyte mitogenesis. Cortisol
3.5.2 Beta Glucans
also decreases the activity of B cells and circulating titers of
Glucans are potent immunomodulators in both mammals and IgM.(79) The effects of cortisol are mediated via the gluco­
fish. In fish, they can activate macrophages, increasing their corticoid receptor (GR), which has been identified in leukocytes
capacity to kill pathogens. They have also been shown to of coho salmon and common carp (Cyprinus carpio).(81)
reinforce other innate immune factors such as lysozyme and
complement activities. When administered orally, glucans
3.5.4 Growth Hormone and Prolactin
have shown a protective effect in many fish species and against
different bacterial pathogens including Vibrio salmonicida, Vertebrate polypeptides like growth hormone (GH) and pro­
Aeromonas salmonicida, and Pasteurella piscicida.(74) The lactin (PRL) are produced in the adenohypohysis of the pituitary
administration of glucans can increase a protection against gland and showed many structural and functional similarities. In
Aeromonas salmonicida in trout; A. salmonicida in Chinook all vertebrates, GH is involved in the regulation of post-natal
salmon; Aeromonas hydrophila in blue gourami; Edwardsiella somatic growth, but in fish it also influences osmoregulation and
ictalurid in catfish; E. tarda in common carp; Flexibacter stimulates gonadal steroidogenesis. Prolactin is a versatile pep­
columnaris in rainbow trout; and Vibrio anguillarum, Vibrio tide with diverse functions that may be summarised under three
salmonicida, and Yersinia ruckeri in Atlantic salmon.(2) broad categories of growth and development, osmoregulation,
Beta glucans act in two different manners to improve fish and reproduction. Both hormones modulate the immune
health: first, stimulating the beneficial gastrointestinal bacteria responses under in vitro and in vivo conditions. Indeed, GH is
as a typical prebiotic, and second, directly stimulating the structurally similar to a number of cytokines, including inter­
immune system through absorption in the small intestine.(35) leukin (IL)-2, IL-4, IL-5, granulocyte-colony stimulating factor
Consequently, the recognition of beta glucans as a fungus (G-CSF), granulocyte-macrophage colony-stimulating factor
triggers a cascade of events resulting in immunostimulation, as (GM-CSF), and the interferons.(81)
recently discussed by Oliveira et al. (2019).(75) Hisano et al. In fish, GH can stimulate lymphopoiesis and phagocytosis in
(2018) (76) fed pacu with beta glucan of varying concentrations gilt head sea bream (Sparus aurata) and silver sea bream
in combination with mannanoligosaccharides (MOS) for (Sparus sarba). It also promotes leukocyte mitogenesis in
30 days and found that the inclusion of 0.2% of beta glucans chum salmon (Oncorhynchus keta), natural killer cell activity,
and mannanoligosaccharides promoted the best feed efficiency, phagocytosis, antibody production, serum hemolytic activity in
growth response, and intestinal morphology. The same product rainbow trout (Oncorhynchus mykiss), and respiratory burst
and different concentrations were treated in pacu by Soares activity of leukocytes in rainbow trout and Mediterranean sea
et al. (2018),(77) who reported that 0.2 percent and 0.4 percent bass (Dicentrarchus labrax).(81)
diets were sufficient to increase the number of neutrophils, Other studies have shown that the GH enhances the resistance
thrombocytes, and monocytes in the blood, the respiratory of rainbow trout to the bacterial pathogen Vibrio anguillarum
burst of leukocytes, and lysozyme activity after a stressful infection. In addition, transferring brown trout (Salmo trutta)
handling and bacterial challenge. from freshwater to seawater increases plasma titres of GH while
reducing titres of thyroid hormones and this has been correlated
with increased phagocytic activity of head kidney leukocytes
3.5.3 Cortisol
and elevated plasma lysozyme concentrations. Prolactin has
Cortisol is a primary corticosteroid, produced from the interrenal similar effects on fish immune responses including stimulation
gland of teleost and there is small evidence for the significant of leukocyte mitogenesis, respiratory burst activity, phagocy­
formation of cortisone or corticosterone in fish. In mammals, the tosis, and an increase of plasma IgM titer.(81)
36 Immunomodulators in Aquaculture and Fish Health

3.5.5 Vitamins of the Gram-positive bacterial cell wall.(88) Lysozyme is

present in lymphoid tissue, mucus and serum of most of the
[Link] Vitamin C fish species, but not in few species (such as cod and wolfish).
The ability of vitamin C to induce the immune response has been Lysozyme activity is an index of innate immunity of fish.
established in fish. Elevated doses of dietary vitamin C have Lysozyme activity vary depending on the age and size, sex,
been shown to enhance several innate immune parameters such water temperature, season, toxicants, pH, infections, and
as macrophage activities, lysozyme activity, lymphocyte pro­ degree of stressors. Lysozyme, which is a strong bacteriolytic
liferation, complement activity, and natural cytotoxicity. agent, has been demonstrated in a variety of fish tissues and
Immunized fish fed high levels of vitamin C, elevated specific also in fish mucus. It is also present at high concentrations in
antibody level.(74) High levels of dietary vitamin C are reported human milk but at lower concentrations in bovine milk.
to increase resistance to E. tarda and E. ictaluri infections in Lysozyme has the ability, as an enzyme, to bring about the
channel catfish to V. anguillarum, IPN, Ichthyophthirius multi­ breakdown of bacterial cell wall peptidoglycan polysaccharide
filiis, Y. ruckeri and VHS in rainbow trout, and to A. salmonicida and destroy susceptible bacterial cells due to osmotic shock.(89)
and V. salmonicida in Atlantic salmon. The enhanced adaptive Lactoferrin is an iron-binding protein, that displays
immune response has been demonstrated in other fish species both bacteriostatic and bacteriocidal effects. In terms of the
such as Labeo rohita through after fish being fed vitamin C in bacteriocidal effects, lactoferrin has the ability to kill species
their diets.(2) Xu et al., (2016) studied the effect of dietary of bacteria, which action is not associated with its ability to
vitamin C deficiency in carp and reported that dietary vitamin C compete with the bacteria for iron.(89) In the African cichlid
deficiency affects the growth, spleen and head kidney immunity, (Sciaenochromis fryeri), dietary bovine lactoferrin (LF)
and structural integrity by regulating NF-kB signalling in young effects on the haemato-biochemical parameters, growth, stress
grass carp (Ctenopharyngodon idella). The studies on vitamin C responses, and non-specific serum immunity were investigated.
dietary supplementation in Labeo rohita displayed improved The highest lysozyme and bactericidal activities were observed in
survival rate.(82) Vitamin C with its redox catalyst property the fish fed the diets with 200 and 800 mg/kg of LF, respectively.
reduced the production of free radicals and increased the enzy­ Their study results showed that adding 400 mg/kg LF could
matic activity of iNOS by chemical stabilization of (6 R)-5,6,7,8- be useful as an iron discriminator, for liver function as well as
tetrahydrobiopterin or BH4.(83) Gupta et al. (2021) explained the an immune and stress resistance promoter in the African
effect of vitamin C dietary supplementation and its stress ame­ cichlid.(90) Several factors such as nutritional status, seasonal
lioration efficacy against various stressors. variation, sex and sexual maturation, salinity, pH, water
temperature, stresses, and infections could be considered as
possible parameters involved in the variations in lysozyme
[Link] Vitamin E
activity in vertebrates.(91) It has been reported that oral
Vitamin E also enhances both cellular and humoral defenses in administration of LF increases mucus secretion in the
animals.(84) For instance, Atlantic salmon fed vitamin E depleted Japanese flounder and Siberian sturgeon. Moreover, dietary
diets had significantly increased mortality rates following LF did not reportedly affect the stress indicators (serum glu­
A. salmonicida infection compared with fish on a commercial cose and cortisol levels) in the Siberian sturgeon (Acipenser
diet. Vitamin E deficiency also affects the specific immune baeri) during the rest period.(92)
response of fish, as demonstrated by the enhanced antibody titres Esmaeili et al. (2019) studied the effect of the dietary
to E. ictaluri in channel catfish fed high levels of vitamin E for supplementation of bovine LF on hematological and immu­
180 days.(2) The detoxifying ability of vitamin E mediated nological parameters, growth performance, antioxidant en­
by methemoglobin reductase production. Moreover, the use of zymes activity, and disease resistance against Vibrio harveyi
dietary vitamin E also attenuated oxidative damage resulting in yellowfin sea bream (Acanthopagrus latus) fingerling. The
from the production of reactive nitrogen species and modulated study results showed that diet supplementation in A. latus
the expression and activation of signal transduction pathways in especially with 1,200 mg LF per kg improves fish immune
L. rohita.(85) Belo et al. (2014) fed pacu with vitamin E (12, 58, parameters and growth performance, as well as survival rate
and 310 mg/kg) during 18 days and evaluated the inflammatory against Vibrio harveyi.(93)
responses to a foreign body (glass coverslip) on connective tissue
over 15 days.(86) Fish fed with the lower vitamin E dose had a
lower number of lymphocytes and LG-PAS cells and an elevated 3.5.7 Minerals
plasma cortisol level. Vitamin E (α-tocopherols) arrested the [Link] Selenium
chain process of lipid peroxidation by quenching lipid peroxyl
radicals in Rohu (Labeo rohita) has been reported.(87) Selenium (Se) is a vital micronutrient for antioxidant defenses,
and one of its roles is to prevent the immunosuppression caused
by oxidative stress. Selenium deficiency is associated with
3.5.6 Lysozyme and Lactoferrin diminished activity of selenium-dependent glutathione peroxi­
Lysozyme (or muramidase or N-acetylmuramic acid hydrolase dase in neutrophils, and negatively affects neutrophil bacterial
E.C. [Link]) is a protein that exerts its enzymatic activity activity.(89) The oxidative stress impairs the immune response
through the hydrolysis of the β-1,4-glycosidic bonds between and indicates the occurrence of a physiological trade-off
N-acetylmuramic acid (NAM) and N-acetylglucosamide between immune and antioxidant systems in fish.(35) The
(NAG) in the polysaccharide backbone of the peptidoglycans immune response and antioxidant defenses of fish were
Immunomodulators 37

improved by supplementing the diet of pacu with 0.3, 0.6, 0.9, phagocytic activity of neutrophils and the number of phago­
and 1.8 mg Se-yeast/kg for 65 days.(94) Selenium is generally cytic cells and leukocytes increased and the activity of lyso­
available as trace element to the fish mainly due to their dif­ zyme in serum of mammals also increased. The similar effects
ferential bioavailability.(95) The zinc oxide (ZnO) and selenium have also been reported in fish by Siwicki (1987). Olivier et al.
(Se) nanoparticles dietary supplementation stimulates immunity (1985) also proved that levamisole provided the protection
and enhances resistance to bacterial infection in L. rohita.(96) against Aeromonas salmonicida. Protection against infectious
fish diseases was achieved with the help of phagocytic cells.
[Link] Chromium Levamisole activated the fish phagocytic cells and NK cell
activities. Levamisole activated the alternative complement
Trivalent chromium is known to reduce plasma cortisol levels pathway of rainbow trout, which might play as opsonic com­
in stressful situations. In the swim bladder, Castro et al. (2014) plement effects in the nonspecific defense mechanism.(100) To
investigated the effect of the inclusion of chromium carbo­ stimulate the immune function of Cirrhinus mrigala, a dose of
chelate in the diet of pacu on acute inflammation challenged by 250 mg of levamisole per kg diet is optimum.(101) The anti­
injection of inactivated A. hydrophila. Gatta et al. (2001) re­ oxidant action of levamisole is by the enhancing effect on the
ported that chromium is able to modulate the immune response major cellular redox systems like glutathione, enzymes like
of rainbow trout, and this effect appears to be both dose and superoxide dismutase, catalase, and also possible effects on
time dependent. In the study of Castro et al. (2014), fish-fed diets glutathione-related enzymes. On the other hand, the anti-
containing 18 and 36 mg of Cr per kg had plasma cortisol and inflammatory action is by the inhibition of TNF-alpha (tumor
glucose reduced and, thus, an enhancement of the immune necrosis factor) and interleukin 6. Anti-neoplastic action of
response. In addition, pacu fed with chromium carbochelate and/ levamisole is by two ways that is, through its apoptotic action
or in combination with Saccharomyces cerevisiae for 105 days causing cell cycle inhibition and increased endothelial cell
by Bortoluzzi et al. (2017). They reported that fish supplemented adhesion and the second action is through its anti-angiogenic
with chromium and S. cerevisiae had a greater degree of orga­ property.(102) The oral administration of levamisole for 15 days
nization of collagen fibers, higher neovascularization, and faster modulated circulating cortisol levels during the stress response
reepithelialization, when subjected to skin injury.(35,97) Dietary and improved the innate immune response against A. hydro­
supplementation with betaine (Bet) and chromium picolinate phila infection in pacu (Piaractus mesopotamicus).(103)
(Cr-Pic) combination is improving fish immune response and Levamisole showed an increase in circulating leukocytes,
antioxidative status and aquaculture sustainability in Nile tilapia phagocytic rate, and increase protection against P. damselae
(Oreochromis niloticus).(98) subs P. piscicida in European seabass.(104)

