Pediveliger Stage Research Articles

Hedgehog signaling is required for larval muscle development and larval metamorphosis of the mussel Mytilus coruscus

Understanding the developmental processes and signaling pathways involved in larval myogenesis and metamorphosis is crucial for comprehending the life history and adaptive strategies of marine organisms. In this study, we investigated the temporal and spatial patterns of myogenesis in the mussel Mytilus coruscus (Mc), focusing on the emergence and transformation of major muscle groups during different larval stages. We also explored the role of the Hedgehog (Hh) signaling pathway in regulating myogenesis and larval metamorphosis. The results revealed distinct developmental stages characterized by the emergence of specific muscular components, such as velum retractor muscles and anterior adductor muscles, in D-veliger and umbo larvae, which are responsible for the planktonic stage. In the pediveliger stage, posterior ventral, posterior adductor, and foot muscles appeared. After larval metamorphosis, the velum structure and its corresponding retractor muscles degenerate, indicating the transition from planktonic to benthic life. We observed a conserved pattern of larval musculature development and revealed a high degree of conservation across bivalve species, with comparable emergence times during myogenesis. Furthermore, exposure to the Hh signaling inhibitor cyclopamine impaired larval muscle development, reduced larval swimming activity, and inhibited larval metamorphosis in M. coruscus. Cyclopamine-mediated inhibition of Hh signaling led to reduced expression of four key genes within the Hh signaling pathway (McHh, McPtc, McSmo, and McGli) and the striated myosin heavy chain gene (McMHC). It is hypothesised that the abnormal larval muscle development in cyclopamine-treated groups may be an indirect effect due to disrupted McMHC expression. We provide evidence for the first time that cyclopamine treatment inhibited larval metamorphosis in bivalves, highlighting the potential involvement of Hh signaling in mediating larval muscle development and metamorphosis in M. coruscus. The present study provides insights into the dynamic nature of myogenesis and the regulatory role of the Hh signaling pathway during larval development and metamorphosis in M. coruscus. The results obtained in this study contribute to a better understanding of the evolutionary significance of Hh signaling in bivalves and shed light on the mechanisms underlying larval muscle development and metamorphosis in marine invertebrates.

Effects of microalgae and stocking density on growth and survival rate of giant clam (Tridacna squamosa Lamarck, 1819) larvae

The present study aimed to evaluate the impact of microalgae mixture and stocking density on growth and survival rate of giant clam (Tridacna squamosa) at the veliger stage. In the first experiment, giant clam larvae of the D-stage were reared in the conical fiberglass tanks and fed with combinations of microalgae, including Chaetoceros muelleri and Isochrysis galbana (1:1), and C. muelleri, I. galbana and Nannochloropsis oculata, (1:1:1). One group of giant clam larvae without feeding was used as a control. The experiment was lasted for 7 days, until the Pediveliger stage with six replicate tanks. In the second experiment, the larvae (1 day post hatched, DPH) were reared at densities of 3, 5, 7 and 9 larvae mL-1 (so-called D3, D5, D7 and D9, respectively) for a period of 7 days. Each density was randomly assigned to 6 replicate tanks. The larvae were fed once a day with a mixture of C. muelleri, I. galbana and N. oculata, (5,000 cells/ml each type of algae). At 4DPH, extracted zooxanthelle (Symbiodinium microadriaticum) was seeded into the giant clam larvae at the concentration of 5,000cells/ml once a day.The results showed that larvae fed with C. muelleri, I. galbana and N. oculata (1:1:1) obtained the largest shell length (217.5 ± 3.1 µm), specific growth rate (SGR) 7.18 ± 0.34 (mean±SD) and the survival rate (31.5% ± 1.6%) (P<0,05). In addition, rearing densities significantly affected growth in shell length, SGR and survival rate in giant clam larvae. Of which, the larvae at D3 gained the highest shell length, SGR and survival rate. Meanwhile, the larvae at D9 had the lowest shell length, SGR and survival rate. There was no significant difference in larval survival rate between D3 and D5. Therefore, it is suggested that in the hatchery giant clam larvae reared at 5 larvae mL-1 in order to improve productivity.

