Swarming around Shellfish Larvae

Journal of Sea Research, 2005

Despite the importance of the planktonic larval stage in intertidal bivalves, our understanding of this stage is still insufficient. A major obstacle in the quantification of planktonic larval distributions is the identification of sampled larvae. Identification is difficult due to the uniform morphology of many larval species. We evaluated the morphology of bivalve larvae reared in our laboratory (Crassostrea gigas, Cerastoderma edule, Macoma balthica, Mytilus edulis) and literature data on larvae from the 1960s, using image analysis techniques. We used this dataset to compile species-specific dimensions (length-width of the larval shell) and shape parameters (contour of the larval shell). The first method yielded different slopes when length and width were plotted against each other, but regression lines overlapped, which rendered the technique impractical for field identification. Multidimensional scaling of larval shape within one species showed shape development of the larvae during ontogeny. Linear discriminant analysis did not produce results when the whole data set was used. But discriminant analysis on larger individuals (length N 150 Am) was relatively successful for species of which sufficient individuals were available. The identity of up to 74% of the large larvae could be predicted correctly. D

Despite the importance of the planktonic larval stage in intertidal bivalves, our understanding of this stage is not well developed. One of the important limitations in quantification of planktonic larval distributions is the identification of sampled larvae. Identification of larvae is difficult due to the uniform morphology of many larval species. Microscopic identification was mostly used for this purpose in the past. The problem with this technique is that direct identification requires specialist knowledge. Recently, considerable progress has been made in identification of bivalve larvae using immunological techniques or molecular biology. However, not every laboratory doing larval research is equipped for molecular or immunological identification of bivalve larvae and these techniques are time consuming and costly. The recent improvements in imagine analysis technology and the reduction in cost of such equipment make optical techniques for identification of bivalve larvae interesting again. We evaluate bivalve larvae reared in our laboratory (Crassostrea gigas, Cerastoderma edule, Macoma balthica, Mytilus edulis) and literature data of larvae from the 1960s using image analysis techniques. We use this dataset to compile species-specific dimensions (length -width of the larval shell) and shape parameters (contour of the larval shell). The first method yields different slopes when length and width are plotted against each other but regression lines overlap, which makes the technique impractical for field identification. Multidimensional scaling of larval shape within one species shows shape-development of the larvae during ontogeny. Linear discriminant analysis did not produce results when the whole data set was used. But discriminant analysis on larger individuals (length > 150 µm) was relatively successful for species for which sufficient individuals were available. The identity of up to 74 % of the larvae could be predicted correctly.

2005

The identification of effective, nontoxic means for physically marking and tracking marine invertebrate larvae is a necessary step towards meeting a major goal of modern marine population biology, the direct measurement of larval dispersal. An inexpensive, rapid and effective means for marking bivalve larvae would be particularly useful because, as a taxonomic group, bivalves contain many commercially important and exploited species. Likewise, bivalves produce large numbers of propagules for experimental procedures and, for many species, methods for rearing larvae have been well established. Calcein has been used as a marker in numerous studies of adults and juveniles of calcium-carbonate-containing marine organisms, but its effects on small and sensitive life history stages such as embryos and larvae can be detrimental. We show that calcein can be used to rapidly and effectively mark large numbers of larvae from two bivalve species, Argopecten irradians concentricus (Say, 1822) (the Bay Scallop) and Mytilus trossulus Gould, 1850 (the Bay Mussel). Calcein had no detectable negative effects on growth or survivorship of larvae of either species; therefore, this fluorescent mark should serve as a useful tool for directly tracking dispersal of these species in the field. Our marking method is simple and inexpensive and can easily be used to determine the effectiveness and potential toxicity of the calcein mark for other bivalves.

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

Limnology and Oceanography: Methods, 2016

Bivalve larvae are small (50-400 lm) and difficult to identify using standard microscopy, thus limiting inferences from samples collected in the field. With the advent of ShellBi, an image analysis technique, accurate identification of bivalve larvae is now possible but rapid image acquisition and processing remains a challenge. The objectives of this research were to (1) develop a benchtop automated image acquisition system for use with ShellBi, (2) evaluate the system, and (3) create a protocol that would maintain high classification accuracies for larvae of the eastern oyster, Crassostrea virginica. The automated system decreased image acquisition time from 2-13 h to 46 min per slide and resulted in the highest classification accuracies at the lowest tested magnification (7X) and shortest image acquisition time (46 min). Quality control tests indicated that classification accuracies were sensitive to camera and light source settings and that measuring changes in light source and color channel intensities over time was an important part of quality control during routine operations. Validation experiments indicated that under proper settings, automated image acquisition coupled with ShellBi could rapidly classify C. virginica larvae with high accuracies (80-93%). Results suggest that this automated image acquisition system coupled with ShellBi can be used to rapidly image plankton samples and classify C. virginica larvae allowing for expanded capability to understand bivalve larval ecology in the field. Additionally, the automated system has application for rapidly imaging other planktonic organisms at high magnification.

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.

