The Asian lineage of Zika virus (ZIKV), a mosquito-borne pathogen originally from Africa, caused ... more The Asian lineage of Zika virus (ZIKV), a mosquito-borne pathogen originally from Africa, caused an epidemic into Brazil in 2015 and subsequently spread throughout the Americas. Local transmission in the U.S. is a public health concern, especially for Florida where the mosquito vectors Aedes aegypti and Ae. albopictus are widespread, abundant, and there is a high potential for virus introduction due to imported cases. Here we evaluate relative susceptibility to infection and transmission of Zika virus among geographic populations of Ae. aegypti and Ae. albopictus in Florida. Both species have been implicated as ZIKV vectors elsewhere, but both virus and vector genotype are known to influence transmission capacities and, hence, the risk of outbreaks. We test the hypothesis that Ae. aegypti and Ae. albopictus show geographic differences in midgut and salivary gland barriers that limit ZIKV transmission, using local populations of the two vector species recently colonized from three re...
Zika virus (ZIKV) first spread into Brazil in 2013 and is now present throughout the Americas. In... more Zika virus (ZIKV) first spread into Brazil in 2013 and is now present throughout the Americas. In 2016, Florida witnessed the beginnings of local ZIKV transmission. No reports of local transmission have been reported for 2018; however, travel related cases continue to be reported. Recurrence of local transmission in the United States is a major public health risk in Florida where vectors Aedes aegypti (Diptera: Culicidae, Linnaeus) and Aedes albopictus (Diptera: Culicidae, Skuse) are abundant and there is a high potential for virus reintroduction. A dose-response study was used to evaluate susceptibility and transmission potential of Florida Ae. aegypti and Ae. albopictus to ZIKV originating from Puerto Rico. Mosquitoes were orally exposed to one of three doses of ZIKV. Higher doses of infected blood resulted in overall greater infection rates in both mosquito species. Ae. aegypti and Ae. albopictus were susceptible to infection with ZIKV and revealed a significant species by dose interaction. At low doses, Ae. aegypti was significantly less susceptible to infection with ZIKV than Ae. albopictus (6.7% and 44.4%, respectively). In contrast, at high doses, Ae. aegypti was significantly more susceptible to infection than Ae. albopictus (75.8% and 53.8%, respectively). No significant differences were observed between Ae. aegypti and Ae. albopictus in disseminated infection (0-75%) and saliva infection (0-52.4%). These observations suggest greater susceptibility to infection for Ae. albopictus at lower doses likely encountered by viremic humans. However, low disseminated infection and saliva infection for Ae. albopictus, combined with catholic feeding behavior, are likely to limit transmission potential relative to Ae. aegypti.
Zika virus (ZIKV) infection has caused devastating consequences in Brazil as infections were asso... more Zika virus (ZIKV) infection has caused devastating consequences in Brazil as infections were associated with neurological complications in neonates. Aedes aegypti is the primary vector of ZIKV, and the evolution of insecticide resistance (IR) in this species can compromise control efforts. Although relative levels of phenotypic IR in mosquitoes can change considerably over time, its influence on vector competence for arboviruses is unclear. Pyriproxyfen (PPF)-resistant populations of Ae. aegypti were collected from five municipalities located in Northeast of Brazil, which demonstrated different resistance levels; low (Serrinha, Brumado), moderate (Juazeiro do Norte, Itabuna), and high (Quixadá). Experimental per os infection using ZIKV were performed with individuals from these populations and with an insecticide susceptible strain (Rockefeller) to determine their relative vector competence for ZIKV. Although all populations were competent to transmit ZIKV, mosquitoes derived from p...
Recurrence of local transmission of Zika virus in Puerto Rico is a major public health risk to th... more Recurrence of local transmission of Zika virus in Puerto Rico is a major public health risk to the United States, where mosquitoes Aedes aegypti (Linnaeus) and Aedes mediovittatus (Coquillett) are abundant. To determine the extent to which Ae. mediovittatus are capable of transmitting Zika virus and the influence of viremia, we evaluated infection and transmission in Ae. mediovittatus and Ae. aegypti from Puerto Rico using serial dilutions of infectious blood. Higher doses of infectious blood resulted in greater infection rates in both mosquitoes. Aedes aegypti females were up to twice as susceptible to infection than Ae. mediovittatus, indicating a more effective midgut infection barrier in the latter mosquito species. Aedes aegypti exhibited higher disseminated infection (40–95%) than Ae. mediovittatus (<5%), suggesting a substantial midgut escape barrier in Ae. mediovittatus. For Ae. aegypti, transmission rates were low over a range of doses of Zika virus ingested, suggesting ...