3.5.8 Bacterial Lipopolysaccharides (LPS) 3.5.10 Kaempferol 3-a-L-(4-O-acetyl)

Rhamnopyranoside-7-a-
Microbial gut flora is known to modulate fish immunity in dif­
Lrhamnopyranoside (SA)
ferent ways and bacterial lipopolysaccharides (LPS) in particular
may have a highlighting effect on fish immune system.(89) The compound isolated from Crown Wood-Fern (Dryopteris
Bacterial LPS, called endotoxin, are among the most potent crassirhizoma) is kaempferol 3-a-L- (4-O-acetyl)
immunomodulators among the known ones. Despite the less rhamnopyranoside-7-a-Lrhamnopyranoside (SA).(105) It is
sensitivity of aquatic animals to LPS, mammalians are very also present in Zingiber officinale. This compound has the
sensitive to LPS thus, it has no application as immunostimulant ability to inhibit Ribosomal S6 kinase (RSK) activity. It can be
for practical use in mammals because of its toxic effect which used to reduce the proliferation of neoplastic cells or for in­
causes inflammation reactions resulting in fever, reduced feed hibiting the establishment or maintenance of an intracellular
intake, and impaired performance of animals.(99) Although LPS pathogenic infection by pathogens whose pathogenicity
has traditionally been regarded as a B cell mitogen and macro­ derives in part from the pathogen’s ability to impede en­
phage activator, it is now apparent that an interaction between T dosomal or phagosomal maturation in the host cell (106) (Patent
cells and LPS occurs.(99) Bacterial endotoxin LPS potentiates No: US9040673B2) This compound increased the immune
antigen-specific proliferation of T helper cells. This effect is responses like lysozyme activity, phagocytic activity, com­
independent of interleukin I action, the T cell activation factor plement C3, IgM level and SOD activity of fish under in vivo
released by macrophages after LPS stimulation. Thus, through condition. It also induced expression of immune response-
LPS, both specific and non-specific protective immune related genes like TNF-α, MyD88, Mx1and IL-1β in fish of in
responses could be improved.(89) vitro studies.(107)

3.5.9 Levamisole 3.5.11 Herbal Immunomodulators

Levamisole protects fish from A. hydrophila in carp, A. sal­ Herbal immunomodulators are promising among the other im­
monicida infections in coho salmon, V. anguillarum in rainbow munomodulators used in aquaculture because of its propensities
trout, and V. anguillarum in gilthead seabream. Levamisole to improve growth performance and fish immunity. They are
administration to fish also reported to increase the antibody good alternatives to chemical treatments and antibiotics.
titres values.(2) In mammals, levamisole stimulates the Medicinal herbs and plant-based immunomodulators gained
38 Immunomodulators in Aquaculture and Fish Health

attention in the prevention and treatment of fish diseases due to Disclosure statement: The authors declare that they have no
being ecofriendly and cost effective and have minimal side ef­ conflicts of interest.
fects.(108) Details of herb/plant immunostimulatory effects in fish
are given in Chapter 7.
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4
Immunomodulators and Stress Oxidative

Tamilselvan Gokul1, Paulraj Balaji2, Karthikeyan Venkatachalam3, Subramanian Ramya4,

Ramaraj Jayakumararaj5, Chinnathambi Pothiraj5, and Kamatchi Ramesh Kumar1
1
PG and Research Department of Zoology, Vivekananda
College, Tiruvedakam (West), Madurai, Tamil Nadu,
India
2
PG and Research Centre in Biotechnology, MGR
College, Hosur, Tamil Nadu, India
3
Faculty of Innovative Agriculture and Fishery
Establishment Project, Prince of Songkla, University
Surat Thani Campus, Makham Tia, Mueang, Surat
Thani, Thailand
4
Department of Zoology, Yadava College (Men),
Madurai, Tamil Nadu, India
5
Department of Botany, Government Arts College,
Melur, Madurai, Tamil Nadu, India

may explain the reduced levels of antioxidant enzymes in var­

4.1 Introduction ious fish tissues (Ghazanfar et al., 2018; Latif et al., 2020). The
ecosystem of aquatic life is threatened by the formation of
Research is increasingly focusing on the development of new free radicals and ROS (Lushchak et al., 2011). When a cell is
immunomodulators as a means to either prevent or treat illness exposed to a stressor, it makes ROS, leading to oxidative stress
(Safriani et al., 2022). Compounds that may influence the (Sopinka et al., 2016). Environmental stressors can also cause
immune response in either a positive or negative manner can be oxidative stress, which leads to cell ageing (Monaghan et al.,
called immunomodulators. These compounds can be obtained 2009), and an increase in the level of protein damage (Iwama
from natural or synthetic sources and can modulate the immu­ et al., 1998). Several studies have looked into the possibility that
nological response via specific or non-specific routes (Nair et al., the production of free radicals and oxidative stress is a major
2019). Fish experience stress when confronted with a particular cause of DNA damage in various organismal tissues (Sharma
scenario that threatens their survival (Schreck et al., 2016). et al., 2019; Ali et al., 2020). In addition, many other substances
Several factors may cause stress, but oxidative stress is the most present in microorganisms can cause oxidative stress; for ex­
significant factor affecting fish on a molecular level. These ample, the lipopolysaccharide found on the outer surface of
factors can come from many different places. One kind of stress Gram-negative bacteria is a well-known oxidative stress inducer
that is known to disrupt the equilibrium between oxidants and (Swain et al., 2008; Giri et al., 2020). Also, other chemicals
antioxidants is referred to as oxidative stress (Dontha et al., like polycyclic aromatic hydrocarbons (Madison et al.,
2016). Oxidative stress indicates cellular and molecular stress 2015), potassium dichromate, melanosomes, cortisol, melatonin
(Sopinka et al., 2016). Fish erythrocytes are easily damaged by (Gozdowska et al., 2022), microplastics (Solomando et al.,
oxidative stress, so they are used as models to study how oxi­ 2022), and other chemical substances can cause oxidative stress
dative stress causes cell death (Li et al., 2013). It can change how in fish (Figure 4.1). There are various signs in fish to understand
a fish’s immune system works. This shows that there is a the oxidative stress level; fish with higher levels of oxidative
physiological trade-off between the immune and antioxidant stress have higher levels of reactive oxygen metabolites
systems (Urbinati et al., 2020). Higher energy demand in fish has (d-ROM) (Hossain et al., 2017). Various antioxidants have been
been linked to poor conditions and elevated oxidative stress, shown in numerous studies to improve stress tolerance and
mainly when the immune system is activated to act against a mitigate oxidative stress’s negative effects (Choi et al., 2017).
pathogenic infection (Kurtz et al., 2006). During the stress Fish must have a healthy equilibrium between oxidative stress
condition, there is an increase in the production of reactive and antioxidant defence mechanisms to adjust physiologically to
oxygen species (ROS) and the development of an imbalance their aquatic system (Wu et al., 2017). There are numerous
between the formation of ROS and antioxidant capacity (Pohl developments and discoveries to mitigate oxidative stress, the
et al., 2018; Lee et al., 2017; Aniya et al., 2018). A decrease in body’s antioxidant defences consist of enzymatic and non-
antioxidant enzymes and subsequent increased oxidative stress enzymatic components that eliminate oxidative stress-inducing

DOI: 10.1201/9781003361183-5 43
44 Immunomodulators in Aquaculture and Fish Health

FIGURE 4.1 Different stress conditions responsible for causing oxidative stress and their effects.

free radicals while keeping ROS at a normal physiological level discovered. This chapter discusses the immunomodulators,
(Ondreicka et al., 1998). In general, feed additives act as im­ stress indicators, oxidative stress, and responses of fish in the
munomodulators, and many natural plant-based phytochem­ aquatic system.
icals, especially phenolic compounds, act as feed additives for
fish and are antioxidative (Beltrán, et al., 2022) For several
4.1.1 Concept of Immunostimulants
aquatic animals, β-glucans have shown usefulness as an im­
munostimulant (Dawood et al., 2015), it has been found that Many pathogens infect fish causing a systemic infection that
glutamate and glutamine protect against oxidative damage leads to disease and death. Several strategies have been used
(Dawood et al., 2018), antioxidant supplements like vitamins in aquaculture, including using various chemicals to strengthen
C and E help fish cope better with oxidative stress (Gao et al., or enhance the body’s immune system of cultured fish and
2013). Farmers are willing to employ these immunostimulants shrimp. Vaccination against certain infections has recently
and other treatments and vaccines. Immunostimulants appear been created with different levels of success depending on the
to have superior efficacy and safety compared to chemotherapy disease, it is an excellent preventive treatment for infections in
and vaccinations (Table 4.1), making them appropriate for fish culture, although it can be costly and traumatic for the fish.
use in aquaculture. As knowledge grew, understanding of Due to the complex antigenic nature, a single vaccination is
immunomodulatory mechanisms increased, and more novel ineffective against many pathogens and only protects against a
strategies for regulating oxidative stress in fish remain to be specific disease (Ardo et al., 2008). Fish and shrimp have been

TABLE 4.1
Comparison of Vaccines and Immunostimulants in Fish Health Management
S. No. Vaccine Immunostimulants
1. Prevention of disease will be for long duration with only one Prevention of disease will be for short duration, require more treatments
or two treatments
2. Highly effective Effective
3. Reduced range of activity Wide range of activity
4. No toxic effects/no accumulation of toxic residues No toxic effects/no accumulation of toxic residues
5. Negligible effects on the environment Negligible effects on the environment
6. Increase the body’s immune response, both innate and Mainly enhance adaptive immune system of larvae before innate immune
adaptive system develops
7. Difficult to vaccinate Easy to vaccinate
8. Expensive Economically viable
Immunomodulators and Stress Oxidative 45

treated and prevented with chemical additives and drugs in illnesses, or inhibit latent or sub-lethal pathogens. These are
addition to vaccination. believed to be an attractive and potential disease prevention
Nonetheless, the rise of multidrug-resistant infections poses an treatment for fish and crustacean. The prophylactic injection of
increasing global danger to veterinary medicine. Consequently, immunostimulants is seen as a feasible alternative to chemo­
innovative antimicrobial medications are always required to limit therapy and vaccinations in aquaculture because of their
the likelihood of increased antimicrobial resistance and wide effectiveness, relatively inexpensive, and environmentally
undesirable properties on the native microbiota of juvenile or friendly properties. The use of natural immunostimulants for the
mature fish (Misra et al., 2006). Antibiotics have been used for prevention and/or control of fish diseases in aquaculture has
many years to treat bacterial infections in fish production. Due to been endorsed by both the WHO and the FAO of the United
the inappropriate use of antibiotics, a worldwide problem has Nations. This is intended to strengthen and stimulate fish’s
emerged in aquaculture: the emergence of microorganisms immune systems while averting future environmental, animal,
resistant to antibiotics associated with fish diseases. and human health problems (Reverter et al., 2014; Cunha et al.,
An immunostimulant is a substance, medication, or activity 2018; Magnadottir et al., 2010). For the further expansion of the
that boosts the non-specific defence mechanism or the specific global aquaculture industry, immunostimulants that improve the
immunological response among the several disease control innate defences of aquatic animals and increase their resistance
strategies. It has been demonstrated to promote phagocytic cell to infection are a feasible alternative to antibiotic treatments
activity and enhance their bactericidal and fungicidal effects and (Fuchs et al., 2015). As a result of their ability to increase
they are substances that stimulate leukocytes and aid in recovery system efficiency, stimulate output, lessen chemical use, and
from stress-induced immunosuppression. When used in aqua­ make aquaculture products more palatable and secure, im­
culture, it can be used to reverse the immunomodulatory prop­ munostimulants are likely to become increasingly widely used
erties of stimuli, avert periodic resurgence of recognized chronic as feed additives in the aquaculture industry (Figure 4.2).

FIGURE 4.2 Functions of immunostimulants.

