Water eDNA metabarcoding is effective in detecting non-native species in marinas, but detection errors still hinder its use for passive monitoring.

Marinas are high-priority targets for marine non-indigenous species (NIS), where they compose a large portion of the biofouling communities. The practicality of water samples collection makes environmental DNA (eDNA) metabarcoding an interesting tool for routine NIS surveys. Here the effectiveness of water-eDNA-metabarcoding to identify biofouling NIS, in 10 marinas from western France, was examined. Morphological identification of specimens collected in quadrats brought out 18 sessile benthic NIS beneath floating pontoons. Water-eDNA-metabarcoding detected two thirds of them, failing to detect important NIS. However, sampling and bioinformatics filtering steps can be optimized to identify more species. In addition, this method allowed the detection of additional NIS from neighboring micro-habitats. Caution should, however, be taken when reporting putative novel NIS, because of errors in species assignment. This work highlights that water-eDNA-metabarcoding is effective for active (targeted) NIS surveys and could be significantly improved for its further use in marine NIS passive surveys.

High-throughput sequencing on preservative ethanol is effective at jointly examining infraspecific and taxonomic diversity, although bioinformatics pipelines do not perform equally.

High-throughput sequencing of amplicons (HTSA) has been proposed as an effective approach to evaluate taxonomic and genetic diversity at the same time. However, there are still uncertainties as to how the results produced by different bioinformatics treatments impact the conclusions drawn on biodiversity and population genetics indices.We evaluated the ability of six bioinformatics pipelines to recover taxonomic and genetic diversity from HTSA data obtained from controlled assemblages. To that end, 20 assemblages were produced using 354 colonies of Botrylloides spp., sampled in the wild in ten marinas around Brittany (France). We used DNA extracted from preservative ethanol (ebDNA) after various time of storage (3, 6, and 12 months), and from a bulk of preserved specimens (bulkDNA). DNA was amplified with primers designed for targeting this ascidian genus. Results obtained from HTSA data were compared with Sanger sequencing on individual zooids (i.e., individual barcoding).Species identification and relative abundance determined with HTSA data from either ebDNA or bulkDNA were similar to those obtained with traditional individual barcoding. However, after 12 months of storage, the correlation between HTSA and individual-based data was lower than after shorter durations. The six bioinformatics pipelines were able to depict accurately the genetic diversity using standard population genetics indices (HS and FST), despite producing false positives and missing rare haplotypes. However, they did not perform equally and dada2 was the only pipeline able to retrieve all expected haplotypes.This study showed that ebDNA is a nondestructive alternative for both species identification and haplotype recovery, providing storage does not last more than 6 months before DNA extraction. Choosing the bioinformatics pipeline is a matter of compromise, aiming to retrieve all true haplotypes while avoiding false positives. We here recommend to process HTSA data using dada2, including a chimera-removal step. Even if the possibility to use multiplexed primer sets deserves further investigation to expand the taxonomic coverage in future similar studies, we showed that primers targeting a particular genus allowed to reliably analyze this genus within a complex community.

Vertical distribution of brittle star larvae in two contrasting coastal embayments: implications for larval transport.

The ability of marine invertebrate larvae to control their vertical position shapes their dispersal pattern. In species characterized by large variations in population density, like many echinoderm species, larval dispersal may contribute to outbreak and die-off phenomena. A proliferation of the ophiuroid Ophiocomina nigra was observed for several years in western Brittany (France), inducing drastic changes on the benthic communities. We here studied the larval vertical distribution in this species and two co-occurring ophiuroid species, Ophiothrix fragilis and Amphiura filiformis, in two contrasting hydrodynamic environments: stratified in the bay of Douarnenez and well-mixed in the bay of Brest. Larvae were collected at 3 depths during 25 h within each bay. In the bay of Brest, all larvae were evenly distributed in the water column due to the intense vertical mixing. Conversely, in the bay of Douarnenez, a diel vertical migration was observed for O. nigra, with a night ascent of young larvae, and ontogenetic differences. These different patterns in the two bays mediate the effects of tidal currents on larval fluxes. O. fragilis larvae were mainly distributed above the thermocline which may favour larval retention within the bay, while A. filiformis larvae, mostly concentrated near the bottom, were preferentially exported. This study highlighted the complex interactions between coastal hydrodynamics and specific larval traits, e.g. larval morphology, in the control of larval vertical distribution and larval dispersal.

Abnormalities in bivalve larvae from the Puck Bay (Gulf of Gdansk, southern Baltic Sea) as an indicator of environmental pollution.