Gel-electrophoresis and visualization of RT-LAMP products with LED blue light (excitation at 470 ... more Gel-electrophoresis and visualization of RT-LAMP products with LED blue light (excitation at 470 nm) through orange filter. (A) Detection of Zika (ID # 3 and 4) and chikungunya (ID # 320 and 328) in 3-plex format with infected mosquito legs or bodies. Zika infected mosquitos generated bright green fluorescence (FAM-labeled probe) whereas chikungunya infected mosquitoes generated yellow-green fluorescence (HEX-labeled probe). Gel electrophoresis analysis showed that in the presence of target viral RNA, ladder like amplicons were generated. (B) Visualization of Zika-infected (ID # 7 and 9) and chikungunya-infected (ID # 191) mosquito samples in 3-plex format on Q-paper after RT-LAMP run at 65 °C for 30 min. Zika samples generated bright green signal due to FAM-labeled probes whereas chikungunya containing samples generated more like yellow-green signal due to the use of HEX-labeled probes (JPEG 56 kb)
Limit of detection for 1-plex chikungunya and dengue-1 RT-LAMP experiments. Substrates for this e... more Limit of detection for 1-plex chikungunya and dengue-1 RT-LAMP experiments. Substrates for this experiment were extracted viral RNA from Vero cell cultures. (A) Varying titers of chikungunya viral RNAs (~189 to 18 copies) were included in RT-LAMP reagents and run real-time using Light cycler (channel 523-568). For chikungunya detection, 80 nM of HEX-labeled probes were used, and about 38 copies of chikungunya viral RNA could be detected in less than 30 min. (B) Varying titers of dengue-1 viral RNAs (~2.44 to 0.12 pfu equivalent RNA copies) were included in RT-LAMP reagents and run real-time using Light cycler (channel 558-610). For dengue-1 detection, 80 nM of TAMRA-labeled probes were used, and about 1.22 pfu equivalent copies of dengue-1a viral RNA could be detected within 35 min (JPEG 58 kb)
Gel electrophoresis of RT-LAMP primers tested on small subunit rRNA of female Ae. aegypti mosquit... more Gel electrophoresis of RT-LAMP primers tested on small subunit rRNA of female Ae. aegypti mosquitoes. Crushed specimens were either put directly into RT-LAMP mixture, or first crushed on Q-paper and then went through ammonia treatment prior to RT-LAMP. In either case, set 2 failed to go to completion where as for set 1, most of the primers were consumed within 30 min of incubation at 65 °C. No template control experiments did not produce any amplicon as expected (JPEG 44 kb)
Document_Point of Sampling Detection of Zika Virus within a Multiplexed Kit Capable of Detecting ... more Document_Point of Sampling Detection of Zika Virus within a Multiplexed Kit Capable of Detecting Dengue and Chikungunya (DOCX 111 kb)
Table S1. PCR primers and Luminex probes designed for this study. R, mixed A and G bases; Y, mixe... more Table S1. PCR primers and Luminex probes designed for this study. R, mixed A and G bases; Y, mixed C and T bases. Oligonucleotides s selected to assemble the diagnostics panel are in Italic Bold. (DOCX 13 kb)
Lethal and nonlethal effects of predation are likely to impact phenotypic traits of potential pre... more Lethal and nonlethal effects of predation are likely to impact phenotypic traits of potential prey that elude capture. Nonlethal effects of predation on prey can include alterations in behavior or morphology in response to predation risk which may impair prey growth and fitness. The application of pesticide in mosquito control during aquatic stages can interact with predation stress and alter density and phenotypic traits of prey. Insect growth regulator (IGR) pyriproxyfen is a pesticide that mainly prevents pupal-adult metamorphosis by mimicking juvenile hormone, whereas the larval stage is not targeted. The use of IGR can therefore act in conjunction with natural aquatic predators that target the larval stage to affect population of prey. In this study, we assessed the invasive mosquito prey Aedes aegypti responses to lethal and nonlethal effects of a combination of IGR and predatory mosquito larvae of Toxorhynchites rutilus. The combination of IGR and Tx. rutilus heavily lowered Ae. aegypti metamorphosis to adulthood more than the independent effects of IGR or Tx. rutilus. Exposing Ae. aegypti larvae to the combination shortened life span of adults after metamorphosis for both males and females, whereas control and numerical density reduction "removals" treatments lengthened life span. Our results show strong lethal and nonlethal outcomes of the combination on Ae. aegypti. These findings suggest an additional benefit, decreases adult life span, of the use of an IGR when combined with a natural predator of mosquitoes that may be exploited to improve mosquito control strategies to reduce the risk of disease transmission.
