Identification of Larvae of Intertidal Bivalves

Marine Ecology Progress Series, 2007

Accurate and efficient identification to the species level of early larval stages has long been a problematic step in the study of marine invertebrates, due to the extremely small size of the larvae and their lack of diagnostic morphological characters. It is nonetheless, a prerequisite for any ecological study. As a consequence, a number of molecular approaches, mostly based on the PCR technique, have been developed over the last decade. We developed a method relying on specific rRNA-targeted oligonucleotide probes for in situ molecular hybridization using whole larvae. A colorimetric reaction following the hybridization allows signal detection with a light microscope. A total of 9 probes have been developed, targeting the species that are major components of benthic communities in several European bays. The method can be applied to roughly sorted wild plankton samples, and some steps could be automated. It is relatively inexpensive to implement and does not require costly equipment or expensive staff training. The overall larval morphology is preserved, allowing visual inspection. The quantitative aspect of the approach is another asset. Field-based studies of larval distribution and behaviour are possible applications of the method, as large numbers of samples can be screened to meet the requirements of adequate spatial and temporal coverage. It is also relevant for routine monitoring of target species, such as species of commercial interest, bio-indicators or invasive species.

Folia Zoologica, 2002

A b s t r a c t. The larval period in fishes has several definitions, each one deviating slightly from the other. Small discrepancies in the definitions, especially concerning the end of the larval period, can create misunderstandings when applied in practice. I examine the different definitions of a larva, the larval period, including metamorphosis, and the juvenile period. Various criteria used to identify the transition from the larval to juvenile periods are contradictory and refer mostly to the length of the larval period. Ignorance of definitions used to identify the juvenile period has caused many larvae to be regarded as juveniles. I discuss the feasibility of various definitions and the nomenclature on the basis of character evaluation. The occurrence of larval morphological characters, i.e. larval fin fold, differentiation of fins, temporary organs, absence of scale cover, non-adult like body proportions and pigmentation, is examined and some features are compared in eleve...

The polymerase chain reaction (PCR) was used to produce species-speci¢c DNA markers (RAPDs) from two sibling cockle species and ¢ve other co-occurring intertidal bivalves. Ampli¢cation reactions with one single primer readily distinguished larvae and adults of Cerastoderma edule from larvae and adults of C. lamarcki, and from adults of Mya arenaria, Macoma balthica, Scrobicularia plana, Venerupis pulastra and Mytilus edulis. Random ampli¢ed polymorphic DNA (RAPD) is suggested as a simple and quick method to determine species identity in taxa that are di¤cult to identify on the basis of morphological characters alone, such as marine bivalve larvae.

PLoS ONE, 2013

Due to insufficient morphological diagnostic characters in larval fishes, it is easy to misidentify them and difficult to key to the genus or species level. The identification results from different laboratories are often inconsistent. This experiment aims to find out, by applying DNA barcoding, how inconsistent the identifications can be among larval fish taxonomists. One hundred morphotypes of larval fishes were chosen as test specimens. The fishes were collected with either larval fish nets or light traps in the northern, southern and northwestern waters of Taiwan. After their body lengths (SL) were measured and specimen photos were taken, all specimens were delivered, in turn, to five laboratories (A-E) in Taiwan to be identified independently. When all the results were collected, these specimens were then identified using COI barcoding. Out of a total of 100 specimens, 87 were identified to the family level, 79 to the genus level and 69 to the species level, based on the COI database currently available. The average accuracy rates of the five laboratories were quite low: 80.1% for the family level, 41.1% for the genus level, and 13.5% for the species level. If the results marked as ''unidentified'' were excluded from calculations, the rates went up to 75.4% and 43.7% for the genus and species levels, respectively. Thus, we suggest that larval fish identification should be more conservative; i.e., when in doubt, it is better to key only to the family and not to the genus or species level. As to the most misidentified families in our experiment, they were Sparidae, Scorpaenidae, Scombridae, Serranidae and Malacanthidae. On the other hand, Mene maculata and Microcanthus strigatus were all correctly identified to the species level because their larvae have distinct morphology. Nevertheless, barcoding remains one of the best methods to confirm species identification.

Journal of Shellfish Research, 2018

From the 1980s through 1995, scientists at numerous marine, coastal, estuarine, and freshwater laboratories spawned bivalves to provide larvae for use in identifying species based on larval hinge structures and gross shell morphometry. These larvae were preserved in 95% ethanol and stored in sample vials, many of which dried out over the years. Advantage was taken of 50 of 56 species from this collection (and two additional species that were not in the collection for a total of 52 species) to explore the use of optical techniques (polarized light and a full-wave compensation plate) to highlight birefringence patterns of larval shells to discriminate individual species. Representative images of various developmental stages of 77% (40/52) of the larval bivalve species in the collection were successfully imaged. Similarities across birefringence patterns were observed at the taxonomic ordinal and familial level. Molecular polymerase chain reaction techniques were used in an effort to sequence many of the dried-out specimens and they successfully identified 19% (10/52) of the larval bivalve species with matches in GenBank. Here it has been demonstrated that optical techniques are efficient for imaging dried-out larval bivalve shells for classification purposes and we present successful sequences of 10 species of bivalve larvae from the preserved collection.