This study described the occurrence of abnormalities in bivalve larvae from the Puck Bay. Analyses of plankton samples collected in 2012-2013 showed that larval Mytilus trossulus, Mya arenaria, and Cerastoderma glaucum exhibited abnormalities that could indicate adverse environmental impacts. The deformities were mainly in shells, but missing soft tissue fragments and protruding vela were also noted. In addition to larval studies, we analyzed benthic postlarvae of Mytilus trossulus. Interestingly, grooves and notches at different locations of the prodissoconch, dissoconch, and shell margin were observed. Some of these deformations were reminiscent of the indentations found on the shell edge of larvae. Comparing the proportion of abnormal postlarvae to larvae with shell abnormalities suggested that the survival of larvae with shell abnormalities was low. Overall, our results suggested that the ratio of abnormal bivalve larvae could be used as an indicator of the biological effects of hazardous substances in the pelagic environment.

Unexpected collective larval dispersal but little support for sweepstakes reproductive success in the highly dispersive brooding mollusc Crepidula fornicata.

In many marine invertebrates, long-distance dispersal is achieved during an extended pelagic larval phase. Although such dispersal should result in high gene flow over broad spatial scales, fine-scale genetic structure has often been reported, a pattern attributed to interfamilial variance in reproductive success and limited homogenization during dispersal. To examine this hypothesis, the genetic diversity of dispersing larvae must be compared with the postdispersal stages, that is benthic recruits and adults. Such data remain, however, scarce due to the difficulty to sample and analyse larvae of minute size. Here, we carried out such an investigation using the marine gastropod Crepidula fornicata. Field sampling of three to four larval pools was conducted over the reproductive season and repeated over 3 years. The genetic composition of larval pools, obtained with 16 microsatellite loci, was compared with that of recruits and adults sampled from the same site and years. In contrast to samples of juveniles and adults, large genetic temporal variations between larval pools produced at different times of the same reproductive season were observed. In addition, full- and half-sibs were detected in early larvae and postdispersal juveniles, pointing to correlated dispersal paths between several pairs of individuals. Inbred larvae were also identified. Such collective larval dispersal was unexpected given the long larval duration of the study species. Our results suggest that each larval pool is produced by a small effective number of reproducers but that, over a reproductive season, the whole larval pool is produced by large numbers of reproducers across space and time.

Does encapsulation protect embryos from the effects of ocean acidification? The example of Crepidula fornicata.

Early life history stages of marine organisms are generally thought to be more sensitive to environmental stress than adults. Although most marine invertebrates are broadcast spawners, some species are brooders and/or protect their embryos in egg or capsules. Brooding and encapsulation strategies are typically assumed to confer greater safety and protection to embryos, although little is known about the physico-chemical conditions within egg capsules. In the context of ocean acidification, the protective role of encapsulation remains to be investigated. To address this issue, we conducted experiments on the gastropod Crepidula fornicata. This species broods its embryos within capsules located under the female and veliger larvae are released directly into the water column. C. fornicata adults were reared at the current level of CO2 partial pressure (pCO2) (390 µatm) and at elevated levels (750 and 1400 µatm) before and after fertilization and until larval release, such that larval development occurred entirely at a given pCO2. The pCO2 effects on shell morphology, the frequency of abnormalities and mineralization level were investigated on released larvae. Shell length decreased by 6% and shell surface area by 11% at elevated pCO2 (1400 µatm). The percentage of abnormalities was 1.5- to 4-fold higher at 750 µatm and 1400 µatm pCO2, respectively, than at 390 µatm. The intensity of birefringence, used as a proxy for the mineralization level of the larval shell, also decreased with increasing pCO2. These negative results are likely explained by increased intracapsular acidosis due to elevated pCO2 in extracapsular seawater. The encapsulation of C. fornicata embryos did not protect them against the deleterious effects of a predicted pCO2 increase. Nevertheless, C. fornicata larvae seemed less affected than other mollusk species. Further studies are needed to identify the critical points of the life cycle in this species in light of future ocean acidification.

Seasonal variations in maternal provisioning of Crepidula fornicata (Gastropoda): fatty acid composition of females, embryos and larvae.

Recruitment success of marine invertebrate populations not only depends on the number of recruits but also on their quality which affects their survival. In species characterized by a mixed development (encapsulated embryonic development and release of planktotrophic larvae), the offspring quality depends on both maternal provisioning and larval feeding. Here, we investigated potential changes of maternal provisioning over the whole reproductive period in a gastropod with a mixed development: Crepidula fornicata. In its introduction area, C. fornicata reproduces from February to October, which implies that both adults and larvae are exposed to different food availabilities. Maternal provisioning was assessed by measuring the fatty acid (FA) composition of females, encapsulated embryos and larvae, in February, May, July and September 2009. FA are essential resources for the development of embryos and larvae, and are key biomarkers of offspring quality. Our results showed differences in FA composition between muscles, visceral masses, and encapsulated embryos. In particular, FA composition of embryos was similar to that of the visceral mass. Seasonal variations in FA composition were observed: in the middle of the reproductive season (May and July), female tissues and embryos showed a higher proportion of polyunsaturated fatty acids and especially ω3, as compared to the beginning and end of the reproductive season (February and September). This showed that through maternal provisioning the quality of C. fornicata offspring was higher in the middle of the reproductive season. Whether this would result in an increase of recruitment success and juvenile performance would require further investigations.