Zika virus (ZIKV) is an emerging mosquito-borne pathogen that can cause global public health thre... more Zika virus (ZIKV) is an emerging mosquito-borne pathogen that can cause global public health threats. In the absence of effective antiviral medications, prevention measures rely largely on reducing the number of adult mosquito vectors by targeting juvenile stages. Despite the importance of juvenile mosquito control measures in reducing adult population size, a full understanding of the effects of these measures in determining mosquito phenotypic traits and in mosquito-arbovirus interactions is poorly understood. Pyriproxyfen is a juvenile hormone analog that primarily blocks adult emergence, but does not cause mortality in larvae. This mechanism has the potential to work in combination with other juvenile sources of mortality in nature such as predation to affect mosquito populations. Here, we experimentally evaluated the effects of juvenile exposure to pyriproxyfen and predatory mosquito Toxorhynchites rutilus on Aedes aegypti phenotypes including susceptibility to ZIKV infection a...
The Asian genotype of Zika virus (ZIKV) emerged in Brazil in 2015 and subsequently spread through... more The Asian genotype of Zika virus (ZIKV) emerged in Brazil in 2015 and subsequently spread throughout the Americas. In July 2016, Florida experienced its first locally acquired ZIKV infection in the continental U.S. Concerns about health risks from ZIKV infection have increased the need to investigate the interactions between potential mosquito vectors and ZIKV. The time it takes for an arbovirus to propagate within a mosquito, and become transmissible, is the extrinsic incubation period (EIP). The EIP for potential mosquito vectors in Florida is unknown. To address this gap in the understanding of ZIKV epidemiology, Florida Aedes aegypti (L.) and Ae. albopictus (Skuse) were orally exposed to ZIKV infected blood meals and fully engorged mosquitoes were held at a constant temperature of 28 °C through the duration of the experiment. Saliva expectorates were collected from cohorts of mosquitoes and tested for the presence of ZIKV at three-day intervals over a period of 24 days to allow ...
The vector competence of mosquitoes for pathogens has been shown to be influenced by the status o... more The vector competence of mosquitoes for pathogens has been shown to be influenced by the status of insecticide resistance in the mosquito population. However, to date, only two studies has explored the impact of insecticide resistance on arbovirus transmission. The global and widespread use of pyrethroids has led to the development of insecticide resistance in many mosquito species, including Aedes aegypti (Linnaeus) (Diptera: Culicidae), the primary vector of Zika virus. Strains of Ae. aegypti that were genetically similar, but responded differently to pyrethroid exposure, were developed using backcrossing techniques. These populations were orally infected with Zika virus and susceptibility to infection, disseminated infection, and transmission potential were evaluated. Analyses revealed differences in susceptibility to infection and disseminated infection between the pyrethroid susceptible and resistant strains of Ae. aegypti during the infection period. Here, we identify an addit...
Parental (transgenerational) effects occur when the conditions experienced by a mother or father ... more Parental (transgenerational) effects occur when the conditions experienced by a mother or father contribute to offspring phenotype. Here we show that parental larval diet in mosquitoes, Aedes aegypti, results in differential allocation of resources in offspring of parents depending on the nutritional condition (quality) of their mate. Maternal effects influenced the number of eggs produced by females as well as their lipid investment. Low nutrient females mated with high nutrient males laid eggs with significantly higher lipid content than those laid by high nutrient females. Paternal effects showed that when high nutrient males mated with low nutrient females, resulting eggs had higher lipid content than when low nutrient males mated with low nutrient females. Overall, our results are consistent with a pattern predicted by the differential allocation of resources hypothesis, when females experience nutritional deprivation, which asserts that mate quality directly influences reproductive allocation.
Mosquito and predatory larvae often share the same habitat. Predators may influence mosquito prey... more Mosquito and predatory larvae often share the same habitat. Predators may influence mosquito prey populations through both lethal effect and non-lethal pathways. A series of experimental manipulations were used to distinguish between lethal (density-mediated interaction) and non-lethal (trait-mediated interaction) effects in a model system comprised of invasive prey mosquito, Aedes aegypti, and a predatory mosquito Toxorhynchites rutilus. Treatments with predators present or manipulations mimicking daily mortality (density reduction) reduced developmental time and recruitment to the adult stage. Daily records of adult survival of A. aegypti showed that exposure to predators during the juvenile stage shortened the lifespan of adults. This was also observed in treatments, where A. aegypti were replaced at the rate of consumption by T. rutilus. In contrast, numerical reductions in A. aegypti that mimicked daily rate of predation led to adults with the longest lifespan. These observations suggest strong effects of density and trait-mediated interactions in the influence of predators on mosquito biology relevant to their ability to transmit pathogens. These results have potentially important implications for disease control strategies. The primary approach to reduce risk of mosquito-borne diseases is through population reduction of the vectors. We show an unanticipated benefit of biological control by predation for the control of juvenile stages of mosquitoes. Specifically, mosquitoes that are exposed to predators but survive to adulthood will have compromised life expectancy, a key parameter in determining risk of disease transmission.