Exploring the feasibility of cryopreserving larvae of the common cockle (Cerastoderma edule) for hatchery production

The decline of natural populations of the common cockle (Cerastoderma edule) through the European coast is posing a threat to local small-scale fisheries. These declines are primarily attributed to the prevalence of several pathogens and the disseminated neoplasia in cockle populations. The institution of a biobank of cryopreserved larvae could enhance hatchery production and help the restocking. The present work aimed at the development of a cryopreservation protocol for larvae of the common cockle using the mollusk cryopreservation protocols designed in our laboratory. Toxicity bioassays and short-term cryopreservation experiments were performed for protocol optimization according with cellular tolerance. Once settled, the viability of cryopreserved larvae was studied long term. Toxicity tests evidenced high tolerance of larvae against detrimental effects of Cryoprotecting Agents (CPAs). Cryopreservation of 48 h-old D-larva showed a 100% survival when increasing the equilibrium time from 15 to 60 min and using Propylene-Glycol (PG) + 0.4 M Trehalose (TRE) in Filtered Sea Water (FSW) and 60 min of exposure to CPA solution before slow-cooling. However, when cryopreserving the older larvae, the variation in equilibrium times hardly showed any effect but 10% Ethylene-Glycol (EG) + 0.4 M TRE and 60 min of exposure yielded the best relative survivorship (100%). Cryopreservation caused a significant delay on the growth rate of the latest larval stage. However, cryopreserved larvae survived to day 4–6, while 30 ± 12.17% of control larvae developed into pediveliger stage, of which 50% settled and transformed into juvenile cockles. These results demonstrated the role of the cell-type specificity in cryopreservation and highlight the importance of studying potential long-term effects of this tool to ensure the viability of the protocols.

Improving efficiency of methods for hatchery production of the limpet Patella candei (Patellogastropoda: Patellidae)

AbstractPatella candei d'Orbigny, 1840 is a valuable resource from the European Macaronesia. Limpet aquaculture is a recent research field that could contribute to reducing anthropogenic impact and developing appropriate restocking technologies. This study presents the results of 14 assays performed under laboratory culture conditions aimed at: (1) improving the larval production methodologies for limpets through the optimization of the artificial maturation of the oocytes and the sperm density required for fertilization and incubation, (2) validating a larviculture protocol for P. candei, and (3) evaluating rearing density effect on larvae performance until the pediveliger stage. Results obtained in the present study indicate that NH4OH is more efficient than KOH and NaOH to enhance the artificial maturation of the oocytes. The optimal combination was NH4OH at pH 9.0 for a duration of 20–30 min. The optimal sperm density was approximately 105 sperm cells mL−1 as in other limpet species. The larviculture method described hereby demonstrated to be efficient to obtain viable pediveligers, that is, the larval survival was high, and the artificial maturation did not influence negatively the posterior larval development. The rearing density, within a range from 2.5 to 30 larvae mL−1, showed minimal influence on survival and development.

Stage-Specific Transcriptomes of the Mussel Mytilus coruscus Reveals the Developmental Program for the Planktonic to Benthic Transition.

Many marine invertebrate larvae undergo complex morphological and physiological changes during the planktonic-benthic transition (a.k.a. metamorphosis). In this study, transcriptome analysis of different developmental stages was used to uncover the molecular mechanisms underpinning larval settlement and metamorphosis of the mussel, Mytilus coruscus. Analysis of highly upregulated differentially expressed genes (DEGs) at the pediveliger stage revealed enrichment of immune-related genes. The results may indicate that larvae co-opt molecules of the immune system to sense and respond to external chemical cues and neuroendocrine signaling pathways forecast and trigger the response. The upregulation of adhesive protein genes linked to byssal thread secretion indicates the anchoring capacity required for larval settlement arises prior to metamorphosis. The results of gene expression support a role for the immune and neuroendocrine systems in mussel metamorphosis and provide the basis for future studies to disentangle gene networks and the biology of this important lifecycle transformation.