Marine Ecology Progress Series, 2012

Physical and biological conditions impact recruitment and adult population structure of marine invertebrates by affecting early life history processes from spawning to post-settlement. We investigated how temperature, salinity and phytoplankton influenced larval abundance and larval size structure for three species of bivalves over two non-consecutive years in Waquoit Bay, MA. Abundance and size of Mercenaria mercenaria (quahog), Anomia simplex (jingle clam), and Geukensia demissa (ribbed mussel) larvae were compared between locations in the bay and with environmental conditions. Shell birefringence patterns using polarized light microscopy were used to distinguish species. Larval abundances for all three species were higher in 2009 than in 2007 and were positively correlated with temperature in both years. Differences in larval abundance and size structure between bay sites were attributed to salinity tolerances and potential source locations. Higher survival in 2009 than in 2007, as determined by number of pediveligers, was likely due to higher temperatures and greater food availability during the peak abundance months of July and August in 2009. Yearly differences in larval growth and survival can have a large impact on recruitment. Knowing the optimal periods and locations for larval abundance and survival can be useful for isolating species-specific patterns in larval dispersal and to aid resource managers in enhancing or restoring depleted populations.

Journal of Plankton Research, 2007

We report the application of a recently developed molecular method, single step nested multiplex PCR (SSNM-PCR) assay and microscopy to identify and investigate temporal patterns of bivalve larvae in a Danish estuary, Isefjord. All samples were collected during the SUSTAINEX program from June to November 2001. Using the molecular assay, larvae could be categorized into six groups: the blue mussel, Mytilus edulis, Ensis spp., species of the Myoidae superfamily (Mya spp.), the common cockle (Cardiidae family), members of the Abra and Macoma genera of the Tellinoidae superfamily and members of the surf clam genera, Spisula spp. A seventh group was composed of unknown larvae. Greater resolution was possible by microscopy, but only for relatively large and intact individuals (.150-200 mm). The molecular approach was capable of differentiating between larvae regardless of shell size. Where it was possible to directly compare identifications based on both methods, concordance was high for M. edulis, Macoma balthica/Abra alba and E. americanus, whereas identification of Myoidae spp. and Cardiids was less consistent. Over the course of the study, two patterns of larval occurrence were observed. Larvae from species known to exhibit a protracted annual spawning period (M. edulis, Myoidae spp., Mysella bidentata and Cardiids) were present in the water column throughout the sampling period, whereas larvae of Abra alba, Barnea candida, E. americanus, Macoma balthica, Musculus marmoratus Scrobicularia plana and Tapes pullastra appeared at clearly defined periods.

Marine Biology, 1997

Epifluorescence microscopy was used to analyze the stomach contents of bivalve larvae collected in the Baie des Chaleurs (western Gulf of St. Lawrence, Canada) in order to document food-particle sizes, compare feeding among taxa, and compare the diet with the in situ phytoplankton community. Stomach contents were mainly composed of small autotrophic flagellates (<5 m) and cyanobacteria (<2 m), reflecting the microbial food web which characterizes these waters. More than half (55%) of all veligers examined contained algal cells of 5 to 15 m, whereas only 3% had cells of 15 to 25 m. Differences in the size ranges of ingested algal cells among similar-sized larvae of different species suggests that veligers actively selected food particles. Among the smallest veligers (185 to 260 m), scallops (Placopecten magellicanus) and mussels (Mytilus edulis) ingested more <5 m and 5 to 15 m algae than clams (Mya arenaria). Among larger veligers (261 to 405 m), clams contained significantly more <5 m cells than mussels, whereas mussels contained significantly more 5 to 15 m algae than clams. Algal cells of 15 to 25 m were preferentially ingested by mussel veligers. Feeding also differed between different-sized veligers within taxa, i.e. the smallest clam veligers ingested fewer of 5 to 15 m algae than the larger size classes. Mussel veligers ingested significantly more 15 to 25 m and fewer <5 m cells as their size increased. The dominance of ultra-plankton in the nearshore waters of Baie des Chaleurs and in the stomach contents suggests that veliger larvae may be an important export path for carbon produced by small phytoplankton.

2012

Physical and biological conditions impact recruitment and adult population structure of marine invertebrates by affecting early life-history processes from spawning to post-settlement. We investigated how temperature, salinity, and phytoplankton influenced larval abundance and larval size structure for 3 species of bivalves over 2 non-consecutive years in Waquoit Bay, Massachusetts, USA. Abundance and size of Mercenaria mercenaria (quahog), Anomia simplex (jingle clam), and Geukensia demissa (ribbed mussel) larvae were compared between locations in the bay and with environmental conditions. Shell birefringence patterns using polarized light microscopy were used to distinguish species. Larval abundances for all 3 species were higher in 2009 than in 2007 and were positively correlated with temperature in both years. Differences in larval abundance and size structure between bay sites were attributed to salinity tolerances and potential source locations. Higher survival in 2009 than in 2007, as determined by number of pediveligers, was likely due to higher temperatures and greater food availability during the peak abundance months of July and August in 2009. Yearly differences in larval growth and survival can have a large impact on recruitment. Knowing the optimal periods and locations for larval abundance and survival can be useful for isolating species-specific patterns in larval dispersal and to aid resource managers in enhancing or restoring depleted populations.