Chikungunya virus is a vector-borne alphavirus transmitted by the bites of infected female Ae. ae... more Chikungunya virus is a vector-borne alphavirus transmitted by the bites of infected female Ae. aegypti and Ae. albopictus. In Brazil between 2014 and 2016 almost 320 thousand autochthonous human cases were reported and in Florida numerous imported CHIKV viremic cases (> 3,800) demonstrate the potential high risk to establishment of local transmission. In the present study, we carried out a series of experiments to determine the viral dissemination and transmission rates of different Brazilian and Florida populations of Ae. aegypti and Ae. albopictus at 2, 5, and 13 days post-infection for the emergent Asian genotype of CHIKV. Our results show that all tested populations of Ae. aegypti and Ae. albopictus have a high proportion (> 0.80) of individuals with disseminated infection as early as 2 dayspost exposure. We found no significant treatment effects of mosquito population origin effects on viral dissemination rates. Transmission rates had a heterogeneous pattern, with US Ae. aegypti and Brazilian Ae. albopictus having the highest proportion of individuals with successful infection (respectively 0.50 and 0.82 as early as 2 days-post infection). Model results found significant effects of population origin, population origin x species, population origin x days post-infection and population origin x species x days post infection.
Aedes aegypti (L.) is the primary vector of many emerging arboviruses. Insecticide resistance amo... more Aedes aegypti (L.) is the primary vector of many emerging arboviruses. Insecticide resistance among mosquito populations is a consequence of the application of insecticides for mosquito control. We used RNA-sequencing to compare transcriptomes between permethrin resistant and susceptible strains of Florida Ae. aegypti in response to Zika virus infection. A total of 2459 transcripts were expressed at significantly different levels between resistant and susceptible Ae. aegypti. Gene ontology analysis placed these genes into seven categories of biological processes. The 863 transcripts were expressed at significantly different levels between the two mosquito strains (up/down regulated) more than 2-fold. Quantitative real-time PCR analysis was used to validate the Zika-infection response. Our results suggested a highly overexpressed P450, with AAEL014617 and AAEL006798 as potential candidates for the molecular mechanism of permethrin resistance in Ae. aegypti. Our findings indicated that most detoxification enzymes and immune system enzymes altered their gene expression between the two strains of Ae. aegypti in response to Zika virus infection. Understanding the interactions of arboviruses with resistant mosquito vectors at the molecular level allows for the possible development of new approaches in mitigating arbovirus transmission. This information sheds light on Zika-induced changes in insecticide resistant Ae. aegypti with implications for mosquito control strategies.
Commercially available assays utilizing antigen or nucleic acid detection chemistries provide opt... more Commercially available assays utilizing antigen or nucleic acid detection chemistries provide options for mosquito control districts to screen their mosquito populations for arboviruses and make timely operational decisions regarding vector control. These assays may be utilized even more advantageously when combined with honey-soaked nucleic acid preservation substrate ('honey card') testing by reducing or replacing the time- and labor-intensive efforts of identifying and processing mosquito pools. We tested artificially inoculated honey cards and cards fed upon individually by West Nile virus (WNV) and Zika virus (ZIKV)-infected mosquitoes with three assays to compare detection rates and the limit of detection for each platform with respect to virus detection of a single infected mosquito and quantify the time interval of virus preservation on the cards. Assays evaluated included CDC protocols for real-time reverse transcriptase polymerase chain reaction (RT-PCR) for WNV an...
Aedes aegypti (L.) is a vector of chikungunya, dengue, yellow fever and Zika viruses. These virus... more Aedes aegypti (L.) is a vector of chikungunya, dengue, yellow fever and Zika viruses. These viruses encounter a variety of induced defense responses from the innate immune system of the mosquito. We cloned defensin A from Ae. aegypti using laboratory populations originating from Key West and Orlando, Florida. To characterize inducible immune defensin peptides, we examined the defensin A (DefA) and defensin C (DefC) expression through time course studies using quantitative real-time PCR. We observed that ingestion of chikungunya virus (CHIKV) and Zika virus (ZIKV) infected blood triggered early upregulated expression of DefA and DefC at 3 h after blood feeding. At 10-d post infection, there was significant downregulation of DefA and DefC in CHIKV-infected females and significant upregulation of DefA and DefC in ZIKV-infected females compared with control mosquitoes fed uninfected blood. Our studies demonstrate that the relative activity of DefA and DefC changed depending on whether A...