The embryonic and larval development of the long-lived, keystone mussel Modiolus modiolus: Implications for its restoration using conservation aquaculture

Biogenic reefs formed by bivalves are biodiversity hotspots globally endangered due to anthropogenic pressures. In Strangford Lough (Northern Ireland) bottom trawling caused extensive damage to reefs formed by the horse mussel Modiolus modiolus, compromising their conservation status. Full cycle aquaculture has been proposed as a potential restorative strategy whereby hatchery grown horse mussel juveniles can be used to reseed impacted beds, but this approach requires a thorough knowledge of the species biology. For M. modiolus, a long-lived, cold-water mussel, there is no clear consensus on the reproductive strategies and duration of the larval development cycle.This study presents the results of the first successful, full cycle aquaculture hatchery for M. modiolus in Atlantic Europe, from fertilized egg to spat. The use of air exposure was the only successful method to induce spawning in M. modiolus overcoming a known obstacle in the captive breeding of this species. Spawning lasted five to eight days until broodstock mussels were spent. The duration of the larval cycle from fertilized egg to the pediveliger stage was 34 days while juvenile mussels reached a maximum length of 5.5 mm after six months. All larval stages were recorded: trochophore (t0 + 24 h), D-larva (t0 + 36 h), early umbonated veliger (t0 + 20 days), late veliger (t0 + 32 days) and pediveliger with active foot (t0 + 36 days). Although settlement trials using inert substrates were unsuccessful, juvenile spat were recorded six months after fertilization when live adult M. modiolus were introduced in the rearing tanks. Growth was significantly faster when the larvae were fed with the microalga Chaetoceros calcitans, on its own or combined with Tetraselmis suecica.The results of these breeding trials demonstrated that hatchery culture of M. modiolus is possible, with up to 2 million D-larvae obtained just 24 h after successful spawning of hatchery-kept broodstock. High mortalities and poor settlement indicate that further trials need to be undertaken to streamline protocols if captive breeding is to be considered a viable restoration option for the species. In the interim, a combination of strict protection and brood stock supplementation from healthy beds is the recommended restoration strategy.

Growth and survival of the hatchery larvae of pacific oyster &lt;i&gt;Crassostrea gigas&lt;/i&gt; under different concentrations of microalgae and salinity in conditions of southern Primorye

Growth and survival rates for larvae of pacific oyster Crassostrea gigas (Thunberg, 1793) hatched in artificial conditions are estimated for the stages of development from D-veliger to pediveliger. The experiment was conducted in the Mariculture Center located on Popov Island (Peter the Great Bay, Japan Sea) for 2 regimes of feeding and water salinity of 26 and 32 ppt. Microalgae Isochrysis galbana, Chaetoceros muelleri and Phaeodactylum tricornutum cultivated in the Mariculture Center were used as a feed in both regimes, in different concentrations. The maximum concentration was 4-fold higher than the minimum one: daily doses of food were 20 and 5 thousand cells/mL for veligers, 40 and 10 thousand cells/mL for early veliconkhes, and 80 and 20 thousand cells/mL for late veliconkhes, respectively, whereas 80 thousand cells/mL for all larvae in transition to pediveliger stage. Statistically significant difference of the growth rate was found for cases with different food concentration (p &lt; 0.05). The larvae with better feeding had higher growth rate under salinity of both 26 and 32 ppt. Besides, the lowered salinity (26 ppt) had some positive effect for growth in the regime of better feeding. Survival rate of the larvae from D-veliger to pediveliger was high under all regimes of the experiment and was estimated for the minimal diet as 77.4 and 64.7 % under salinity of 26 and 32 ppt, respectively, and for the maximum diet as 81.2 and 80.7 % under salinity of 26 and 32 ppt, respectively. According to the experiment results, deficit of food at early stages of the oyster larval development affects negatively on their growth but does not have significant impact on their survival.