46 Immunomodulators in Aquaculture and Fish Health

homeostasis following a stressful event. Nevertheless, it is

4.2 Stress and Disease in Fish known that an animal’s adaptive reaction might be insufficient,
excessive, or prolonged, which can result in disease. In reaction
Fish and other animals experience stress as a general physio­
to a stressor, the general adaptation syndrome develops in three
logical response to harmful occurrences. When a stressor is
stages. Hormones are released rapidly during the “alarm stage,”
seen, a stress response is generated very immediately. Even
the first step of the “fight or flight” response. The fight-or-flight
under low-stress levels situations, adaptive reactions can be
reaction has been resolved and the normal condition has been
elicited, but they can also lead to maladaptive or unfavourable
restored during the resistance stage, which takes place within
results (distress). Both corticosteroids (mainly cortisol) and
48 hours of contact with the stressor. It is during this period of
catecholamines are involved in the stress response (adrenaline,
tiredness that animals lose their ability to withstand stress and
non-adrenaline, and its pioneer dopamine). As a whole, these
surrender owing to the return of alarm-stage traits to their en­
components regulate the body’s secondary stress response,
vironment. The so-called chronic stress repercussions are now
which in turn affects the flow of vital nutrients and oxygen
included in later definitions of stress as a result of the over­
throughout the body, putting the immune system at danger. A
stimulation of physiological and psychological reactions.
stressor’s response to a fish differs widely among taxa, stocks,
The animal’s survival, the onset of a diseased state, or even
and species because of the genetic diversity among these
death are all possible outcomes of a stressor. When it comes to
groups. The way a fish reacts to stress depends on a number of
the stress response, the amplitude and duration of physiolog­
factors, including its past experiences, its current environment,
ical reactions are largely dictated by the type of stressor,
and its current physiological state. Because unstressed fish can
whether acute or chronic (Davis, 2006). When faced with an
exist even in the absence of clinical stress markers, break­
acute stressor, the body’s fight-or-flight reaction kicks in
throughs in fish physiology have made it possible to swiftly
quickly, and the stress response is characterised by its extreme
identify stressed fish. Stress-related clinical indications may
severity, brief duration, and rapid onset. Because of their
have a tendency to falsely indicate a bad outcome. A stressor’s
longevity, chronic stressors induce more discomfort and mal­
severity cannot be determined with any precision or accuracy
adaptation than short-term ones. This puts people’s lives in
from clinical data.
jeopardy because of the greater energy costs (Ellis et al., 2012;
Adding fresh, oxygenated drinkable freshwater can increase
Martnez-Porchas et al., 2009). Chronic strain in farmed fish
solubility, but it can also introduce pollutants and deprive the
must be taken into account because acute stress frequently
plants and animals being farmed of oxygen (in this example,
leads in a positive stress response (Davis, 2006). The impacts
fish). Physical and chemical factors, such as those already
of the stress response are often seen as negative since farmed
mentioned, are common contributors to stress. For decades,
fish cannot avoid the difficulties of aquaculture (Davis, 2006).
doctors have been at odds about the origins of the stress syn­
Many environmental and physical stressors, as well as social
drome, but now they agree. The specific effects of stress on a
(competitiveness, crowding) and behavioural (aggressiveness)
human being’s body and mind are that it can slow down or even
factors are involved in the production process of farmed fish
stop the body’s natural functions, growth, and resistance to
compared to wild fish that are not subjected to these stressors
disease, and even cause death. It’s well knowledge that immu­
(Braithwaite and Ebbesson, 2014). Disruption in the fish’s
nosuppression can have severe effects, but viruses are more
welfare can be difficult to identify because it might be mis­
prone to flourish in water. It’s no secret that stress contributes to
taken for normal and adaptive reactions to stress (Huntingford
health problems and has been the subject of a lot of research.
et al., 2006; Segner et al., 2012; Ellis et al., 2012). Reduced
appetite, irregular swimming, and repetitive behaviours are
some of the behavioural and physiological signs of persistent
4.2.1 Stress and Its Impact on Aquaculture
stress, along with stunted growth, inability to reproduce, and
The concept of stress is both all-encompassing and universally poor nutrition (Huntingford and Kadri, 2014). Aquaculture
known, although its scientific definition has been heavily dis­ aims to maximise output by improving growth, resistance to
puted. Selye (1936, 1973) invented the term “stress” and disease, reproductive success, and quality in farmed fish by
described by way of “the non-specific response of the organism controlling stress. With this fundamental difficulty in aqua­
to any need for change”; however, he likewise stated that culture, a wide range of experiments on stress physiology and
“everyone understands what stress is, but nobody truly its effects on fish well-being have been published in recent
knows.” Selye (1936) comprised ideas in his attempt to char­ decades, with several reviews of these studies (Pavlidis and
acterise the response of the stress, calling it a “generalised Mylonas, 2011). Stress markers have also contributed to
effort of the organism to adjust itself to new situations, called improvements in fish husbandry as a result of the rising focus
the general adaptation syndrome,” and contrasting it with other on fish well-being.
forms of generic self-defence. According to one definition,
stress is “a state in which the dynamic equilibrium of the
4.2.2 Cellular and Molecular Indicators of Stress
species, known as homeostasis, is threatened or altered as a
result of an action of intrinsic or external stimuli that serve as Stress responses at the cellular and molecular level function in
stressors.” The “disruption of homeostasis” by the threatening tandem with those that occur at higher levels of biological
stimuli is a critical factor shared by most definitions of stress. structure to provide a temporary resistance to stressors (Kultz,
These definitions suggest that the stress response is an adap­ 2005). Heat shock factors (HSFs) interact with cortisol, which
tation that helps an organism survive by restoring a state of raises the production of metallothionein, ubiquitin, and HSPs
Immunomodulators and Stress Oxidative 47

as part of stress reactions Kassahn et al., 2009). The c-Jun Stress factors such as low water and air exposure raise the
element of transcription factor activation protein-1 (AP-1) and level of JUNB in rainbow trout (Momoda et al., 2007). In
glucocorticoid receptors are also affected by cortisone. rainbow trout, stress-induced increases in the transcription
Cortisol has different transcriptional effects depending on factor NUPR1, which regulates cell development and death,
tissue type, HSP 90 expression levels, and cortisol binding to were seen in a similar way (Momoda et al., 2007). As a result
glucocorticoid receptors. Damaged DNA and proteins are of stress, rainbow trout (O. mykiss) livers have already
repaired, the cell cycle is stopped, waste products are elimi­ revealed gene expression alterations connected to wound,
nated, and the body shifts from an anabolic to a catabolic amino acid degradation, and the host–pathogen response
metabolic state as a response to various stresses. These cellular (Momoda et al., 2007). According to the findings of Krasnov
and molecular processes are caused by minimum-stress pro­ et al. (2005) a netting stressor alters gene levels of genes
teins (Faught et al., 2016; Kultz, 2005). involved to immunological responses, cell growth, cell death,
When a cell is subjected to oxidative stress, it releases more and synthesis of protein in rainbow trout, as well as gene ex­
reactive oxygen species (ROSs). Oxidative stress contributes to pression in the kidney. Gene expression in the liver that is
protein damage. The degree of protein damage can be gauged by involved in anaerobic glycolysis, glycolysis, and glucose
measuring the amount of ubiquitin-labelled protein in the metabolism may be an indicator of post-stress recuperation
cell. Because of the defective and ubiquitinated proteins, (Momoda et al., 2007). Both male and female fish display
the cell’s heat shock response focuses on protein repair. When a unique genetic responses to an exercise stressor, according to
cell is exposed to a stressor that is both large and long-lasting, Donaldson et al (2014). Stress has been tough to study because
they are more likely to suffer from heat shock. When the of this, and it’s been difficult to figure out how different spe­
hypothalamic–pituitary–interrenal (HPI) axis is activated, HSF1 cies, methods, and fish (Krasnov et al., 2005) all influence the
regulates HSP expression by dissociating HSPs from HSF1. activation of specific genes in the fish (Kassahn et al., 2009).
After making its way to the nucleus, HSF1 begins transcription
of HSP (Kassahn et al., 2009). It’s possible that ROS can speed
4.2.3 Primary and Secondary Indicators of
up the telomere shortening that occurs as a result of oxidative
stress. Telomeres, or the protective caps on the ends of chro­
Physiological Function
mosomes, are critical for genome stability and can hasten cell The whole animal’s physiology is impacted by stress in­
ageing if damaged too quickly. Growing data suggest that dicators, which include cellular and molecular reactions and
telomeres may be an indicator of cell and, possibly, organismal systemic effects. The HPI axis is only one of the many phys­
ageing (Monaghan et al., 2009). Stress, such as emotional iological responses triggered by stress (Schreck and Tort,
straining or increased generative exertion, may impact telomere 2016). Researchers may utilise a variety of basic indicators (for
length (Kotrschal et al., 2007). Oxidative stress, which can lead example, glucose levels or ion balances) and secondary
to cell senescence and eventually organism senescence, has been reactions (such as changes in acid–base balance and immu­
linked to environmental stress (Monaghan et al., 2009). nological processes) to measure stress in fish (for example,
Gene expression (quantitatively and qualitatively, as well as catecholamines or stress hormones). The pace of subsequent
in terms of response coefficients) has been connected to a adjustments slows. Many stress indicators have advantages and
variety of stresses (Krasnov et al., 2005). It is currently disadvantages depending on the stressor that is being studied
common practise to employ genotyping and expression levels and how much contextual evidence is obtainable about the
analysis to learn more about fish responses to environmental population being researched. Short-term field disturbances can
stresses. These well-known stressors include hypoxia (tem­ have a major impact on stress indicators, including main or
perature) and hypoxic stress management (toxicants), which secondary stress markers, depending on the aquaculture and
have been examined in several research (Jeffries, 2012). handling practises used. Catecholamines are the first stress
Clusters of differentially expressed genes associated to a wide hormones to be released, but because of their fast release, it is
range of metabolic pathways can be identified utilising cDNA difficult to detect them. As a result, it is extremely difficult to
microarray technology, which examines thousands of genes measure catecholamine levels in the field. Also known as the
simultaneously. Compared to other stress indicators, such as stress response marker cortisol, this hormone is one of the most
primary, secondary, and tertiary, acute stressor exposure has often used in stress testing.
been researched in a very small number of studies (Caipang In contrast to catecholamines, cortisol is stable and can be
et al., 2008). To understand better how environmental influ­ found in either labs or the field, provided animals are analysed
ences regulate expression of genes is rather one of the primary within minutes of capture. Secondary stress indicators include
goals of functional genetic analysis (Buckley, 2007). Migrating glucose catabolism and an increase in anaerobic metabolism
salmon have an unfavourable profile that includes signs of bad after hypoxia or exercise stressors, in addition to glucose and
health, such as decreased expression of blood clotting proteins lactate increases. Three factors can be affected by an increase
and genetic factor linked to aerobic respiration and immuno­ in heart rate and the permeability of the gills: (1) osmolality,
logical function (Miller et al., 2011). Several genes and gene (2) specific ions, and (3) leukocytes. As well as indicating
families are difficult to understand, and the ramifications of stress responses, these factors can also be used to predict
their responses to stimuli are not always evident. A wide outcomes such as a person’s survival or performance. It is
variety of fish genes have been studied as a possible signal of essential that these markers be assessed in plasma in order to
various stresses (Jefferies et al., 2012). acquire an accurate picture of their concentrations in the
48 Immunomodulators in Aquaculture and Fish Health