The Asian lineage of Zika virus (ZIKV), a mosquito-borne pathogen originally from Africa, caused ... more The Asian lineage of Zika virus (ZIKV), a mosquito-borne pathogen originally from Africa, caused an epidemic into Brazil in 2015 and subsequently spread throughout the Americas. Local transmission in the U.S. is a public health concern, especially for Florida where the mosquito vectors Aedes aegypti and Ae. albopictus are widespread, abundant, and there is a high potential for virus introduction due to imported cases. Here we evaluate relative susceptibility to infection and transmission of Zika virus among geographic populations of Ae. aegypti and Ae. albopictus in Florida. Both species have been implicated as ZIKV vectors elsewhere, but both virus and vector genotype are known to influence transmission capacities and, hence, the risk of outbreaks. We test the hypothesis that Ae. aegypti and Ae. albopictus show geographic differences in midgut and salivary gland barriers that limit ZIKV transmission, using local populations of the two vector species recently colonized from three re...
Zika virus (ZIKV) first spread into Brazil in 2013 and is now present throughout the Americas. In... more Zika virus (ZIKV) first spread into Brazil in 2013 and is now present throughout the Americas. In 2016, Florida witnessed the beginnings of local ZIKV transmission. No reports of local transmission have been reported for 2018; however, travel related cases continue to be reported. Recurrence of local transmission in the United States is a major public health risk in Florida where vectors Aedes aegypti (Diptera: Culicidae, Linnaeus) and Aedes albopictus (Diptera: Culicidae, Skuse) are abundant and there is a high potential for virus reintroduction. A dose-response study was used to evaluate susceptibility and transmission potential of Florida Ae. aegypti and Ae. albopictus to ZIKV originating from Puerto Rico. Mosquitoes were orally exposed to one of three doses of ZIKV. Higher doses of infected blood resulted in overall greater infection rates in both mosquito species. Ae. aegypti and Ae. albopictus were susceptible to infection with ZIKV and revealed a significant species by dose interaction. At low doses, Ae. aegypti was significantly less susceptible to infection with ZIKV than Ae. albopictus (6.7% and 44.4%, respectively). In contrast, at high doses, Ae. aegypti was significantly more susceptible to infection than Ae. albopictus (75.8% and 53.8%, respectively). No significant differences were observed between Ae. aegypti and Ae. albopictus in disseminated infection (0-75%) and saliva infection (0-52.4%). These observations suggest greater susceptibility to infection for Ae. albopictus at lower doses likely encountered by viremic humans. However, low disseminated infection and saliva infection for Ae. albopictus, combined with catholic feeding behavior, are likely to limit transmission potential relative to Ae. aegypti.
Zika virus (ZIKV) infection has caused devastating consequences in Brazil as infections were asso... more Zika virus (ZIKV) infection has caused devastating consequences in Brazil as infections were associated with neurological complications in neonates. Aedes aegypti is the primary vector of ZIKV, and the evolution of insecticide resistance (IR) in this species can compromise control efforts. Although relative levels of phenotypic IR in mosquitoes can change considerably over time, its influence on vector competence for arboviruses is unclear. Pyriproxyfen (PPF)-resistant populations of Ae. aegypti were collected from five municipalities located in Northeast of Brazil, which demonstrated different resistance levels; low (Serrinha, Brumado), moderate (Juazeiro do Norte, Itabuna), and high (Quixadá). Experimental per os infection using ZIKV were performed with individuals from these populations and with an insecticide susceptible strain (Rockefeller) to determine their relative vector competence for ZIKV. Although all populations were competent to transmit ZIKV, mosquitoes derived from p...
Recurrence of local transmission of Zika virus in Puerto Rico is a major public health risk to th... more Recurrence of local transmission of Zika virus in Puerto Rico is a major public health risk to the United States, where mosquitoes Aedes aegypti (Linnaeus) and Aedes mediovittatus (Coquillett) are abundant. To determine the extent to which Ae. mediovittatus are capable of transmitting Zika virus and the influence of viremia, we evaluated infection and transmission in Ae. mediovittatus and Ae. aegypti from Puerto Rico using serial dilutions of infectious blood. Higher doses of infectious blood resulted in greater infection rates in both mosquitoes. Aedes aegypti females were up to twice as susceptible to infection than Ae. mediovittatus, indicating a more effective midgut infection barrier in the latter mosquito species. Aedes aegypti exhibited higher disseminated infection (40–95%) than Ae. mediovittatus (<5%), suggesting a substantial midgut escape barrier in Ae. mediovittatus. For Ae. aegypti, transmission rates were low over a range of doses of Zika virus ingested, suggesting ...