Metabolic Cost of the Immune Response During Early Ontogeny of the Scallop Argopecten purpuratus.

The scallop Argopecten purpuratus is an important resource for Chilean and Peruvian aquaculture. Seed availability from commercial hatcheries is critical due to recurrent massive mortalities associated with bacterial infections, especially during the veliger larval stage. The immune response plays a crucial role in counteracting the effects of such infections, but being energetically costly, it potentially competes with the physiological and morphological changes that occur during early development, which are equally expensive. Consequently, in this study, energy metabolism parameters at the individual and cellular levels, under routine-basal status and after the exposure to the pathogenic strain bacteria (Vibrio splendidus VPAP18), were evaluated during early ontogeny (trochophore, D-veliger, veliger, pediveliger, and early juveniles) of A. purpuratus. The parameters measured were as follows: (1) metabolic demand, determined as oxygen consumption rate and (2) ATP supplying capacity measured by key mitochondrial enzymes activities [citrate synthase (CS), electron transport system (ETS), and ETS/CS ratio, indicative of ATP supplying efficiency], mitochondrial membrane potential (ΔΨm), and mitochondrial density (ρm) using an in vivo image analysis. Data revealed that metabolic demand/capacity varies significantly throughout early development, with trochophores being the most efficient in terms of energy supplying capacity under basal conditions. ATP supplying efficiency decreased linearly with larval development, attaining its lowest level at the pediveliger stage, and increasing markedly in early juveniles. Veliger larvae at basal conditions were inefficient in terms of energy production vs. energy demand (with low ρm, ΔΨm, enzyme activities, and ETS:CS). Post-challenged results suggest that both trochophore and D-veliger would have the necessary energy to support the immune response. However, due to an immature immune system, the immunity of these stages would rely mainly on molecules of parental origin, as suggested by previous studies. On the other hand, post-challenged veliger maintained their metabolic demand but decreased their ATP supplying capacity, whereas pediveliger increased CS activity. Overall, results suggest that veliger larvae exhibit the lowest metabolic capacity to overcome a bacterial challenge, coinciding with previous works, showing a reduced capacity to express immune-related genes. This would result in a higher susceptibility to pathogen infection, potentially explaining the higher mortality rates occurring during A. purpuratus farming.

Dietary Exposure of Pacific Oyster (Crassostrea gigas) Larvae to Compromised Microalgae Results in Impaired Fitness and Microbiome Shift.

The Pacific oyster Crassostrea gigas is the world’s most cultivated oyster and seed supply is heavily reliant on hatchery production where recurring mass mortality events are a major constraint. Outbreaks of bacterial infection via microalgal feed are frequently implicated in these mortalities. This study assessed the effects of feeding compromised microalgae to developing oyster larvae. Intentionally ‘stressed’ (high pH) or non-stressed microalgae were fed to 11 day-old oyster larvae at two feeding rations for 96 h, followed by a recovery period. Biological endpoints of larval performance were measured following the 96 h exposure and subsequent recovery. Bacterial communities associated with the microalgae feed, rearing seawater, and the oyster larvae, were characterized and correlated with effects on oyster fitness parameters. Feeding stressed algae to oyster larvae for 96 h increased the occurrence of deformities (>70% vs. 20% in control), reduced feeding and swimming ability, and slowed development. Following the recovery period, fewer larvae reached pediveliger stage (2.7% vs. 36% in control) and became spat (1.5% vs. 6.6% in control). The quantity of stressed algae supplied to oyster larvae also influenced overall larval performance, with high feeding rations generally causing greater impairment than low rations. Bacterial profiling using 16S rRNA showed that most bacterial families characterized in larval tissue were also present in larval rearing seawater and in the microalgae feed (98%). The rearing seawater showed the highest bacterial richness compared to the larval and the microalgal compartments, regardless of feeding regime. In larval tissue, bacterial richness was highest in stressed and high-feed treatments, and negatively correlated with larval fitness parameters. These results suggest significant dysbiosis induced by compromised feed and/or increased feed ration. Several bacterial genera (e.g., Halomonas, Marinomonas) were strongly associated with impaired larval performance while the presence of genera in larvae including Vibrio was closely associated with overfeeding. Our research demonstrated that metabarcoding can be effectively used to identify microbiota features associated with larval fitness.