bloodstream. It is possible to collect plasma (or red blood cells investigate other parts of behaviour, such as swimming, as well
for measurements like hematocrit) without injuring the fish, as the heart and metabolism as a whole (both the performance
which is a benefit (Beecham et al., 2006). Not all signs make of swimming and general locomotion). Metabolic and loco­
sense when seen in plasma, even if it is the most prevalent. motor activity have a significant impact on energy consump­
Plasma-signal detection has this as its primary drawback. tion. Energy budgets dictate somatic growth rates and
Glycogen buildup in the liver as a result of chronic stressor circumstances, and behaviour. The metabolic and cognitive
exposure cannot be detected in a plasma sample alone. Muscle functions of fish are more closely linked to their survival than
tissue is preferred for measuring PCr and ATP since these previously thought (Davis, 2010).
markers are acute exercise stress indicators. Thus, the choice of Markers of homeostasis as a whole (such as neuroendocrine
tissue is determined by the nature of the question being inves­ activity) make it far more challenging to infer a stressed
tigated and the primary or secondary physiological signal that is condition than tests that concentrate on secondary or primary
to be analysed. Environmental obstacles cause stress in both determinants of homeostasis (such as temperature or blood
people and animals, and these creatures‘ stress levels can be pressure). A fish’s death isn’t always an indication that it was
measured using secondary and main stress markers (Breuner stressed; ageing is an inevitable part of life, and fish can be
et al., 2008). preyed on even if they weren’t stressed. Fish’s previous
feeding histories and metabolic demands, as well as the
availability of food at different times of the year and heredity,
4.2.4 Tertiary/Whole-Organism Indicators of
all influence their feeding habits. In the case of stress signals
Physiological Function
like cortisol and osmolality, reference ranges and thresholds
Just a few of the various whole-organism (or tertiary) reactions are well defined. If a single fish’s cortisol levels are greater
to stress can be detected, such as changes in development, than the standard, but there are no clear goals at the organism
health, and health-related behaviours. Environmentally rele­ level, it’s feasible. This may or may not endanger the fish, one
vant stress indicators are often referred to as this species. cannot be sure. It’s vital to compare tertiary outcomes to
Bioenergetics and population models are affected by growth acceptable controls while stressing out a system. However,
rates, fitness, and survival, although fitness and survival are despite these difficulties, collecting and analysing tertiary in­
also relevant to demographic processes. Everything from food dicators is simpler than other laboratory-based physiological
intake to predator avoidance to habitat selection has a physi­ stress assessments. Survival and response indicators (such as
ological basis. There are several condition-related metrics that the presence of a given reaction) are two simple, obvious,
rely on simple measures of mass or length (or both). Their fish critical measurements of total stress in an organism (e.g., did
condition measurements have been criticised for being too an organism live or die).
simplistic and lacking in detail. Despite their relative com­ In the field, researchers frequently use the presence or absence
plexity, organosomatic indices (such as the ratio of organ mass of certain reflex dysfunctions as an indicator of an animal’s
to body mass) are commonly utilised as a stress and condition health and a possible death forecast (Raby et al., 2012; Davis,
assessment. 2010). This reflex dysfunction has yet to be linked to a specific
Researchers can tailor their HAI selections to their indi­ molecular aetiology. In free-swimming fish, secondary stressors
vidual needs and the various environments in which their can be measured. Examples of this type of data include loco­
studies are conducted. Custom-built swim tunnels and flumes motor activity and heart activity at small scales (Clark et al.,
are the most precise means for assessing a swimmer’s swim­ 2010). Either wirelessly (using biotelemetry/biosensors) or on
ming ability, but other methods are available. Despite its the animal’s body for future retrieval, electronic tags can be used
present lack of significance, critical swimming speed is still to assess blood glucose levels in animals. Biologging is a term
important for evaluating the swimming ability of fish subjected used in the field of biometrics to describe this process (Raby
to various stressor treatments. In 1978, tail-pinning data col­ et al., 2012).
lected in a round tank and compared to more formal studies of
essential swimming speed were shown to be statistically sig­
nificant. Resilience to stress is measured by the “recovery 4.2.5 Considerations for Measuring and
ratio,” which has been applied to a wide range of organisms. Interpreting Stress
Measures of cardiac activity (such as vital signs or myocardial
[Link] Interspecific Differences
contractility), metabolic activity (oxygen intake), and effective
swimming activities are all necessary in studies examining the To be expected, different fish species react differently to the
effects of stress on ecologically relevant metrics of organism same pressures in the environment. Given the evolutionary,
performance. ecological, environmental, and life cycle variables that con­
An accurate technique for predicting mortality in the field tribute to species divergence, it is anticipated that HPI activity
can be established by scaling metabolic, cardiovascular, and and other stress markers will vary among species. It is difficult
swimming capacity from laboratory data. In comparison to to compare the HPI stress response amplitude across species
primary or secondary stress reactions, tertiary stress indicators because of the large diversity of stressor types, severity, and
have access to a broader range of data. Tertiary stress response duration in numerous investigations. Cortisol and glucose
data may be collected and analysed by field ecologist, ethol­ levels, gene expression, immune function, habitat preference,
ogist or physiologist. In contrast, the physiological basis of and avoidance behaviour are all affected simultaneously by
tertiary stress markers is clearer. However, physiologists also stress (Jefries et al., 2014), as well as other factors such as gene
Immunomodulators and Stress Oxidative 49

expression, immune function, and habitat preference (Jacobsen Tort, 2016). An individual can have physiological and beha­
et al., 2014). Stress responses might be similar amongst var­ vioural changes if they are confronted with a predator, com­
ious animals in some circumstances. Sablefish juveniles petition for resources or a lack of food. Stress assessments vary
Anoplopoma fimbria are as vulnerable to capture mortality as depending on the environment in which they are taken at all
walleye pollock juveniles Theragra chalcogramma. organisational levels. Stressor type influenced the concentra­
tions of brain monoamines (e.g., norepinephrine and serotonin)
in three-spined sticklebacks (Bell et al., 2010). Plasma
[Link] Intraspecific Differences
responses may not be affected by minor changes in stressor
Stress analysis is typically inspired by the emergence of type (Ros et al., 2014). Identical reactions can be triggered by
diverse populations. Populations from different parts of the two stressors that couldn’t be more dissimilar in terms of en­
world are likely to have different stress responses due to a vironmental significance and severity. The three-spined stick­
variety of environmental and biological factors. Indeed, there leback’s plasma cortisol levels rise in reaction to an implanted
was a longitudinal gradient in the stress-induced plasma cor­ elastomer tag, which is visible to the naked eye, much as they
tisol responses of killifish. If the fish were caught in an area would after experiencing a real predator attack (Furtbauer et al.,
with low or high levels of predators, the outcomes would be 2015). The stressor’s circumstances also have an effect on
different. After stressor exposure, fitness outcomes can differ secondary, physiological processes. Galaxias maculatus showed
between groups even when there are no differences in physi­ varying degrees of metabolic changes when exposed to visual or
ological stress markers (e.g., plasma cortisol and lactate) olfactory predator cues (Milano et al., 2010). Stressors affect
(Donaldson et al., 2012). From an evolutionary vantage point, whole-animal breathing rates differently. Stressor intensity is
it is important to account for population variability in stress most likely linked to environmental and ecological context when
response before drawing species-wide generalisations about determining which indicator to employ, predicting how the
the sensitivity of an organism to a given stressor. indicator will respond, and interpreting the results.
Gender, size, social rank, and domestication are among the If stress is to be judged scientifically, it is best to expose
other intraspecific characteristics that influence stress indicator animals to a variety of stressors. Experimentation with various
variance. When it comes to some animals, it is easy to see the stressors can be utilised to determine the magnifying power of
variations between sexes. High stress-induced plasma cortisol stress markers (Jacobsen et al., 2014; Marcogliese et al., 2005).
levels and an increased risk of death were seen in adult female Fish, like humans, are subjected to repeated stressors, and
Pacific salmon (Oncorhynchus spp.) exposed to high temper­ much like humans, fish may exhibit different stress responses
atures (Donaldson et al., 2014). Size matters when it comes to depending on how much experience they’ve had with a certain
European sea bass’s maximal stressor-induced plasma cortisol stressor and their potential for learning (Barcellos et al., 2010).
response (Fatira et al., 2014). HPI activity varies between Before exposure to stressors (e.g., isolation versus groups),
dominant and subordinate rainbow trout (Jeffrey et al., 2014). housing environments (e.g., olfactory indications of con­
Fish in captivity or put back into the wild may not do as well if specific stress) may also alter stress indicators (Giacomini
they are stressed out to the same degree as hatchery or wild et al., 2015; Barcellos et al., 2014). Fishermen may prefer to
trout or salmon. At long last, there is growing recognition of test individual fish rather than groups because of the species’
the heritability of stress-responsiveness and the differences schooling and social behaviour. If biological significance of
between individuals and families, as well as the connection stress markers is the research priority, such intricacies of
between physiological and behavioural stress indicators within ecological and environmental context can be investigated. In
an individual (Kittilsen and Pottinger, 2009; Hori and other cases, a wide range of acceptable indicators can be used.
Pottinger, 2012; Castanheira et al., 2017). To develop geneti­ Gene expression, gamete quality, and a number of physio­
cally distinct strains of rainbow trout (O. mykiss) that respond logical processes and behaviours are all affected by toxico­
differently to a three-hour confinement stressor, aquaculture logical stresses (Jeffries et al., 2015; Sloman and McNeil,
has been using this variation for more than a decade. This 2012). Responses can vary even within a single class of
variation is now related to a variety of behavioural stress symptoms (for example, rises in lactate concentration sug­
markers that are regularly found in circulation. Tolerance to gesting insufficient oxygen delivery to muscles but no change
stress sampling methodologies may be affected by these in HSP expression indicating no protein degradation). As a
individual factors. Stress indicators can be influenced by a result, when determining which stress indicators are better to
variety of intraspecific variable components, and it is vital to monitor, it is necessary to take the stressor or stressors‘ type
account for and explain this variation. and the interplay of signs into account.

[Link] Context-Specific Differences [Link] Stressor Severity

There may be differences in HPI axis activity and supple­ Acute or single exposure is characterised by physiological
mentary molecular physiological and whole-animal responses adaptations and brief exposure durations. The consequences of
depending on the specific ecological and environmental con­ an acute stressor may be reflected in variations in the levels of
ditions to which an individual is subjected. The HPI axis stress indicators over time (Gesto et al., 2015, 2013).
should always be initiated in a consistent, reliable manner. All Depending on the anticipated level of impairment (e.g., a two-
fitness losses must be accepted and overcome through HPI- minute confinement versus a five-minute net pursuit), acute
mediated physiological and behavioural changes (Schreck and stressors may have varying intensities. It is typical to see
50 Immunomodulators in Aquaculture and Fish Health

relationships between the concentration of pollutant stressors be taken in the wild. It is easy to regulate variables that can skew
and the rate of indicator reaction or mortality (i.e., LC50). stress indicator measurements in controlled laboratory investi­
Similar reactions can be provoked by stressors that are thought gations, but this is not the case in the field.
to be different from one another in terms of severity, but which For studies examining the mechanistic links between
are actually the same save for their duration (Fatira et al., stressors and indicators, this degree of control is essential.
2014). Repetitive, consecutive exposure to an acute stressor Reproducing stresses that fish might experience in the field in a
(such as routine handling over several weeks) or protracted, laboratory environment is insufficient to capture the full
uninterrupted exposure to a stressor both qualify as examples response to stress in an animal under ecologically suitable
of what is known as “chronic stress”. On the reaction of an settings. Stress indicators, for instance, don’t reflect the long-
indicator to various exposures, short-term (e.g., avoidance term effects of stress (e.g., post-release predation after fisheries
behaviour) and long-term (e.g., growth) fitness repercussions capture, a stressor that upsets equilibrium) because they’re
may have different effects. Additionally, several animals are tested in a controlled environment (Danylchuk et al., 2007).
exposed to chronic stressors. Adaptive responses may occur in It’s possible that doing stress tests in the wild would be better
response to indicators of a chronically stressful condition (such to a controlled laboratory setting, or vice versa, depending on
as allostatic overload) (Boonstra, 2013). Thus, while de­ the study’s hypotheses and goals. With the advent of remote
termining whether “disrupted negative feedback” or allostatic sensing tools and the validation of point-of-care devices in
overload is to blame for stress marker modifications, habitua­ fishes, it is now possible to monitor markers that were previ­
tion must be taken into account (Schreck and Tort, 2016). ously only achievable in the lab (Stoot et al., 2014).
Hormonal stress indices, distinguish between habituation, Advances in biologging and biotelemetry now make it pos­
desensitisation, and fatigue. Behavioural/performance in­ sible to track fish before, during, and after they are exposed to
dicators may not be as useful in detecting habituation as stressors. Free-swimming fish experience shifts in their activity,
physiological markers (such as HPI axis activity). Repeated vigour, foraging, and heart rate (Clark et al., 2008; Donaldson
acute stressor exposure may be tracked halfway through by et al., 2010; Raby et al., 2015). There are a number of issues with
monitoring the HPI axis and stress indicator in certain animals mechanical stress detection in the field, including expensive
(ie, immediately following daily handling stressor delivered costs, surgical procedures for tag installation, inefficient detec­
over multiple weeks). It is conceivable to expose a subgroup of tion, and challenging tag retrieval. Studying wild fish that have
animals to an acute stressor in the midst of a longer stressor been subjected to unpredictable, variable pressures (such as
exposure to guarantee the HPI axis and the target stress indi­ cyclones or floods) can be instructive, but it is also quite risky
cator are responsive throughout the exposure (i.e., single, acute (Wingfield, 2013). Multidisciplinary approaches that combine
chase stressor during continuous exposure to increased water field research with laboratory investigations to isolate pathways
temperature over numerous weeks). Many chronically stressed provide the fullest picture of the stress response possible.
species have a lower endocrine response to stress (Barcellos
et al., 2006; Jentoft et al., 2005).
[Link] Temporal Aspects
According to Wingfield et al. (2011), stress becomes mal­
adaptive after a certain threshold. When thresholds are crossed, There are numerous levels of timing to consider when looking at
a stressor’s resistance potential rises due to attenuated stress animal stress. First, what age group is the stress indicator
response (Wingfield et al., 2011). It’s feasible to determine appropriate for? Several species have well-developed hypor­
thresholds at which stress indicators no longer respond or esponsive periods in the early stages of development.
revert to pre-stressor values by taking multiple samples during Hyperresponsive phases can be seen in smolting salmon as well.
chronic exposure (exhaustion). It’s vital to incorporate phe­ Stress-induced plasma cortisol peaks were shown to be different
notypic plasticity when analysing stress in wild populations in concentration and onset time in fingerling, juvenile, and adult
because it’s a chronic stressor in environments with changing jundia (Rhamdia quelen). Cortisol, a stress hormone, has been the
conditions (Crozier and Hutchings, 2014). Due to coping primary focus of research into the impacts of ageing up until
mechanisms, lab-based stress markers may not be reliable in recently (Schreck and Tort, 2016). Changing stress signals during
wild animals. In the lab, wild populations may show indica­ life may make it difficult to accurately measure stress. Some
tions of chronic stress, but these may not affect the population physiological performance and behavioural symptoms of stress
as a whole. The round goby population is steady in contami­ might change over time. The fact that some species may be able to
nated regions but dropping in reference areas (McCallum et al., change their circadian rhythms should be kept in mind, though.
2014). These new stable states in organisms can be viewed as There will be a variation in induction and recovery timeframes
stress while not influencing population dynamics. based on the diagnosis (Gesto et al., 2015). Cortisol takes longer
to climb above prestressed levels than catecholamines (and other
sympathetic nervous system activity like pulse rate and respi­
[Link] Field versus Laboratory
ration). An interplay between stress and variations in mRNA
Both in-lab and on-site stress testing have advantages and dis­ abundance is hypothesised (i.e., transcription). If the stressor is
advantages. Because of the complexity of the instruments used mild or severe, the induction and recovery periods may also
to gather samples, certain stress indicators can only be analysed be different. However, when treatment-level repercussions
in a lab (for example, serial blood sampling via cannulations). (for example, stressor kind or severity) are important (e.g., the
Since the behaviour cannot be reproduced in a controlled labo­ stress response’s onset time), temporal influences should also be
ratory setting, additional indicators will require measurements to taken into account.
Immunomodulators and Stress Oxidative 51