Gel-electrophoresis and visualization of RT-LAMP products with LED blue light (excitation at 470 ... more Gel-electrophoresis and visualization of RT-LAMP products with LED blue light (excitation at 470 nm) through orange filter. (A) Detection of Zika (ID # 3 and 4) and chikungunya (ID # 320 and 328) in 3-plex format with infected mosquito legs or bodies. Zika infected mosquitos generated bright green fluorescence (FAM-labeled probe) whereas chikungunya infected mosquitoes generated yellow-green fluorescence (HEX-labeled probe). Gel electrophoresis analysis showed that in the presence of target viral RNA, ladder like amplicons were generated. (B) Visualization of Zika-infected (ID # 7 and 9) and chikungunya-infected (ID # 191) mosquito samples in 3-plex format on Q-paper after RT-LAMP run at 65 °C for 30 min. Zika samples generated bright green signal due to FAM-labeled probes whereas chikungunya containing samples generated more like yellow-green signal due to the use of HEX-labeled probes (JPEG 56 kb)
Limit of detection for 1-plex chikungunya and dengue-1 RT-LAMP experiments. Substrates for this e... more Limit of detection for 1-plex chikungunya and dengue-1 RT-LAMP experiments. Substrates for this experiment were extracted viral RNA from Vero cell cultures. (A) Varying titers of chikungunya viral RNAs (~189 to 18 copies) were included in RT-LAMP reagents and run real-time using Light cycler (channel 523-568). For chikungunya detection, 80 nM of HEX-labeled probes were used, and about 38 copies of chikungunya viral RNA could be detected in less than 30 min. (B) Varying titers of dengue-1 viral RNAs (~2.44 to 0.12 pfu equivalent RNA copies) were included in RT-LAMP reagents and run real-time using Light cycler (channel 558-610). For dengue-1 detection, 80 nM of TAMRA-labeled probes were used, and about 1.22 pfu equivalent copies of dengue-1a viral RNA could be detected within 35 min (JPEG 58 kb)
Gel electrophoresis of RT-LAMP primers tested on small subunit rRNA of female Ae. aegypti mosquit... more Gel electrophoresis of RT-LAMP primers tested on small subunit rRNA of female Ae. aegypti mosquitoes. Crushed specimens were either put directly into RT-LAMP mixture, or first crushed on Q-paper and then went through ammonia treatment prior to RT-LAMP. In either case, set 2 failed to go to completion where as for set 1, most of the primers were consumed within 30 min of incubation at 65 °C. No template control experiments did not produce any amplicon as expected (JPEG 44 kb)
Document_Point of Sampling Detection of Zika Virus within a Multiplexed Kit Capable of Detecting ... more Document_Point of Sampling Detection of Zika Virus within a Multiplexed Kit Capable of Detecting Dengue and Chikungunya (DOCX 111 kb)
Table S1. PCR primers and Luminex probes designed for this study. R, mixed A and G bases; Y, mixe... more Table S1. PCR primers and Luminex probes designed for this study. R, mixed A and G bases; Y, mixed C and T bases. Oligonucleotides s selected to assemble the diagnostics panel are in Italic Bold. (DOCX 13 kb)
Lethal and nonlethal effects of predation are likely to impact phenotypic traits of potential pre... more Lethal and nonlethal effects of predation are likely to impact phenotypic traits of potential prey that elude capture. Nonlethal effects of predation on prey can include alterations in behavior or morphology in response to predation risk which may impair prey growth and fitness. The application of pesticide in mosquito control during aquatic stages can interact with predation stress and alter density and phenotypic traits of prey. Insect growth regulator (IGR) pyriproxyfen is a pesticide that mainly prevents pupal-adult metamorphosis by mimicking juvenile hormone, whereas the larval stage is not targeted. The use of IGR can therefore act in conjunction with natural aquatic predators that target the larval stage to affect population of prey. In this study, we assessed the invasive mosquito prey Aedes aegypti responses to lethal and nonlethal effects of a combination of IGR and predatory mosquito larvae of Toxorhynchites rutilus. The combination of IGR and Tx. rutilus heavily lowered Ae. aegypti metamorphosis to adulthood more than the independent effects of IGR or Tx. rutilus. Exposing Ae. aegypti larvae to the combination shortened life span of adults after metamorphosis for both males and females, whereas control and numerical density reduction "removals" treatments lengthened life span. Our results show strong lethal and nonlethal outcomes of the combination on Ae. aegypti. These findings suggest an additional benefit, decreases adult life span, of the use of an IGR when combined with a natural predator of mosquitoes that may be exploited to improve mosquito control strategies to reduce the risk of disease transmission.
