Juvenile Clams Research Articles

Toxicity of Nitrite to Juvenile Sunray Surf Clam (Mactra chinensis Philippi)

Nitrite is a common pollutant in marine environments and can cause mortality in crustaceans and bivalves. The purpose of the current study is to understand nitrate’s toxicity to juvenile clams due to its potential impact on aquaculture and marine ecosystems. Juvenile sunray surf clams (Mactra chinensis Philippi) (1.00 ± 0.10 cm shell length, 0.75 ± 0.04 cm shell height) were exposed to varying concentrations of nitrite for 96 h and 20 days, respectively. The LC50 for survival at 96 h was 37 mg/L NO2-N. Histological evaluations were made on juvenile clams exposed at 30 mg/L after 20 d of exposure. Epithelial cells and digestive diverticulum are the best sub-lethal effect indicators. Shell length and antioxidant enzyme activities were measured at the beginning of the experiment and then observed 10 and 20 days after exposure. A logarithmic relationship was obtained between the relative growth rate (based on the shell length) of juvenile M. chinensis and the nitrite concentration. Compared to the control, activity suppression of superoxide dismutase and catalase activity was detected from the concentration of 1 mg/L NO2-N. It is recommended that nitrite concentrations remain below 1 mg/L to prevent stress during the early developmental stages of clams.

Ocean acidification will not affect the shell strength of juveniles of the commercial clam species Chamelea gallina: Implications of the local alkalinization of seawater

Ocean acidification (OA) is expected to decrease the strength of bivalves’ shells, especially during the early stages of development, with negative consequences to the resilience of natural populations and the economy. The objectives of the present study were to assess the long-term effect of increasing pCO2 after 217 days of exposure under controlled conditions of pH of ∼8.2, 8.0, and 7.7 on the strength and integrity of shells of juveniles of the commercial striped venus clam Chamelea gallina. Shell strength was estimated through compression tests and integrity through scanning electron microscopy (SEM) and dispersive X-ray analyses (EDX). The results showed that under increasing pCO2 the shell strength of juveniles is unaffected, which could be related to the locally elevated total alkalinity of seawater with respect to other parts of the coastal lagoon. However, despite this, it was also observed that the juvenile clams exposed to elevated pCO2 decreased their shell thickness and increased the porosity of their prismatic layer. Under future OA conditions, these changes could eventually compromise the integrity of the shells, becoming more vulnerable to the attack of predators and breakable during fishing operations. Future studies should address the plasticity of the organisms and the effect of the alkalinization of seawater on the resilience of shellfish juveniles under global change conditions.

Predator-prey interactions in a coastal setting: Linking crab feeding rates to small scale distribution of clams

A coastal predator-prey system, juvenile green crabs (Carcinus maenas) preying upon juvenile hard clams (Mercenaria mercenaria), was used to explore the link between crab predation rates and clam density and small-scale distribution patterns. The channel working area of a racetrack flume was adapted to form a sedimentary arena in a flowing seawater system (5 cm s−1) to assess crab predation rates in relation to clam density and distribution patterns (clams clustered in one patch vs two nearby vs two farther apart). The trials detected significant differences in relation to clam initial density and distribution with strong (∼50%) declines in clam mortality levels among spatial arrangements (one patch > two nearby > two farther apart). Feeding of clams was associated with the time taken by crabs to handle the first clam (first patch), and the frequency of three distinct types of crab behavior (eating, resting, and searching). Altogether these results suggest that small-scale changes in number and distribution of juvenile clams matter and may have unexpectedly strong effects on the outcome of predator-prey interactions.