and folklore transmitted from one generation, and their phys­

4.3 Modulation of the Immune System by iological features are generally analysed in vitro or, at times, in
Immunostimulants animal experiments. An efficient and ecologically acceptable
farming depends on the advantages of immunostimulants. In
We now have a better understanding of fish’s innate immune order to maximise the advantages and minimise immuno­
system because of immunostimulant research, and we also suppression, studies to determine the optimum protocol are
have an alternative technique for preventing common illnesses needed to determine the dose/concentration, duration, and route
in fish farming (Galina et al., 2009). The global impact of of administration of these components. The process of receptor
disease resistance, toxicity, and environmental impact have all identification, extraction structure, and stimulation of fish
contributed to antibiotic resistance; however, dietary variables immune function are yet fragmented, despite certain research
(vitamin C and selenium) may enhance the effects of im­ showing a clear and dose/concentration dependent stimulatory
munostimulants, but no nutritional element can be termed an action of medicinal plants. As a result, reaching an agreement on
immunostimulant on its own. Specific or non-specific pathways this issue is a top priority. As a final step, more studies em­
may be activated by Immunostimulants, which modify the ploying high-throughput screening technologies are required to
immune system and improve the fish’s immunocompetence. better understand the genomic and proteomic responsiveness of
Several immunostimulants, including glucan, have been dem­ fish to immunostimulants in order to fully understand how the
onstrated to establish early immunity, reduce mortality, and dietary supplementation of immunostimulants affects fish.
increase life span after their administration (Rojo-Cebreros et al.,
2018; Petit and Wiegertjes, 2016). The term “trained immunity”
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 Section II

Immunomodulators and sustainable

aquaculture development
5
Immunomodulators to Prevent Diseases and
Minimize Antimicrobial Use

Akshay Thuruthiyil Rajesh1, Sajna Beegum1, Neha Omgy2, Sreeja Lakshmi2, Hethesh Chellapandian3,
Sivakamavalli Jeyachandran3, Einar Ringø4, and Preetham Elumalai1
1
Department of Marine Biology, Microbiology and
Biochemistry, School of Marine Sciences, Cochin
University of Science and Technology (CUSAT), Lakeside
Campus, Cochin, Kerala, India
2
Kerala University of Fisheries and Ocean Studies,
Panangad, Kochi, India
3
PG and Research Department of Biotechnology and
Microbiology, National College (Autonomous),
Tiruchirappalli, Tamil Nadu, India
4
Norwegian College of Fishery Science, Faculty of
Biosciences, Fisheries and Economics, UiT, The Arctic
University of Norway, Tromsø, Norway

biologically active molecules, there are to our knowledge less

5.1 Introduction studies available looking at how these resources can modify the
immune system, only approximately 190,000 of the estimated
Through the diagnosis and extermination of pathogens, ageing 2,210,000 species that are expected in the ocean have been
cells, and tumor cells, the immune response is essential in documented (Grienke et al., 2014). Immunostimulants can be
sustaining human health. Numerous factors, including the pres­ produced using proteins, peptides, and protein hydrolysates that
ence of pathogens, tissue damage, and myocardial infarction, are isolated from marine organisms (algae, fish, shellfish, etc.).
can impact functional efficiency (Villani et al., 2018). “Imm- Current research on the probable immunomodulatory conse­
unomodulation” deals with the immune system’s capacity to quence of marine resources is only emerging.
regulate a slew of potentially severe disease, including the human
immunodeficiency virus, cancer (Naidoo et al., 2014), aging
(Routy et al., 2016) and multiple sclerosis (Havla et al., 2016).
Macrophages are mononuclear leukocytes that serve as the host’s
5.2 Immune System – An Overview
main defense mechanism and have the ability to identify and The immune system, a cornerstone of the host’s defensive
phagocytose cancer cells and microbial infections. These cells are mechanism, eliminates pathogens, or potentially harmful mo­
also involved in the control of the immune system by releasing lecules. Innate and adaptive immune systems are the two
cytokines such as interferon (IFN), tumor necrosis factors (TNFs), subsystems that make up the immune system. Natural or native
and interleukin (IL-1β, IL-6, IL-10, and IL-12) (Figure 5.1). immunity, also referred to as innate(non-specific) immunity
Immunoadjuvants, immunosuppressants, and immunostimulants and acts as the body’s initial line defense employing the
are the clinical categories for immunomodulators. By modifying mucosa, bone marrow, skin, mast cells, neutrophils, mono­
the immune system of the host, immunotherapy treats diseases. cytes, basophils, dendritic cells, eosinophils, macrophages, as
Several drugs, including imiquimod, levamisole, pidotimod, well as other inflammatory components such as defensins,
cyclophosphamide, tilorone, prostaglandin, thiocarbamate, interferons, and cytokines. Important first-line phagocytosis
cyclosporine A, penicillamine, and niridazole, are now used to responders include neutrophils and macrophages.
suppress immunity of humans. Synthetic compounds that alter Specific or adaptive immunity (acquired immunity), uses T
immunity show adverse effects and toxicity, which restricts their cells and B lymphocytes and is extremely specific to deadly
application to conditions like those of chronic disorders (Wang pathogens. Either cells or humoral mechanisms can mediate the
et al., 2010). In contrast, most naturally occurring proteins or adaptive immune response. B lymphocytes discharge antibodies
peptides have less complications and are less expensive, indi­ that are particular for the pathogenic microorganisms, mediate
cating the possibility of using them in therapies of immune humoral immunity. The pathogen or infected cells are bound by
system. Although the compounds from marine source is full of T lymphocytes during cell-mediated reaction, which results in

DOI: 10.1201/9781003361183-7 59
60 Immunomodulators in Aquaculture and Fish Health

FIGURE 5.1 Immunomodulatory effects of marine-derived bioactive compounds (created by [Link]).

the lysis of these cells and the production of immune regulating body’s initial line of defense and reacts more quickly than the
molecules, cytokines. Cytotoxic T cells, Helper T (TH) cells, specialized (adaptive) system but cannot retain memory of past
cytotoxic T cells, and regulatory T (suppressor T)cells are the responses. If the innate immune defenses fail to eliminate the
distinct subgroups of T lymphocytes. Cluster of differentiation pathogen, adaptive immune system is recruited. Innate immune
(CD)8+, a surface receptor on cytotoxic T lymphocytes, rec­ system consists of all elements that were there in the body before
ognizes endogenous antigens linked to major histocompatibility the pathogenic agent appeared. Physical barriers include scales,
complex class I and eliminates the infected cells. The major mucous surfaces of the skin, gills, and epidermis. Additionally,
histocompatibility complex class II-complexed foreign antigens the complement system, antimicrobial enzymes, interleukins,
are recognized by TH cells that have CD4+ as their surface interferon, and organic defense cells such granulocytes, mono­
receptors. Cytokines secreted by TH cells aid in the activation of cytes, macrophages, and natural killer cells are present. The
B and T lymphocytes and other immune cells, by the prevention complicated interplay between cellular and humoral components
of harmful immune activation the immune response is controlled that cause inflammation are also considered to be an innate
with the help of regulatory T cells and also helps to maintain the immunological reaction. When tissue becomes infected, media­
self-tolerance to prevent autoimmune diseases. tion factors are released to stretch and make blood capillaries
more permeable, allowing defense cells to travel across them. The
first cell type to arrive at the site of inflammation are granulocytes,
5.2.1 Fish Immune System
which are responsible for eliminating pathogens. Macrophages,
The changes in the cultural methods in fish farming accelerated on the other hand, phagocytose the remaining potentially dan­
the outburst of many infections. A more practical way to decrease gerous cells and cellular waste (Magnadottir et al., 2006).
or keep away from chemicals and antibiotics is to increase the use The nonspecific immune system, the simplest mechanism,
of compounds like β-glucans, chitin, prolactin, etc. which boosts most likely developed in single-celled creatures early in evolu­
immune system, but this urges the need to understand the tion. The innate immune system is capable of detecting areas in
immune system’s composition and operation in fish. molecules known as PAMPS (Pathogen Associated Molecular
The immune system is an intricate web of organs, tissues, and Patterns) from pathogens or members of the normal microbiota,
cells. The compounds they create aid the body in its defense such as lipopolysaccharide (LPS), peptidoglycan, bacterial
against poisons, cancerous cells, and diseases. Both endogenous DNA, viral RNA, or other molecules known as “non-self”
and exogenous factors stimulate immune system. Innate (non- compounds, which are not typically reported on the surface of
specific) and acquired (specific) immune systems are the two multicellular organisms. The PAMPS are well-preserved sec­
types of immune system. These two systems jointly work to tions of a species evolutionary history and exists in most path­
eliminate intruders, thus activating defense mechanism. Fish ogens. The specific system originally arose 450 million years
have both innate and adaptive (memory) immune systems, which ago and are located in all animals, with the exception of
are further separated into humoral and cell-mediated defense Agnatha-class fish. The specific system receptors, also known as
mechanisms (soluble substances). The innate system works as the membrane immunoglobulin or free antibodies, are found on the
Immunomodulators to Prevent Diseases and Minimize Antimicrobial Use 61