Zika virus (ZIKV) is an emerging mosquito-borne pathogen that can cause global public health thre... more Zika virus (ZIKV) is an emerging mosquito-borne pathogen that can cause global public health threats. In the absence of effective antiviral medications, prevention measures rely largely on reducing the number of adult mosquito vectors by targeting juvenile stages. Despite the importance of juvenile mosquito control measures in reducing adult population size, a full understanding of the effects of these measures in determining mosquito phenotypic traits and in mosquito-arbovirus interactions is poorly understood. Pyriproxyfen is a juvenile hormone analog that primarily blocks adult emergence, but does not cause mortality in larvae. This mechanism has the potential to work in combination with other juvenile sources of mortality in nature such as predation to affect mosquito populations. Here, we experimentally evaluated the effects of juvenile exposure to pyriproxyfen and predatory mosquito Toxorhynchites rutilus on Aedes aegypti phenotypes including susceptibility to ZIKV infection a...
The Asian genotype of Zika virus (ZIKV) emerged in Brazil in 2015 and subsequently spread through... more The Asian genotype of Zika virus (ZIKV) emerged in Brazil in 2015 and subsequently spread throughout the Americas. In July 2016, Florida experienced its first locally acquired ZIKV infection in the continental U.S. Concerns about health risks from ZIKV infection have increased the need to investigate the interactions between potential mosquito vectors and ZIKV. The time it takes for an arbovirus to propagate within a mosquito, and become transmissible, is the extrinsic incubation period (EIP). The EIP for potential mosquito vectors in Florida is unknown. To address this gap in the understanding of ZIKV epidemiology, Florida Aedes aegypti (L.) and Ae. albopictus (Skuse) were orally exposed to ZIKV infected blood meals and fully engorged mosquitoes were held at a constant temperature of 28 °C through the duration of the experiment. Saliva expectorates were collected from cohorts of mosquitoes and tested for the presence of ZIKV at three-day intervals over a period of 24 days to allow ...
The vector competence of mosquitoes for pathogens has been shown to be influenced by the status o... more The vector competence of mosquitoes for pathogens has been shown to be influenced by the status of insecticide resistance in the mosquito population. However, to date, only two studies has explored the impact of insecticide resistance on arbovirus transmission. The global and widespread use of pyrethroids has led to the development of insecticide resistance in many mosquito species, including Aedes aegypti (Linnaeus) (Diptera: Culicidae), the primary vector of Zika virus. Strains of Ae. aegypti that were genetically similar, but responded differently to pyrethroid exposure, were developed using backcrossing techniques. These populations were orally infected with Zika virus and susceptibility to infection, disseminated infection, and transmission potential were evaluated. Analyses revealed differences in susceptibility to infection and disseminated infection between the pyrethroid susceptible and resistant strains of Ae. aegypti during the infection period. Here, we identify an addit...
Parental (transgenerational) effects occur when the conditions experienced by a mother or father ... more Parental (transgenerational) effects occur when the conditions experienced by a mother or father contribute to offspring phenotype. Here we show that parental larval diet in mosquitoes, Aedes aegypti, results in differential allocation of resources in offspring of parents depending on the nutritional condition (quality) of their mate. Maternal effects influenced the number of eggs produced by females as well as their lipid investment. Low nutrient females mated with high nutrient males laid eggs with significantly higher lipid content than those laid by high nutrient females. Paternal effects showed that when high nutrient males mated with low nutrient females, resulting eggs had higher lipid content than when low nutrient males mated with low nutrient females. Overall, our results are consistent with a pattern predicted by the differential allocation of resources hypothesis, when females experience nutritional deprivation, which asserts that mate quality directly influences reproductive allocation.
Mosquito and predatory larvae often share the same habitat. Predators may influence mosquito prey... more Mosquito and predatory larvae often share the same habitat. Predators may influence mosquito prey populations through both lethal effect and non-lethal pathways. A series of experimental manipulations were used to distinguish between lethal (density-mediated interaction) and non-lethal (trait-mediated interaction) effects in a model system comprised of invasive prey mosquito, Aedes aegypti, and a predatory mosquito Toxorhynchites rutilus. Treatments with predators present or manipulations mimicking daily mortality (density reduction) reduced developmental time and recruitment to the adult stage. Daily records of adult survival of A. aegypti showed that exposure to predators during the juvenile stage shortened the lifespan of adults. This was also observed in treatments, where A. aegypti were replaced at the rate of consumption by T. rutilus. In contrast, numerical reductions in A. aegypti that mimicked daily rate of predation led to adults with the longest lifespan. These observations suggest strong effects of density and trait-mediated interactions in the influence of predators on mosquito biology relevant to their ability to transmit pathogens. These results have potentially important implications for disease control strategies. The primary approach to reduce risk of mosquito-borne diseases is through population reduction of the vectors. We show an unanticipated benefit of biological control by predation for the control of juvenile stages of mosquitoes. Specifically, mosquitoes that are exposed to predators but survive to adulthood will have compromised life expectancy, a key parameter in determining risk of disease transmission.