Modelling early growth of the pearl oyster Pteria sterna under pilot‐commercial production

The pearl oyster Pteria sterna produces pearls from animals gathered from the wild but spat collection is increasingly unreliable. Therefore, hatchery support is required to supply spat for aquaculture operations. In this study is described the hatchery processes to produce spat of P. sterna in a pilot-commercial grade. Growth and survival (relative and absolute) are described for the different developmental stages and critical aspects identified. Growth (height and length) was analysed using depensatory hypothesis and the multi-model inference (MMI). Spat production cycle is described (egg to spat in size and time); spat ≥ 3 mm = 70 days. Average absolute growth rate for shell height was 7.8 µm/day during embryonic phase, 14.5 µm/day during metamorphosis and 71.4 µm/day for post-larvae phase. Survival was 75% after 48 hr (D-larvae stage), 25% at day 14 (umbo stage) and 12% at day 28 (pediveliger stage). Metamorphosis was the main critical phase since survival drop to 0.32%. From 32.5 million eggs, 80,000 commercial spat were produced. Early growth in P. sterna showed depensatory pattern since coefficient of variation in height data increased (2%–28%), and this was supported by MMI. The hatchery procedures presented here guarantee the production of spat for aquaculture activities. Growth depensation leads to the application of a high performance function to analyse individual size-at-age variability and to parametrize the models without sub underestimation of values. The best fitted model to describe early growth in P. sterna is the Schnute model case 1.

Improving the quality of tridacnid clam juveniles through crossbreeding broodstock from different zones across the Spermonde Archipelago

Tridacnid clam populations have declined, and they are considered as endangered. There is increasing concern over their status in the Spermonde archipelago, South Sulawesi, where some species can no longer be found, including larger species such as Tridacna gigas and T. derasa as well as the shallow-water clam Hippopus hippopus. This research aimed to produce high-quality juvenile tridacnid clams by crossbreeding distantly related broodstock from different zones as a contribution to the effective conservation of endangered species. The research was conducted in the Spermonde archipelago and at the Hasanuddin University Marine Station Hatchery in Barrang Lompo Island. Broodstock of T derasa and T. squamosa were collected from zones III and IV of the Spermonde Archipelago. Juvenile clam production followed established methods for inducing spawning and larval rearing through trocophore, veliger, and pediveliger stages to produce juveniles that were reared in land-based and ocean nurseries. Parameters measured were the juvenile survival rate and the quality of zooxanthellae (density, chlorophyll content, and mitotic index). Data were analyzed descriptively. The mean density of zooxanthellae in juvenile clams was 3.17 x 106 cells/ind (range 3.15x106 - 5.27x106 cells/in). The mean chlorophyll-a content of these zooxanthellae was 2.7 mg/m3 (range 1.1 mg/m3 - 4.1 mg/m3), which is consistent with the mean phaeopigment concentration of 5.5 mg/m3 (range 4.2 mg/m3 - 6.2 mg/m). The mitotic index peaked at 8.5% between 09:00 and 12:00.

Effects of Water Quality, Stocking Density, Water Exchange Frequency, and Food, on Growth and Survival of the Green Mussel, Perna viridis Larvae

Experiments were conducted to determine the optimum conditions for growth and survival of the green mussel, Perna viridis larvae. Effects of various temperatures and salinities, stocking densities, water exchange frequency, and natural food preference of the larvae from D-hinged to pediveliger stage were investigated. The green mussel broodstocks were collected from the natural source, and spawning occurred in captivity. All experiments were conducted in triplicate. The results demonstrated that larvae from D-hinged to pediveliger stage had better growth and survival when the temperature was between 29ºC and 30ºC, and salinity ranging from 30-33ppt. Stocking density from 10-20 larvae/ml did not affect growth and survival of the larvae. Likewise, frequency of water replacement from daily to every 5 day interval did not influence the growth and survival until the pediveliger stage. During this stage, the larvae preferred a combination of Isochrysis galbana and Chaetoceros calcitrans as food in terms of better growth, and either Isochrysis galbana, Chaetoceros calcitrans, or their combination in terms of higher survival rate.