cell membranes of T lymphocytes (TCR- T cell receptor) and B thus promoting immune memory. The liver produces humoral
lymphocytes (BCR- B cell receptor), which are responsible for substances such as complement system proteins and acute
detecting harmful substances. The presence of an antigen is phase proteins of inflammatory reaction.
necessary for the specific system of defense. A molecule or cell
known as an antigen can trigger reactions, boost the circulation
5.2.3 Humoral- and Cellular-Mediated Immunity
of particular antibodies, and promote immunological memory.
Antigen-presenting cells (APCs) such as macrophages, dendritic Fish are susceptible to infections with agents like virus, fungus,
cells, and B lymphocytes identify and process foreign antigens bacteria, and parasites. Because of specialized and intrinsic
to break down microorganisms into their component molecules components with coordinated humoral and cellular processes,
and initially elicit an immune response of proliferation and they can, to some extent, defend against pathogens. The
immunological response of memory. humoral-specific system only uses antibodies, whereas the
T lymphocytes (the cells of the specific system) will be pre­ humoral innate mechanism functions through a variety of
sented with the APC-processed antigen. T cells can only detect the soluble components in bodily fluids. The elements of the
antigen,when a certain humoral component known as the major humoral innate system include antiproteases, lysozyme, trans­
histocompatibility complex (MHC) is present. The major histo­ ferrin, C-reactive protein, and complement system. Phagocytes,
compatibility complex (MHC) is a group of genes that codes for neutrophils, and macrophages are the cells of humoral innate
glycoprotein receptors. Once the invader has been identified, the T immunity (Takahashi et., 2014).
cell releases cytokines, which are proteins that encourage the Antiproteases cause the proteolysis of blood proteins. Amino
activation of other cells such as B lymphocytes (cells that produce acids obtained from proteolysis form the source of energy.
antibodies), cytotoxic lymphocytes, macrophages, and other cells. Monocytes and neutrophils are the principal cells that secrete
Phagocytosis (component of innate immunity) is activated as a lysozymes, which are prevalent in mucus, eggs, blood, and tissues,
result of the antibody’s recognition and connection to specific Lysozymes cause lysis of peptidoglycan on pathogens cell wall.
microorganisms. This clearly demonstrates the interaction bet- The pentraxin family member C-reactive protein has been reported
ween the nonspecific and specific immunity. Antibodies can aid in in blood, eggs, and mucus. It can identify and attach to the phos­
agent neutralization or opsonization in addition to binding the phoryl colin on the pathogen’s cell wall. Along with mannose-
complement system and potentially binding to foreign antigens. binding lectin they assist in opsonization, complement activation,
When the antigen is within the cell, cytotoxic T cells are in charge and phagocytosis. The inflammation-acute proteins known as C-
of providing the defense. reactive proteins and mannose-binding lectin are also known as
receptors of soluble microbial components. Acute phase proteins
5.2.2 Organs of Immune System are produced mostly by the liver, brain, and leukocytes and are
concentrated more when there are pathogens present and high
Lymphoid immune system is seen in bony fish. Since they lake temperatures. For illness diagnosis, the most often utilized tests
bone marrow and lymph nodes, myeloid classification is absent include the C-reactive protein, serum amyloid A, transferrin, C3
in bony fish. The lymphoid organs include the kidneys (the complement, lysozyme, and lectins. The primary mechanism in
biggest lymphoid organ), the thymus, the spleen, and the gut- fish is the complement system, which secretes inflammatory
associated lymphoid tissues (GALT). It is crucial for the for­ mediators, has lytic action, and encourages chemotaxis, pro-
mation of immune system components such as lymphocytes, inflammation, and opsonization. Sequential pathway activates
monocytes, granulocytes, thrombocytes, mast cells, NK cells, the complement system, which is made up of membrane and
cytotoxic cells, and dendritic cells that lymphoid tissues and soluble proteins that are inactive or present in low concentra­
organs be arranged in a reticular cell network. tions. The three mechanisms that activate the complement
In thymus, development and maturation of T lymphocytes system are the lectin pathway, which is activated by pathogen’s
take place. The kidney is crucial for fish immunity and surface carbohydrates, the alternative pathway, which is acti­
hemopoiesis. Red and white blood cells are produced and vated by PAMPS from bacteria, and the classical pathway,
matured in the kidney. For the production of defense cells, the which is activated by an antibody-dependent activation. The
anterior kidney region is particularly crucial. Additionally, the alternative pathway, which is thought to be crucial, is quickly
anterior portion of the kidney is where leukocytes including activated by gram-negative bacteria and LPS and results in cell
granulocytes, macrophages, monocytes, and lymphocytes dif­ lysis. The classical route mediates the interaction of the innate
ferentiate and mature. The fish spleen is composed of red and immune system with the specialized immune system. Two
white pulp and is not as highly organized as the spleen in complement systems C3b and C5a are essential in inflammation
mammals. Red pulp aids in the phagocytosis of damaged or and phagocytosis. For neutrophils and macrophages, C5a and
aged cells, while white pulp promotes hematopoiesis and the C3b are chemotactic proteins that initiate opsonization, phago­
development of defense cells. Antigens are held on macro­ cytosis, and leukocyte chemotaxis. The complement system is
phages grouped in centers when blood passes through the frequently utilized to determine immunological state.
spleen so they can be processed and given to T lymphocytes. Lymphoid tissues such as the kidney, thymus, spleen, and
Gut-associated lymphoid tissues (GALTs), comprised of GALT produce defense cells. Hematopoiesis, or the generation
gills, skin, and mucosa, secrete mucus that act as first barrier and differentiation of erythrocytes, granulocytes, monocytes,
against pathogens. Mucus consists of defense components such lymphocytes, mast cells, and thrombocytes, is the process of
as mast cells, granulocytes, macrophages, and lymphocytes. producing lymphoid cells. Cytokines that operate as pluripotent
Antigens which are to be processed are captured by these cells, cell receptors affect the survival, proliferation, differentiation,
62 Immunomodulators in Aquaculture and Fish Health

FIGURE 5.2 Immune system of fish.

maturation, and function of hematopoiesis. The evaluation of the last few decades and extensively studied. Amino acids,
hematological parameters may serve as a marker for fish disease peptides, proteins, glycoproteins, lipids, and polysaccharides
outbreaks and physical health. produced from marine sources possess the potential to modu­
The cells of the adaptive immune system are T and B lym­ late the immunity (Kiewiet et al., 2018). This chapter gives an
phocytes, that differ from natural killer cells or T cytotoxic cells overview of immunomodulatory compounds from marine
and destroys injured somatic cells. Dendritic cells and macro­ sources and from different organisms as an alternative for
phages correlate the function of the innate immune system with antibiotics (Table 5.1).
that of the acquired immune system (antigen-presenting cells).
Major histocompatibility complex (MHC) class 2 receptors 5.3.1 Immunomodulatory Peptides, Amino Acids,
assist antigen-presenting cells digest the pathogen and deliver and Proteins
the processed molecule to T lymphocytes, generating cell-
mediated acquired immune reaction. MHC molecules are gly­ The described marine biomaterials such as proteins, biopolymers,
coprotein receptors that lack specificity and can recognize both fatty acids, enzymes, pigments, and minerals are obtained from
internal and external antigens. They are encoded by a gene natural marine sources. Large numbers of different proteins with
complex. As opposed to this, antibodies (Immunoglobulins Ig) a range of bioactivities and functions are present in marine bio­
are glycoproteins that are expressed on the B lymphocyte materials (10–47% (w/w)). Many proteins in hemolymphs and
membrane (BCR) or are free in body fluids and released by hemocytes serve important roles in the innate immune system by
plasma cells. Immunoglobulins IgM, IgD, IgZ, and IgT are protecting diverse immunological components like glycopro­
detected in fish. The well-known fish protein immunoglobulin teins, antimicrobial peptides, metalloproteins, amino sulfonic
IgM is a tetrameric protein with four sites for antigen acid, coagulation factors, and protease inhibitors.
recognition. Immunoglobulins are reported in bodily fluids, Lectin, a glycoprotein that regulates immune activity, has
mucous, eggs, and gastrointestinal mucosa. Among the roles that been implicated in innate immune responses that result in
antibodies can play are the anti-adhesin function, anti-toxins pathogen identification and endocytosis as well as adaptive
functions, and anti-invasive functions. Anti-adhesin function immunological responses that include B- and T-cell activation
happens in digestive systems epithelial surface, gills, and skin and apoptosis (Sharon, 2007). Many different plants and ani­
and prevents bacterial adherence. Anti-toxins antibodies make mals have been found to contain lectins. Lectins from clam are
bacterial toxins inert, and anti-invasions antibodies prevent similar, and influence the innate immune response(Medzhitov
bacterial invasion of unprotected cells. et al., 2000). C-type that are Ca2+-dependent, R-type, and
Immunoglobulin receptors on B lymphocytes that recognize metal-independent (Galectins) have all been classified as lec­
an antigen promote the cellular uptake and multiplication of B tins. C-type lectins serve a critical function in carbohydrate
memory cells, which in turn activates T lymphocytes. This recognition during the immune response by recognizing a wide
triggers the production of cytokines and activates macrophages range of endogenous ligands and pathogens that control
and B cells. Species, age, sexual maturity, physiological intercellular connections (Brown et al., 2018). These proteins
events, artificial immunization, and persistent infection can all have a significant role in autoimmune disorders as well.
affect the serum antibody concentration (Figure 5.2). Tachylectin is a pathogen-recognition protein that was identi­
fied from the hemocytes of the mangrove horseshoe crab
(Carcinoscorpiusrotundicauda). Phagocytosis or the lectin
5.3 Immunomodulatory Compounds from pathway in the complement system is used to recognize microbes
(Ma et al., 2004). Phagocytosis or the lectin pathway in the
Marine Organisms
complement system are used to recognize microbes. In human
Immunomodulatory compounds from more than 16,000 marine peripheral blood cells, the lectin from the mussel Mytilus tros­
species have been isolated from the seas and oceans during sulus increases the production of proinflammatory cytokines
TABLE 5.1
Immunomodulatory Compounds and its Mechanism of Action
Organism Obtained from Bioactive Compound Concentration Mechanism/Activity References
Filamentous marine fungi PolatuzumabVedotin In vitro administration with Induction of human mesenchymal stem cell (hMSC) Sehn et al., 2020
125 µg/mL differentiation
Marine fungus (Phomaherbarum D-glucan In vivointraperitoneal injection Regulation of T lymphocytes and dendritic cells Newman et al., 2019
YS4108) (40 mg/kg)
Microalgae β-carotene In silico study Anti-inflammatory mechanism Costa et al., 2021
Phycocyanobilin
9-cis- β-carotene
Microalga β-glucans β-glucans 0.6 g/kg of feed Promotes the growth of commensal microbiota Speranza et al., 2012
(Phaeodactylumtricornutum) Antioxidant, anti-inflammatory and
immunomodulatory reactions in fishes
Microalga (Porphyridiumcruentum) Exopolysaccharides (EPS) Treated with increasing conc. Antibacterial and immunostimulatory effects with Wasana et al., 2021
of EPS elevation in total hemocyte value, phagocytotic
activity
Alga (Ulva ohnoi) Ulvan In vitro: 100 µg/mL Immunomodulatory activity Hafting et al., 2015
Alga (Gracilarialemaneiformis) Sulfated oligosaccharides – Immunomodulatory activity Cumashi et al., 2007
(GLSO)
Alga (Caulerpa cupressoides) Sulfated pyruvates (SP1 and In vitro: 100 µg/mL Immunostimulatory activity Lauritano et al., 2018
SP2) and galactans, sulfated
polysaccharides
Algae (Ecklonia cava, Fucus Fucoidan In vitro: 100 µg/mL Pro-proliferative activity Yang et al., 2008
Immunomodulators to Prevent Diseases and Minimize Antimicrobial Use

vesiculosus, Macrocystis pyrifera Immunostimulatory activity

and Underia pinnatifida)
Coral (Pseudopterogorgia PPA polysaccharide In vitro: 10 µg/mL Immunomodulatory activity Xiang et al., 2022
Americana)
Mollusk (Megathuracrenulata, Haemocyanin Immunostimulatory activity against several cancer Del Campo et al., 2011
Concholepasconcholepas, types with no side effects
Fissurellalatimarginata)
Mollusk (Scapharcabroughtonii) Protein HPCG2 In vitro: 250 µg/mL and Immunomodulatory activity and promotes Paolino & Penninger 2010
500 µg/mL phosphorylation
Mollusk (Mytilus edulis and Mytilin A, B, C, D, G1, – Antimicrobial activity and prevents the breach of Mitta et al., 2000
M. galloprovincialis) Defensin, Myticin, Mytimycin microbes to circulatory system
Mollusk (Mytilus coruscus) Polysaccharides In vitro: 300 µg/mL and Immunomodulatory activity Gunathilake et al., 2020
600 µg/mL Anti-inflammatory, promotes abundance of probiotics
in the colon
Mollusk (Arca subcrenata Lischke) ASPG-2 polysaccharide In vitro: 250 µg/mL and Immunomodulatory activity De Almeida Leone et al., 2000
500 µg/mL for 24 h
Sponge (Astrosclerawilleyana) Ageliferins extracts 10-50 µM Immunomodulatory activity by inhibiting ubiquitin Altmann et al., 2001
ligase activity
(Continued)
63
64 Immunomodulators in Aquaculture and Fish Health

including TNF-α and IFN-γ but inhibits the anaphylaxis of the

anti-inflammatory cytokine IL-10 (Chikalovets et al., 2013).
Lectins, which are generated from the hemolymph of crustaceans,
References

are regarded as active antibodies‘ progenitors since they are

Acosta et al., 2019

Acosta et al., 2019

Talley et al., 1967

Ding et al., 2012

crucial for both immune recognition and antimicrobial activity.

Lin et al., 2016

In mollusks, hemocyanins, the metalloproteins are present that
use copper-binding sites to bind and transport oxygen. Similar to
hemoglobin, hemocyanins are multi-component molecules with
an oxygen-binding functional subunit. In a rat colon carcinoma
model, hemocyanins derived from a variety of marine gastropods
exhibit strong immunostimulatory activity against some malig­
Immunomodulatory activity and increases neutrophil:

Antimicrobial, wound-healing, and antitumor activity

byAeromonashydrophilaby elevating Antimicrobial

nancies with manageable side effects (Del Campo et al., 2011).

Extension of the survival rate of zebrafish infected

Immunomodulatory activity, antibacterial activity

Hemocyanins have been tested in clinical trials for treating
superficial bladder cancer, whereas hemocyanins derived from
activity and immunomodulatory activity

Concholepasconcholepas have been employed as innate im­

Mechanism/Activity

munostimulators to decrease the development of superficial

bladder cancer (Lammers et al., 2012). An immunostimulator
called keyhole limpet hemocyanin is made from the sea mol­
lusk’s circulating glycoproteins (Megathuracrenulata). Keyhole
limpet hemocyanin enhances the host immune response by
Antimicrobial activity

interacting with T cell monocytes, macrophages, and poly­

lymphocyte ratio

morphonuclear lymphocytes (Mora Roman et al., 2019).