Inhibitory function of nitric oxide on the onset of metamorphosis in competent larvae of Crepidula fornicata: A transcriptional perspective.

In diverse invertebrate species characterized by a biphasic life cycle, metamorphosis represents a fundamental biological transition which determines the fate of benthic population dynamics through settlement and recruitment. Within this context, nitric oxide (NO) is thought to act as an endogenous inhibitor of metamorphosis. While attention has been focused on the mechanisms of this inhibitory pathway with pharmacological agents and immunohistochemistry tools, relatively few studies have investigated transcriptional process at the origin of NO synthesis. In this paper, we report the isolation of a 218-bp cDNA fragment of an ortholog of the neuronal nitric oxide synthase (nNOS) gene in the invasive marine species Crepidula fornicata. By the use of quantitative real-time PCR, we examine the transcription of this gene throughout larval development and in response to two inducers [K+ excess (20 mM), dibromomethane (DBM) (1000 ppm)] that are known to potentiate metamorphosis in invertebrate species. The level of transcription increased constantly during the larval development, suggesting an increased repressive effect over metamorphosis as larvae aged. Interestingly, maximum values were reached 6 h post-treatment, before declining within 20 h for all the tested conditions. Overall, our results are in agreement with the involvement, at a molecular level, of the NO signalling pathway in metamorphosis. The decrease in nNOS gene transcription post-induction could support the inhibitory effect of NO upon the onset of metamorphosis in competent larvae, although further studies are needed to fully describe the pathways triggered by K+ ions and DBM induction. Furthermore, results indicate that metamorphosis could occur after termination of endogenous inhibition, bringing support to the hypothesis of spontaneous metamorphosis.

Viral gametocytic hypertrophy of Crassostrea gigas in France: from occasional records to disease emergence?

Viral gametocytic hypertrophy was reported for the first time in 2001 in Pacific oyster Crassostrea gigas in France. Since this date, the number of reported cases and the distribution area have increased every year; however, the cases are not associated with macroscopic signs or increased mortality rates. Both male and female gametes were hypertrophied and basophilic inclusions were observed in gamete nuclei. Transmission electron microscopy revealed the presence of viral particles in these intranuclear basophilic inclusions. These particles had characteristics similar to those of the Papillomaviridae and Polyoma viridae families: they were small, non-enveloped, icosahedral, and 44 to 56 nm in diameter. The viral particles were found in male, female and hermaphrodite oysters and no significant difference in viral infection was observed between those groups. The frequency of detection and the intensity of infection were low and no host defence reaction was recognised, suggesting that the viral particles had a weak impact on C. gigas. The viral particles described in the present study seem to be similar to these described in C. virginica in the USA and Canada and in C. gigas in Korea, but further studies are required to confirm their identity. The issue of a possible emergence of this infection is discussed.

First record of the microsporidian parasite Steinhausia mytilovum in Mytilus sp. (Bivalvia: Mytilidae) from France.

Steinhausia mytilovum is a globally distributed microsporidian parasite which infects the oocytes of the blue mussels Mytilus edulis and M. galloprovincialis. Despite the intensive monitoring effort made on mussel populations, the parasite has not previously been reported in France. We report herein on the occurrence of S. mytilovum in Mytilus sp. from 1 cultured and 2 natural populations on the northern coast of France, thus extending the parasite's known distribution northwards. We also report on the observation in 1989 of S. mytilovum in M. galloprovincialis from the Golfe de Fos area in the Mediterranean Sea (South of France). S. mytilovum was observed in the European hybrid zone between M. edulis and M. galloprovincialis, which therefore renders the exact taxonomic status of the infected hosts unknown. The prevalence of the parasite was low, which suggests that its effect on mussel populations was probably limited.

Infection of the cockle Cerastoderma edule in the Baie des Veys (France) by the microsporidian parasite Steinhausia sp.

We report the occurrence of the microsporidian parasite Steinhausia sp. in the oocytes of the common cockle Cerastoderma edule in a natural population in France, where high mortalities occurred. Steinhausia sp. appeared primarily as sporocysts containing many small spores, and putative earlier developmental stages were also observed. Both its prevalence and infection intensity were low, and no host defence reaction was recognized, suggesting that Steinhausia sp. had no detrimental effect on C. edule. Its prevalence was higher in cockles lying on the sediment surface, but the significance of this observation could not be explained given the poor knowledge of the Steinhausia life cycle. The present data did not allow specific identification of the parasite, and further studies are required to determine whether Steinhausia sp. in the cockle is a new species, or a microsporidian infecting multiple host species.