Development stage of cryopreserved mussel (Perna canaliculus) larvae influences post-thaw impact on shell formation, organogenesis, neurogenesis, feeding ability and survival

Cryopreservation of genetic material from farmed aquatic species is a valuable technique to advance selective breeding programs for stock improvement. In this study, effects of cryopreservation on development of trochophore and D-stage larvae of Greenshell™ mussel (Perna canaliculus) were evaluated through histology, light microscopy, scanning electron microscopy, and confocal microscopy. Larvae of both life stages were motile immediately post-thawing, but survival declined rapidly from 4 days post-fertilisation (dpf). At 18 dpf, ~23% of non-cryopreserved control larvae had progressed to the pediveliger stage, while <1% of cryopreserved larvae had survived. Control larvae grew faster and larger, and consumed more food than larvae cryopreserved at either life stage (trochophore or D-stage). Settlement competency was achieved in the control larvae at 21 days post-fertilization, with most remaining individuals developing eye spots. Organogenesis was delayed in all cryopreserved larvae, and eyespots did not appear at all. Neurogenesis was stunted in cryopreserved trochophore larvae but seemed to progress almost normally in their cryopreserved D-stage counterparts. Developing abnormalities in shell morphology rapidly became apparent in all mussels post-thaw, with trochophore larvae being most highly afflicted. These delays in organogenesis and overall development are indicative of cryo-injuries sustained at a cellular level. Our results show that D-stage larvae are somewhat more resilient to cryopreservation than trochophore larvae. D-larvae are good life-stage candidates for cryobanking genetic resources in this species because there is generally an excess of larvae from selective breeding family crosses and these can be banked and stored for later use. Further on-going research aims to improve the long-term viability of cryopreserved D-larvae for successful rearing.

Novel Insights into the Role of Copper in Critical Life Stages of Oysters Revealed by High-Resolution NanoSIMS Imaging.

Copper (Cu) is a key trace element for many biological processes, but there is little available information regarding its requirements and functions in critical life stages of marine bivalves, during which dramatic morphological and physiological changes occur. In this study, the ontogeny pattern of Cu accumulation across the life history of oysters was explored for the first time and the distributions of Cu in oysters at critical life stages (pediveliger and early settled spat) were in situ mapped by nanoscale secondary ion mass spectrometry (NanoSIMS) with high lateral resolution. We first demonstrated that the late pelagic stage to early settled stage was the critical stage requiring Cu during oyster development. NanoSIMS imaging revealed a significant elevation of intercellular Cu levels along with mitochondrial calcium overload and obvious structural degradation of mitochondria in velar cells at the pediveliger stage, implying the possible role of Cu in cell apoptosis of the velum during larval metamorphosis. Furthermore, an obvious enrichment of Cu together with calcium was observed in the nucleus and mitochondria of ciliated cells of gill tissue at the early settled stage. Their accumulation in the gill cells was significantly higher than that in the juveniles, indicating the potential role of Cu in sustaining the fast growth of the gill filament and the concomitant acceleration of energy metabolism during early benthic development. Our findings offer new insights into the understanding of the interactions between trace metals and marine bivalves.