Hemocyaninfrom Fissurellalatimarginata (gastropod), is a
novel immunostimulator with stronger immunostimulatory
activity than other isolated gastropod hemocyanins, like keyhole
limpet hemocyanin and Concholepasconcholepas.
An amino acid called taurine, also known as 2-amino ethane
sulfonic acid, is found in large quantities in animal tissues,
In vivo: injection of 1.0 μg/g.

including those of saltwater clams. Taurine is produced

In vivo: 5 mg/kg; 10 mg/kg;

In vivo: 5 mg/kg; 10 mg/kg;

by numerous different creatures in addition to being a rich source

In vitro: 250 µg/mL and
Concentration

in marine organisms. Lymphocytes, monocytes, and neutrophils

In vitro: 1.0 μg/mL

are among the immune cells that are enriched in taurine, which
has cytoprotective and immunomodulatory properties (Marcin-
500 µg/mL

kiewicz & Kontny et al., 2014). This implies that phagocytes that
15 mg/kg

15 mg/kg

are mediating inflammatory lesions have high taurine levels,

which may be related to innate immunity. Taurine builds up in
–

phagocytes; upon coming into touch with a pathogen, activated

cells (neutral and macrophages) release harmful oxidants and a
variety of antibacterial chemicals via the peroxidase system, and
Salmo salar natural killer(NK)-
Immunomodulatory Compounds and its Mechanism of Action

Phosvitin-derivedpeptide Pt5

act at inflammatory areas to kill germs. By eradicating pathogens,

Tilapia piscidin 3 (TP3)and
Bioactive Compound

toxic oxidants—part of the innate immune system—protect the

tilapia piscidin4 (TP4)

host from infections. However, they may also cause tissue damage
to the host. Additionally, taurine functions as a scavenger to flush
out undesired or dangerous compounds from cells and safeguard
BpNK-Lysine
Crude extract

them from oxidative [Link] activating NF-κB, a strong signal

transducer for inflammatory cytokines, taurine affects the immune
lysin

system. Additionally, taurine in the liver activates the peroxisome

proliferator-activated receptor-γ (PPAR-γ) to regulate adiposity
and glucose metabolism. Therefore, taurine’s stimulation of
PPAR-γ prevents diabetic retinopathy’s retinal neuronal damage
(Song et al., 2011).
Fish (Oreochromis niloticus)
TABLE 5.1 (Continued)

Organism Obtained from

Sponge (Haliclona sp.)

5.3.2 Antimicrobial and Immunomodulatory

Fish (Beleophthalmus
Fish (Salmo salar)
Fish (Danio rerio)

Compounds
pectinirostris)

Active research is being done on the peptides that regulate

immune responses. Although it is known that immunomodulatory
peptides boost immunological responses like lymphocyte prolif­
eration, NK cell activity, and cytokine modulation, the precise
Immunomodulators to Prevent Diseases and Minimize Antimicrobial Use 65

mechanisms behind these actions are yet unknown. For instance, AMP
little is known about how immunomodulatory peptides affect the
human neurological, digestive, cardiovascular, and immunolog­
ical systems. Therefore, in order to create unique im­ Direct action Indirect
munomodulatory peptides, it is essential to comprehend how action
peptides interact with these systems. Antimicrobial peptides B-lymphocytes
(AMPs) from marine sources are defensive chemicals that may Bacteria
Dendritic Cells
improve innate immunity. Marine AMPs’ promise as therapeutic Virus
candidates was reinforced by the finding that they target particular Neutrophils
cells. Therefore, AMPs with immune regulating qualities origi­ Fungi
T-lymphocytes
nating from marine sources are crucial.
Specificity of marine-derived AMPs to target cells makes them Monocytes
a potential drug candidate. The rationale for the targeting speci­ Mast cells
ficity of AMP is the variation in cellular membrane makeup
among pathogens and host cells (Zhang et al., 2021). By selec­ FIGURE 5.4 Immunomodulatory activity of AMP on different cell types.
tively reacting with the negative charges on the membrane
through electrostatic interaction, cationic AMPs exert antibac­ in the microenvironment, completing the inflammatory process
terial activity. This increases porosity, causes cell membrane (Figure 5.4).
rupture and substance leakage and ultimately results in cell death. Cathelicidins, the AMPs in neutrophils and mucosal epithelia,
In order to interact with molecules inside cells, AMPs can exhibit a broad spectrum of antimicrobial activity. Defensins are
potentially penetrate the membrane and enter the cytoplasm. The AMPs that function as peptides for host defense. Insects, plants,
mechanism of action of anionic AMPs is not clearly known. In and animals all have defensin. Humans have two different forms
addition to penetration and pore formation AMPs have other of defensins, known as α defensins and β defensins, depending
mechanisms to kill bacteria. AMPs show their action on nuclei upon their sequence homology and cysteine residues. Defensins
acids, protein synthesis, on enzyme activity, on cell wall syn­ destroy microbial membrane and activate immunological
thesis. Bacterial resistance to AMPs is prevented by the quick response. Defensins adjuvant activity facilitated the concept of
death and intracellular effects, which may not focus on any par­ “defensin vaccine” as a novel vaccine development.
ticular molecules or pathways. Hence, AMPs are good candidates The AMP, phosvitin-derived peptide Pt5 from zebrafish has
in the management of antibacterial resistance (Figure 5.3). equivalent in vitro antibacterial and immunomodulatory action
In spite of antibacterial activity AMPs also show antiviral, to phosvitin. Pt5 significantly improved the survival rate of in­
antiparasitic, tumor-modulatory activity, and immunomodulatory fected zebrafish by reducing the amount of Aeromonas hydro­
activity. AMPs, also known as host defence peptides, work by phila in the spleen, blood, liver, kidney, and muscles. Pt5 also
modulating the immune system and preventing pathogen growth. decreases the expression of proinflammatory cytokine genes
AMPs activate innate immunity by acting directly on dendritic (IL-1, IL-6, TNF-, and IFN-), while boosting the expression of
cells, neutrophils, T-lymphocytes, monocytes, and mast cells. B- anti-inflammatory cytokine genes (IL-10 and IL-14) in the head
lymphocytes are indirectly activated by AMPs, thus switching on kidney and spleen of zebrafish infected with A. hydrophila (Ding
adaptive immunity. AMPs immunomodulatory activity incorpo­ et al., 2012). Because they boosted the expression of pro-
rates effects on the recruitment and activation of other cells inflammatory cytokines (IL-1 and IL-8) in the head renal leu­
kocytes, NK-lysin fish from Atlantic salmon (Salmo salar)
strengthened immune responses (Acosta et al., 2019).
Cell AMP research is of at most significance in studying and
membrane analyzing the evolution of immunity of different organisms.
lysis Numerous peptides with antimicrobial function can be used to
develop as potent drug in the current situation of developing
Cell wall Penetrationa antibiotic resistance in microorganisms.
synthesis nd pore
formation
5.3.3 Immunomodulatory Protein Hydrolysates
AMP
Protein hydrolysates made from various proteins have been
shown to exhibit a wide range of biological activities, notably
Interacts anti-inflammatory, anticancer, antioxidant, antibacterial, anti-
Enzyme
with Nucleic
activity hypertensive, and immunomodulatory actions (Kang et al.,
acid
2019). Bioactive peptides of various lengths are produced
because of peptide bond cleavage during protein hydrolysis.
Protein Numerous immunomodulatory protein hydrolysates were
Synthesis produced using a number of proteolytic enzymes. Pancreatin,
KojizymeTM, trypsin, Alcalase®, Flavourzyme®, ProtamexTM,
α-chymotrypsin, pepsin, Neutrase®, and thermolysin are some
FIGURE 5.3 Mechanism of antibacterial activity of AMP on different
molecules.
of the enzymes in this group (Ahmad et al., 2019).
66 Immunomodulators in Aquaculture and Fish Health

TABLE 5.2
Immunomodulatory Marine Protein Hydrolysates
Hydrolysate Source Mechanism of Action Ref.
Cyclina sinensis protein Venusclam: Cyclina sinensis Boosting NO, TNF-α, IL-6, and IL-1β, and increased protein Li et al., 2019
hydrolysate levels of iNOS, NF-κB, and NLRP3 in RAW 264.7 cells;
activation of NF-κB signalling pathway.
Shellfish Mytilus Shellfish: Mytilus coruscus Inhibited lipopolysaccharide (LPS) - NO production is Kim et al., 2013
Protein hydrolysate induced in macrophages of RAW264.7 cells
Alaska Pollock protein Alaska pollock: Theragra Increased the action of specific, and non-specific immune Hou et al., 2016
hydrolysate chalcogramma system in mice with immunosuppression
Salmon fish protein Fish: Atlantic salmon fish IgM, IgG, and IgA and CD4/CD8 ratios changes were Nesse et al., 2011
hydrolysate observed in Indian children with malnutrition
Tuna fish protein Fish: Tuna:Thunnasalbacares Elevation in the levels of NO, TNF-α, IL-6, and IL-1β, and Cai B et al., 2022
hydrolysate overexpression protein levels of TLR2 and TLR4, in
RAW264.7 cells; activation of the NF-κBsignalling pathway

Antimicrobial peptides play a crucial role in the initial line

of the human defense system against harmful bacteria which
are ingested by the host through the environment. The immune Fucoidan
system is stimulated by protein hydrolysates on both the
humoral and cellular levels. Oyster and salmon protein
hydrolysates increase NK cell activity in the spleen. Protein Agar Carrageenan
hydrolysates from algae modulate cytokine action by inhibiting
TNF-α and IFN-γ (pro-inflammatory cytokines), boosting IL- Sea weed
10 in rat splenocytes. Table 5.2 summarizes the mechanism of polysacch
action of marine protein hydrolysates. arides

Alginate Ulvan

5.4 Polysaccharides
Laminaran
A large part of algal biomass is made up of marine poly­
saccharides. It primarily functions as a structural storage com­
pound. Algal polysaccharides as immunomodulators have
gained more attention because of their nontoxicity and varia­
FIGURE 5.5 Seaweed polysaccharides.
bility in structure. Sulfated polysaccharides are produced by the
association of sulfur and carbon by photosynthetic organisms.
There are three major forms of sulfated polysaccharides, fu­ Nitric oxide (NO), tumour necrosis factor-α (TNF-α), and
coidans, carrageenans, and ulvan based on their sources. interleukin-1 (IL-1) production, as well as macrophage cell pro­
Rhodophyceae, Phaeophyceae, and Chlorophyceae are three liferation and phagocytic activity, are all stimulated by
major classes of seaweeds containing photosynthetic pigments: U. intestinalis. U. ohnoi exhibited immune system functional
red, brown, and green, respectively (Figure 5.5). changeson Senegalese sole (Solea senegalensis) (Sasikala &
Some echinoderms(sea urchin and sea cucumber) and marine Geetha Ramani et al., 2017). Ulvan enhanced the inflammation
brown algae (Phaeophyceae) are used to make fucoidan. brought on by LPS and raised IL-1, IL-6, and IL-10 levels.
Fucoidan is a complex sulfated polysaccharide, containing Carrageenan, a polymer of repeating galactose units and
fucose residues and other monosaccharides. It is a polymer of 3,6-anhydrogalactose is used as an additive to emulsify
α-l-fucose units possesses strong antibacterial activity and is an and preserve foods. According to Zhu et al. (2017), oxidized-
immunostimulant. Fucoidan shows bacteriostatic effects in carrageenan inhibits the growth of both Gram-positive and
Escherichiacoli, S. epidermidis, S. aureus, and B. licheniformis. Gram-negative bacteria and kills bacteria including S. aureus,
E. coli is the most delicate (Ayrapetyan et al., 2021). Interleukin- L. monocytogenes, E. coli, and P. aeruginosa by destroying
2 (IL-2) and tumour necrosis factor-α (TNF-α) were secreted their cell walls and cytoplasmic membranes. An amino-
more readily as a result of the immunostimulatory activity of polysaccharide called chitin is derived from the exoskeleton of
two polysaccharides, SCP1 and SCP2, which were isolated from marine creatures, primarily crabs and shrimp. The naturally
Sargassum carpophyllum. Ulvan from sea lettuce, or Ulvasp, is occurring chitin is deacetylated to produce chitosan. Chitosan
the polymer of glucuronic acid and sulfated rhamnose or iduronic directly causes the inflammasome to activate and the release of
acid and sulfated rhamnosehas antibacterial, antiviral, and the pro-inflammatory molecules IL-1 and PGE2 by activating
immunomodulatory activity. Ulvan has shown inhibition NLRP3. COS causes cytokines and NO to be released from
against Staphylococcus sp., Streptococcus sp., and Bordetella sp. dormant macrophages. A significant amount of NO produced
Immunomodulators to Prevent Diseases and Minimize Antimicrobial Use 67

TABLE 5.3
Polysaccharides from Marine Sources with Immunostimulant Activity
Name of the Compound Name of the Source Mode of Action References
Fucoidans (Sulphated Phaeophyceae Improved CD3+ T cell expression, increased GM-CSF, Ayrapetyan et al., 2021
polysaccharides) TNF-α, interleukin (IL)-4, and IL-10 secretion Peng et al., 2019
Ulvan (Sulphated Chlorophceae Pro-inflammatory cytokines, including nitric oxide (NO), Sasikala, & Geetha Ramani
plysaccahrides) tumor necrosis factor-α (TNF-α), and interleukin-1β et al., 2017
(IL-1β) are produced
Carrageenan (Sulphated Rhodoph-yceae Damages bacterial cell wall and cytoplasmic membranes. Zhu et al., 2017
polysaccharides) Inhibits bacterial growth.
IFN-γ, IL-12, and IL-1β are secreted
Chitosan Exo-skeleton of Crabs Stimulatesdorment macrophages to secrete NO and Laurienzo, 2010;
and shrimps cytokines. Wu et al., 2019
Secretes IL-1β and PGE2
Laminaran Pheoph-yceae Influences the up-regulation of immune-related genes and Selvaraj et al., 2005
proteins.