2011

Project Hypotheses We hypothesized that differences in the distribution of adult mussels are the result of differences in the selection pressures on their larvae, such that the vertical distribution of larvae from the inner bay species (M. senhousia) facilitates bay retention, while that of the open-coast species (M. californianus) enhances alongshore dispersal. Project Goals and Objectives The preliminary research to be conducted was designed to address the following questions: (1) Can new imaging and particle analysis techniques (fluorescence in situ hybridization and a large-particle cell sorter-FISH-LPCS) be used to successfully isolate and distinguish among three mytilid species in field-collected plankton samples? (2) Do larvae from the three mytilid species display different vertical distribution patterns that may influence their horizontal transport? (3) Does vertical distribution change with tide, light or ontogenetic stage? Briefly describe project methodology (1) LAB-SPAWNED LARVAE. Adult M. californianus, M. galloprovincialis, and M. senhousia were collected and brought back to the lab where spawning was induced. After quantifying larval densities, test mixtures of known concentrations of mussel larvae were created and used to test the accuracy of the large-particle cell sorter to quantify samples containing mixed species.

Proceedings Ninth IEEE International Conference on Computer Vision, 2003

This paper describes a novel application of support vector machines and multiscale texture and color invariants to a problem in biological oceanography: the identification of 6 species of bivalve larvae. Our data consists of polarized color images of scallop and other bivalve larvae (between 2 and 17 days old) collected from the ocean by a shipboard optical imaging system of our design. Larvae of scallops, clams, and oysters are small (100 microns) with few distinguishing features when observed under standard light microscopy. However, the use of polarized light with a full wave retardation plate produces a vivid color, bi-refringence pattern. The patterns display very subtle differences between species, often not discernable to human observers. We show that a soft-margin support vector machine with Gaussian RBF kernel is a good discriminator on a feature set extracted from Gabor wavelet transforms and color distribution angles of each image. By constraining the Gabor center frequencies to be low, the resulting system can attain classification accuracy in excess of 90% for vertically oriented images, and in excess of 80% for randomly oriented images.

Copeia, 2000

We investigated the use of geometric morphological shape differences to differentiate laboratory-reared larval (4-22 days posthatch, Ͻ 10 mm SL) Morone chrysops, Morone saxatilis, and Morone chrysops & ؋ M. saxatilis ( hybrids. We also examined impacts of allometry on descriptions of individual shape. For validation of a shapebased taxonomic discrimination model, we used cellulose acetate electrophoresis to establish a ''known species'' test group of field-collected larvae based on banding patterns for the enzyme system esterase. Geometric shape was described with Cartesian coordinates of 16 anatomical landmarks located along the midsaggital outline of laboratory-reared (n ‫؍‬ 373) and field-collected (n ‫؍‬ 29) larvae. Coordinate data were reoriented and rescaled to uniform centroid size and analyzed as landmark displacements from a reference form. Discriminant function analysis resulted in 100% separation of M. chrysops and M. saxatilis larvae based on geometric shape. Discrimination success dropped to 87% when more variable data from hybrids were included in the discriminant function. Seventy percent and 87%, respectively, of field collected larvae (n ‫؍‬ 29) and a randomly selected laboratory subgroup (n ‫؍‬ 40) were correctly classified to taxon. Results suggest that taxonomic separation of early-stage larvae based on shape data is not affected by allometry but is sensitive to larval nutritional condition and handling. Landmark-based morphometrics may provide an improved, simple method of discrimination among species in sympatric populations of closely related larval fishes.

Aquaculture, 2009

The method currently used for accurate identification of mussel larvae is based on the study of morphological traits under an optical microscope, which is a tedious and time-consuming procedure. It also requires considerable taxonomic experience, because of the similarities in the larvae of different bivalves present in the plankton. The introduction of specific monoclonal antibodies (mAbs) directed against mussel larvae, such as M22.8 and M36.5 mAbs developed by our group, may allow an easier and more specific identification. Handling conditions and sample preservation were optimized for using these antibodies in the monitoring of mussel larvae in the Galician rías. Bivalve larvae can be isolated very efficiently from plankton samples by centrifugation in sugar solution. Samples can be maintained at 4°C on the boat and during transport to the laboratory, and then preserved for longer periods at − 80°C or in liquid nitrogen until staining. In an attempt to minimize the time required for immunodetection, different incubation periods were tested, which showed that only 5 min of incubation with the primary monoclonal antibody and 60 min with the secondary antibodies are sufficient to stain over 98% of the larvae. Here, we show that the use of mAbs allows a rapid and specific recognition of mussel larvae, with clear advantages over the traditional method, particularly for large-scale field studies.

Limnology and Oceanography: Methods, 2012

The larvae of many coastal benthic invertebrates have complex life cycles beginning with a pelagic larval stage lasting from a few days to weeks. During development, larvae are passively transported by ocean currents that determine their fates (Thorson 1950; Scheltema 1986). Studies of invertebrate larval dispersal have been met by challenges associated with small sizes of individuals, high mortality, and patchiness over large spatial scales (Boicourt 1988; Garland 2000; Pineda et al. 2007). Particularly for bivalve larvae, it is difficult to perform species-specific field studies because of an inability to accurately identify early stage larvae (Garland 2000; Garland and Zimmer 2002; Gregg 2002). Because bivalve larvae exhibit species-specific behaviors in the field (Shanks and Brink 2006), one cannot accurately assess transport without identifying species. This is especially important when considering populations of commercially important species, as an understanding of larval transport is necessary to address management questions concerning species productivity and decline, shellfish enhancement through seeding, and habitat restoration (Gregg 2002). Once a bivalve larva begins shell mineralization (usually 20 h post-fertilization), most species proceed to a straight-hinge (veliger) stage followed by transformation to a more rounded, umbonate (pediveliger) stage after several days (Chanley and Andrews 1971). It is particularly difficult to distinguish species of straight-hinged larvae by morphological features alone, but as the larva develops, characteristic morphological changes can