Chikungunya virus is a vector-borne alphavirus transmitted by the bites of infected female Ae. ae... more Chikungunya virus is a vector-borne alphavirus transmitted by the bites of infected female Ae. aegypti and Ae. albopictus. In Brazil between 2014 and 2016 almost 320 thousand autochthonous human cases were reported and in Florida numerous imported CHIKV viremic cases (> 3,800) demonstrate the potential high risk to establishment of local transmission. In the present study, we carried out a series of experiments to determine the viral dissemination and transmission rates of different Brazilian and Florida populations of Ae. aegypti and Ae. albopictus at 2, 5, and 13 days post-infection for the emergent Asian genotype of CHIKV. Our results show that all tested populations of Ae. aegypti and Ae. albopictus have a high proportion (> 0.80) of individuals with disseminated infection as early as 2 dayspost exposure. We found no significant treatment effects of mosquito population origin effects on viral dissemination rates. Transmission rates had a heterogeneous pattern, with US Ae. aegypti and Brazilian Ae. albopictus having the highest proportion of individuals with successful infection (respectively 0.50 and 0.82 as early as 2 days-post infection). Model results found significant effects of population origin, population origin x species, population origin x days post-infection and population origin x species x days post infection.
Aedes aegypti (L.) is the primary vector of many emerging arboviruses. Insecticide resistance amo... more Aedes aegypti (L.) is the primary vector of many emerging arboviruses. Insecticide resistance among mosquito populations is a consequence of the application of insecticides for mosquito control. We used RNA-sequencing to compare transcriptomes between permethrin resistant and susceptible strains of Florida Ae. aegypti in response to Zika virus infection. A total of 2459 transcripts were expressed at significantly different levels between resistant and susceptible Ae. aegypti. Gene ontology analysis placed these genes into seven categories of biological processes. The 863 transcripts were expressed at significantly different levels between the two mosquito strains (up/down regulated) more than 2-fold. Quantitative real-time PCR analysis was used to validate the Zika-infection response. Our results suggested a highly overexpressed P450, with AAEL014617 and AAEL006798 as potential candidates for the molecular mechanism of permethrin resistance in Ae. aegypti. Our findings indicated that most detoxification enzymes and immune system enzymes altered their gene expression between the two strains of Ae. aegypti in response to Zika virus infection. Understanding the interactions of arboviruses with resistant mosquito vectors at the molecular level allows for the possible development of new approaches in mitigating arbovirus transmission. This information sheds light on Zika-induced changes in insecticide resistant Ae. aegypti with implications for mosquito control strategies.
Commercially available assays utilizing antigen or nucleic acid detection chemistries provide opt... more Commercially available assays utilizing antigen or nucleic acid detection chemistries provide options for mosquito control districts to screen their mosquito populations for arboviruses and make timely operational decisions regarding vector control. These assays may be utilized even more advantageously when combined with honey-soaked nucleic acid preservation substrate ('honey card') testing by reducing or replacing the time- and labor-intensive efforts of identifying and processing mosquito pools. We tested artificially inoculated honey cards and cards fed upon individually by West Nile virus (WNV) and Zika virus (ZIKV)-infected mosquitoes with three assays to compare detection rates and the limit of detection for each platform with respect to virus detection of a single infected mosquito and quantify the time interval of virus preservation on the cards. Assays evaluated included CDC protocols for real-time reverse transcriptase polymerase chain reaction (RT-PCR) for WNV an...
Aedes aegypti (L.) is a vector of chikungunya, dengue, yellow fever and Zika viruses. These virus... more Aedes aegypti (L.) is a vector of chikungunya, dengue, yellow fever and Zika viruses. These viruses encounter a variety of induced defense responses from the innate immune system of the mosquito. We cloned defensin A from Ae. aegypti using laboratory populations originating from Key West and Orlando, Florida. To characterize inducible immune defensin peptides, we examined the defensin A (DefA) and defensin C (DefC) expression through time course studies using quantitative real-time PCR. We observed that ingestion of chikungunya virus (CHIKV) and Zika virus (ZIKV) infected blood triggered early upregulated expression of DefA and DefC at 3 h after blood feeding. At 10-d post infection, there was significant downregulation of DefA and DefC in CHIKV-infected females and significant upregulation of DefA and DefC in ZIKV-infected females compared with control mosquitoes fed uninfected blood. Our studies demonstrate that the relative activity of DefA and DefC changed depending on whether A...
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