Ingestion, digestion, growth and survival of green mussel Perna viridis pediveliger larvae fed different microalgae

Nutrition plays a critical role in the growth and development of green mussel Perna viridis larvae. In this study, three species of tropical microalgae were investigated to determine their efficacy as feed source for the pediveliger stage. Larvae (2 larvae ml−1) were given Isochrysis galbana (Iso), Chaetoceros calcitrans (Cc), and Tetraselmis tetrahele (Tt) at 100,000 cells ml−1. No statistically significant difference in survival was found among treatments. The Cc diet promoted maximum growth in terms of final shell height and shell length, as well as instantaneous growth rate. Filtration rates were high, but were not significantly different. A significantly higher ingestion rate was observed in Cc-fed compared with Iso- and Tt-fed larvae. Moreover, all diets examined were readily ingested and digested by the larvae, with faster cell digestion in Cc-fed larvae based on epifluorescence microscopy. This study demonstrated that C. calcitrans, I. galbana, and T. tetrahele were all acceptable and digestible for the animals, but in terms of growth parameters and filtration and ingestion rates, C. calcitrans performed best. Information on food digestibility and feeding habits of green mussel will provide us with a better understanding of the appropriate food to be given that will suit their feeding biology and improve their production in captivity.

In Silico Analysis of Pacific Oyster (Crassostrea gigas) Transcriptome over Developmental Stages Reveals Candidate Genes for Larval Settlement.

Following their planktonic phase, the larvae of benthic marine organisms must locate a suitable habitat to settle and metamorphose. For oysters, larval adhesion occurs at the pediveliger stage with the secretion of a proteinaceous bioadhesive produced by the foot, a specialized and ephemeral organ. Oyster bioadhesive is highly resistant to proteomic extraction and is only produced in very low quantities, which explains why it has been very little examined in larvae to date. In silico analysis of nucleic acid databases could help to identify genes of interest implicated in settlement. In this work, the publicly available transcriptome of Pacific oyster Crassostrea gigas over its developmental stages was mined to select genes highly expressed at the pediveliger stage. Our analysis revealed 59 sequences potentially implicated in adhesion of C. gigas larvae. Some related proteins contain conserved domains already described in other bioadhesives. We propose a hypothetic composition of C. gigas bioadhesive in which the protein constituent is probably composed of collagen and the von Willebrand Factor domain could play a role in adhesive cohesion. Genes coding for enzymes implicated in DOPA chemistry were also detected, indicating that this modification is also potentially present in the adhesive of pediveliger larvae.

Embryonic and Larval Development of the Subtropical Geoduck Clam Panopea globosa (Bivalvia: Hiatellidae)

The embryonic and larval developmental stages of the geoduck clam Panopea globosa were described, and the size at different ages post-fertilization were evaluated using both shell length (SL) and shell height (SH). Gametes were obtained from wild ripe broodstock and fertilized under controlled conditions (22.15°C, 35‰, pH8.4). Fertilization and meiosis were confirmed by single membrane formation (after ∼5–10 min) and extrusion of the first polar body (after ∼15–20 min), respectively. Trochophore larvae (94.80 ± 0.74 µm SL and 82.92 ± 4.69 µm SH) were observed 17.5 h after fertilization. Veliger larvae (146.36 ± 17.54 µm SL and 121.78 ± 15.96 µm SH) were observed 43 h after fertilization. Umbo growth was apparent 7 days after fertilization, and pediveliger larvae (355.36 ± 20.99 µm SL and 313.64 ± 21.24 µm SH) developed 21 days after fertilization. The growth of SH and SL were best described by a linear function of time (T = days) from fertilization to the pediveliger stage (SL = 12.04 T + 81.40 and SH = 10.69 T + 67.33). The linear relationship between SL and SH indicates that the rate of SH growth during larval development of P. globosa is smaller than that of SL (SH = 0.875, SL = -2.20). The results demonstrate that highly uniform larvae sizes can be obtained from spawning runs in controlled environmental conditions. This is the first description of the development of P. globosa from the embryonic stage to early juvenile stages.