has the ability to kill tumor cells, parasites, and microbes due When Channa striata was exposed to A. hydrophila, the
to its cytotoxic properties. The immune-regulatory mecha­ N-Oxide-Quaternary Alkaloid Fraction (NOQAF) of the
nisms of COS entail the control of a number of vital pathways, chlorophycean macroalga Caulerpa scalpelliformis signifi­
such as the activation of AMP-activated protein kinase cantly improved the non-specific immune responses by
(AMPK) and the inhibition of nuclear factor kappa B (NF-κB) increasing serum lysozyme, peroxidase, antiprotease, and
and mitogen-activated protein kinases (MAPK). alternate complement activities as well as the production of
Laminaran is a 1,3-glucan found in the cell walls of brown reactive oxygen and nitrogen species and peroxidase activity
seaweed (Phaeophyceae). The seaweed species Durvillaeaanta- by peripheral blood leucocyte. IL-2 production by Jurkat T
rctica has a high concentration of -1,3/1,6-glucan, which acti­ cells is inhibited by the bis-heterocyclic quinolone-imidazole
vates both specific and non-specific immunity and affects the alkaloids (+) and (–) spiroreticulatine from the South China sea
production of immune-related genes and proteins. Glucan sponge Fascaplysinopsis reticulata (Wang et al., 2015).
administration increased survival in carp, perhaps by promoting
both generalized and specialized immune responses. By inter­
acting with particular cell receptors, B-glucans taken from the 5.6 Polyphenols
microalga Phaeodactylumtricornutum can function as pre­
biotics, fostering the development of the commensal microbiota Potent compounds primarily belonging to polyphenols and
and directly triggering the innate immune system (Table 5.3). sulfated polysaccharides are found in marine plants.
Sulfated polysaccharides primarily regulate macrophage Polyphenols have antibacterial and free-radical scavenging
activity, the activity of natural killer cells, T and B lymphocytes, properties. Green seaweeds’ bromophenols and flavonoids have
and complement system activation in order to have immuno­ antioxidant properties. It has been demonstrated through testing
logical activity (Cunha et al., 2016). The RAW264.7 macro­ that some species, including Ulva clathrata, Ulva compressa,
phage cell line’s pinocytic activity has been reported to be Ulva intestinalis, and Chaetomorphamoniligera, exhibit high
promoted by the sulfated polysaccharide UPP-2, which was radical scavenging properties. 5′-hydroxyisoavrainvilleol, a
obtained from the brown alga Undaria pinnatifida. Nitric oxide, bromophenol extracted from the Avrainvillea nigricans is toxic
tumor necrosis factor-α (TNF-α), and interleukin-6(IL-6) to KB cells and has shown some encouraging anti-microbial
secretions have also been shown to be increased. Crude poly­ action. Red seaweeds are the only source of the mycosporine-
saccharides from Gracilaria dura and Chloroidiumellipsoideum like amino acid class of phenolic substances, which possesses
showed similar impacts by stimulating macrophage and immunomodulatory effects.
RAW264.7 cells and generating significant amounts of different The most typical multidrug-resistant cause of nosocomial
cytokines and nitric oxide. Nuclear factor-kappa B (NF-κB) and infection in Europe is methicillin-resistant S. aureus (MRSA).
mitogen-activated protein kinase (MAPK) pathways are acti­ Such hazardous conditions as sepsis, pneumonia, endocarditis,
vated as part of the procedure (Kim et al., 2017). erysipelas, osteomyelitis, and foodborne illnesses are caused
by this bacterium. Penicillin-binding protein 2a (PBP2a),
whose existence determines a decreased affinity for binding
β-lactam antibiotics, is encoded by the mecA gene, which
5.5 Alkaloids causes MRSA to be resistant to β-lactam antibiotics. β-lactam
Alkaloids are made up of heterocyclic nitrogen substances. antibiotic resistance is provided by this protein. According to
Alkaloids in marine algae are rather uncommon when com­ reports, the algae E. bicyclis phlorotannin has potent anti-
pared to those in terrestrial plants. Halogenated indole alka­ MRSA properties. The affected MEC operon mediates this
loids obtained from red algae has antibacterial properties. characteristic of substances.
68 Immunomodulators in Aquaculture and Fish Health

5.6.1 Sterols/Steroids were significantly altered by YCP in the gut microbiota

(Kamada et al., 2013). In order to promote human mesen­
Steroids come from a variety of sources, including those that are
chymal stem cell (hMSC) development, Marchese et al. (2021)
synthetic, marine, and terrestrial and are lipophilic molecules
used a high-throughput drug-screening technique to bioprospect
generated from cholesterol. Steroidal substances extracted from
a sizable library of Irish deep-sea creature extracts. Additionally,
sponges modify NF-κBactivity, that affect the pregnane X
filamentous fungi were present in the library, and they had a 6.8%
receptors (PXRs). Solomonsterol A, a selective PXR agonist
success rate in lowering the inflammation caused by activated
from the marine sponge Theonellaswinhoei, decreased immune
macrophages. In fact, the extracts have demonstrated a consid­
system abnormalities and systemic inflammation in a rat model
erable decrease in the production of pro-inflammatory cytokines
of [Link] A prevents transgenic mice carrying a
like TNF and IL-1, making them promising candidates for the
humanized PXR2 from developing arthritis brought on by anti-
development of anti-inflammatory medications. (Marchese et al.,
collagen antibodies. Solomonsterol A decreases the expression
2021). Excessive use of antibiotics, antiseptics, and chemo­
of the chemokines MIP1-α and RANTES and the inflammatory
therapeutic drugs has negative effects on the environment and
markers TNF-α, IFN-γ, and IL-17, which lessens the inflam­
has contributed to the emergence of bacteria that are resistant to a
matory response (Mencarelli et al., 2014). Steroids extracted
variety of antibiotics. In Hong Kong in 1955, Shigella was shown
from the starfish Astropecten polyacanthus exhibited im­
to be resistant to antibiotics like sulphonamides, streptomycin,
munomodulatory function by reducing the production of IL-6,
chloramphenicol, and tetracycline. The natural balance of flora
IL-12 p40, and TNF-α, when dendritic cells derived from bone
and fauna is being threatened by the buildup of chemical and
marrow are activated by LPS (Thao et al., 2014).
antibiotic residues in the environment. The World Health
Organization defines probiotics as living bacteria that, when
taken insufficiently, promote digestion and food absorption while
5.7 Marine Microorganisms with also improving immunity, disease prevention, stress tolerance,
and disease control. Pseudoalteromonasxiamenensis, which has
Immunomodulatory Compounds from
demonstrated potential as a probiotic candidate to stimulate the
Bacteria and Fungi host’s immune system to fight disease and to improve environ­
It is widely recognized that fungi produce anti-inflammatory mental suitability, has shown to have antimicrobial capabil­
substances (Xu et al., 2019). Some fungi have been regarded ities against a variety of pathogens. This phenomenon was
great candidates for immunotherapy because they have been assessed by using murine macrophages (Wasana et al.,
shown to be able to boost the immune system, have anticancer 2021). According to the definition of the World Health
action, and restore the immune system that has been damaged Organization, probiotics are living microorganisms which,
by chemotherapy and radiotherapy (Ayeka 2018). There are when administered in adequate quantities, confer health
numerous substances that have been isolated from various benefits such as improvement of immunity, disease control,
marine fungi species that have been shown to have an impact tolerance to stress, improved digestion, and improved absorption
on the immune system. Examples include semi-vioxanthin and of food.
azonazine, which have been tested on RAW264.7 cells for
their immunomodulatory and anti-inflammatory activities,
respectively (Wu et al., 2010). According to certain research, 5.8 Cyanobacteria
fungi are also a source of polysaccharides, which are
increasingly valued for their potential probiotic effects and use Cyanobacteria are biologically important group of microorgan­
in the treatment of immunological disorders (Goldring & isms showing an abundant resource of biologically active che­
Pattenden, 2004). The mushroom (Phomaherbarum YS4108) micals, including toxins with a variety of therapeutic uses (Singh
whose mycelium was used to make the α-D-glucan YCP was et al., 2011). Bioactive chemicals made by cyanobacteria have a
purified, and it demonstrated significant immunomodulatory wide range of effects, including antibacterial, antiviral, protease
functions by controlling T lymphocytes and dendritic cells in inhibition, anticancer, and immunomodulatory activity, LPS
vitro, activating macrophages in vitro, and boosting phagocy­ produced by marine cyanobacteria may have TLR4 antagonistic
totic activity in vitro and in vivo (Liu et al., 2020). In a recent properties (Durai et al., 2015). Cyanobacterial LPS (CyP), from
study by Wei Liu et al., (2020), mice with acute colitis brought Oscillatoria planktothrix FP1, demonstrated protective effects in
on by dextran sodium sulphate (DSS) were given the α-D- inflammatory circumstances and inhibits TLR4 activation. In
glucan YCP. After seven days, the YCP administration was both human and mouse dendritic cells, CyP inhibits the MyD88-
successful in alleviating the colitis-related clinical symptoms dependent and MyD88-independent TLR4 signaling pathways,
in rats, reestablishing intestinal immunological homeostasis, potentially inhibiting the inflammatory response brought on by
and reversing mucosal damage. The overexpression of the pro- LPS (Macagno et al., 2006). A group of substances called
inflammatory cytokines IL-1, IL-6, and TNF-αinduced by DSS malyngamides, which are produced from the marine cyano­
in the colon was also inhibited by the YCP. The tissues also bacterium Lyngbyamajuscula, have strong anti-inflammatory
had much higher amounts of IL-10 and IL-22, which led to the properties. Malyngamide F acetate inhibits nitric oxide (NO)
regeneration of injured tissues. Firmicutes, Bacteroidetes, and other inflammatory indicators production in RAW 264.7
Proteobacteria, Clostridiales, and Lachnospiraceae, which are cells. Because LPS stimulation decreases IL-1 and increases
strongly associated to immune modulation and mucus repair, TNF-α production in MyD88 mutant animals via a MyD88-
Immunomodulators to Prevent Diseases and Minimize Antimicrobial Use 69

independent mechanism, it specifically suppresses the MyD88- modulated (Magnani et al., 2000). In this way, microalgae pig­
dependent pathway (Villa et al., 2010). ment may help to modulate the immune system and prevent and
lessen chronic inflammatory diseases (Widyaningrum et al.,
2022). The majority of the microalgae-derived substances
5.9 Microalgae (alginate, fucoidan, ascophyllan, and porphyrin) function as
adjuvants in vaccines by boosting the immune response and
Among the most potential natural source of substances that can activating APCs (Montuori et al., 2022). Mice were used to test
be employed as alternatives to popular pharmaceutical medica­ the Chlorella stigmatophora polysaccharide extract both in vitro
tions is algae, specifically microalgae. They have long evolved and in vivo. Studies showed that the polysaccharide extract
and adapted to a variety of habitats and harsh environments, from Chlorella stigmatophora enhanced the phagocytic
which makes them particularly interesting candidates for use as nature of macrophages from the peritoneal cavity. The im­
immunomodulating agents (Sayed et al., 2020). They are very munostimulatory activity was boosted by the use of microalgae
simple to sample and cultivate in comparison to macroorgan­ as diet. In mice, the spray-dried version of D. salina boosted NK
isms. Numerous microalgae have also demonstrated im­ and macrophage activation. Tetraselmischuii was administered
munomodulatory effects in human and animal models. orally, and the outcome was a high number of immune system-
Polysaccharides that are derived from marine microalgae have related genes such as major histocompatibility complex genes,
potent immune-boosting effects. Due to their high bioactivity IgM, and T-cell receptor beta being expressed. Further research
and relative lack of toxicity, polysaccharides have drawn a lot of is necessary since the variety of microalgal species that produce
interest (Yu et. al., 2018). The microalgae Thraustochytriidae immunomodulating chemicals has not yet been sufficiently
species’ exopolysaccharide (EPS) demonstrated anti-cancer defined (Riccio et al., 2019).
efficacy for changes in gene expression and cell proliferation.
primarily concerned with the gene expression occurring in the
BG-1 ovarian cancer cell line, MCF-7 breast cancer cell line, and
5.10 Macroalgae
SW-620 colon cancer cell line. The impact of EPS is determined
by looking at T-cell cytokine production as well as the accel­ Numerous chemicals found in macroalgae have been shown to
eration of T and B lymphocyte development. Microalgal EPS have positive effects on human health (Biris-Dorhoi, et al.,
decreased the production of