Proteinaceous secretion of bioadhesive produced during crawling and settlement of Crassostrea gigas larvae

Bioadhesion of marine organisms has been intensively studied over the last decade because of their ability to attach in various wet environmental conditions and the potential this offers for biotechnology applications. Many marine mollusc species are characterized by a two-phase life history: pelagic larvae settle prior to metamorphosis to a benthic stage. The oyster Crassostrea gigas has been extensively studied for its economic and ecological importance. However, the bioadhesive produced by ready to settle larvae of this species has been little studied. The pediveliger stage of oysters is characterized by the genesis of a specific organ essential for adhesion, the foot. Our scanning electron microscopy and histology analysis revealed that in C. gigas the adhesive is produced by several foot glands. This adhesive is composed of numerous fibres of differing structure, suggesting differences in chemical composition and function. Fourier transformed infrared spectroscopy indicated a mainly proteinaceous composition. Proteomic analysis of footprints was able to identify 42 proteins, among which, one uncharacterized protein was selected on the basis of its pediveliger transcriptome specificity and then located by mRNA in situ hybridization, revealing its potential role during substrate exploration before oyster larva settlement.

Developmental dynamics of myogenesis in Pacific oyster Crassostrea gigas.

Pacific oyster (Crassostrea gigas) is a sessile bivalve living in the intertidal zone. It has become an attractive model for developmental studies due to its metamorphic transition from a mobile planktonic larvae to a sessile adults. To determine the effect of metamorphosis on muscle development in oyster larvae, we characterized myogenesis during larval development and metamorphosis by phalloidin staining which labels filamentous actin filaments. Our data revealed a dynamic pattern of myogenesis during embryonic and larval development. It appears that simple "U-shaped" muscle ring first developed at the trochophore stage. This was followed by a more complex musculature including an anterior adductor, velum ventral retractors at the veliger stage, and the addition of posterior adductors and foot retractors at the veliger and pediveliger stages. During metamorphosis, muscle structures in the anterior adductor, velum retractors and ventral retractors were degenerated. At the same time, mantle and gill musculature appeared and became the primary muscle system in juveniles together with the posterior adductor. In addition, indirect immunofluorescence with the monoclonal antibody against C. gigas muscle proteins (myosin heavy chains (MYHC) and α-actinin) were used to monitor changes in the developing musculature at different larval stages. The immunofluorescence staining results of muscle proteins were consistent with phalloidin staining. The expression locations of two muscle proteins were similar and mainly located in larval velum retractor and adductor muscle. The α-actinin expression positions were located in Z-lined of velum striated retractors. Data from these studies provide a comprehensive description of myogenesis in C. gigas embryos and larvae. Moreover, our data showed that metamorphosis has a significant impact on remolding the musculature after transition from a mobile planktonic larvae to a sessile mollusk, associated with certain muscle group degradation.

NMR Profiling of Metabolites in Larval and Juvenile Blue Mussels (Mytilus edulis) under Ambient and Low Salinity Conditions

Blue mussels (Mytilus edulis) are ecologically and economically important marine invertebrates whose populations are at risk from climate change-associated variation in their environment, such as decreased coastal salinity. Blue mussels are osmoconfomers and use components of the metabolome (free amino acids) to help maintain osmotic balance and cellular function during low salinity exposure. However, little is known about the capacity of blue mussels during the planktonic larval stages to regulate metabolites during osmotic stress. Metabolite studies in species such as blue mussels can help improve our understanding of the species’ physiology, as well as their capacity to respond to environmental stress. We used 1D 1H nuclear magnetic resonance (NMR) and 2D total correlation spectroscopy (TOCSY) experiments to describe baseline metabolite pools in larval (veliger and pediveliger stages) and juvenile blue mussels (gill, mantle, and adductor tissues) under ambient conditions and to quantify changes in the abundance of common osmolytes in these stages during low salinity exposure. We found evidence for stage- and tissue-specific differences in the baseline metabolic profiles of blue mussels, which reflect variation in the function and morphology of each larval stage or tissue type of juveniles. These differences impacted the utilization of osmolytes during low salinity exposure, likely stemming from innate physiological variation. This study highlights the importance of foundational metabolomic studies that include multiple tissue types and developmental stages to adequately evaluate organismal responses to stress and better place these findings in a broader physiological context.