The stress response of the seagrass Zostera noltei and three commercial clam species to low salinity associated with heavy rainfall

Abstract The maintenance of stocks of estuarine species strongly depends on the ability of the species to cope with environmental stress. In NW Spain, commercial clam beds, which are usually co-occupied by the seagrass Zostera noltei, are often exposed to reduced salinity caused by intense rainfalls. Our goals were to evaluate the effects of low salinity events on both juvenile clams and Z. noltei, including their interactions. A mesocosm experiment was performed to simulate three salinity decreases (35–35, 25–10, and 20–5), and several indicators of clam and seagrass performance were measured after 3 and 6 days of exposure and again after a recovery period of 4 days. No differences were observed in the non-native clam Ruditapes philippinarum, while oxygen consumption, clearance rate and growth decreased significantly in the native clams R. decussatus and Venerupis corrugata in response to low salinity stress. Zostera noltei indicators did not vary in response to low salinity exposure, except the sucrose content, which decreased. Moreover, the seagrass buffered juvenile clams from salinity fluctuations in the short term, although the interactions were weak. The species-specific sensitivity to low salinity should be a major concern in future management plans for the shellfish beds in the context of climate change.

Expression of bone morphogenetic protein 10 and its role in biomineralization in Hyriopsis cumingii

Shells and pearls are the products of biomineralization of shellfish after ingesting external mineral ions. Bone morphogenetic proteins (BMPs) play a role in a variety of biological function, and the genes that encode them, are considered important shell-forming genes in mollusks and are associated with shell and pearl formation, embryonic development, and other functions, but bone morphogenetic protein 10 (BMP10) is poorly understood in Hyriopsis cumingii. In this study, we cloned Hc-BMP10 and obtained a 2477 bp full-length sequence encoding 460 amino acids with a conserved TGF-β structural domain. During the embryonic developmental stages, the cleavage stage had the highest expression of Hc-BMP10, followed by juvenile clams; the expression in the mantle gradually decreased with increasing mussel age. A strong signal was detected on epidermal cells on the mantle edge by in situ hybridization. In both the shell notching and inserting operations of the pearl fragment assay, we found that the expression of Hc-BMP10 increased after the above treatments. RNA interference assays showed that the silencing of Hc-BMP10 resulted in a change in the morphology of the prismatic layer and nacreous layer, with the prismatic layer less closely aligned and the disordered aragonite flakes in the nacreous layer. These findings indicate that Hc-BMP10 is involved in the growth and development of H. cumingii, as well as the formation of shells and pearls. Therefore, this study provides some reference for selecting superior species for growth and pearl breeding of H. cumingii at a molecular level and further investigation of the molecular mechanism for biomineralization of Hc-BMP10.

Effects of dietary carbohydrate/lipid ratios on growth, body composition, amylase activity, oxidative status, and mTOR/autophagy pathway in juvenile clam, Sinonovacula constricta

The nutritional requirements and metabolism of bivalves remain largely unknown. In this study, we investigated the effects of dietary carbohydrate/lipid ratios on growth performance, body composition, digestive enzyme activities, oxidative status, and transcriptome profile in juvenile razor clam Sinonovacula constricta. Five microcapsule diets with varying levels of carbohydrate (30–45%) and lipid (2–15%) were prepared and designated as C45L2 (45% carbohydrate and 2% lipid), C41L5, C36L8, C33L11, and C30L15 groups. Juvenile S. constricta with an initial shell length of 4.9 mm were fed with these microcapsule diets for 2 weeks. The results showed that the clam in the C33L11 group exhibited the fastest growth rate. The body protein content decreased with the decreasing dietary carbohydrate/lipid ratio. The clam in the C33L11 group showed significantly higher glycogen content compared to the C45L2, C41L5, or C30L5 group. The amylase activity of the clam in the C45L2 and C41L5 group was significantly higher than that in the C36L8, C33L11, and C30L15 group. The clam in the C33L11 group showed significantly higher catalase and superoxide dismutase activities compared to other microcapsule groups. The malondialdehyde level of the clam in the C30L15 group was significantly higher than that in the C45L2, C41L5, and C33L11 group. Transcriptome analysis showed that the autophagy pathway in the clam fed C41L5 was upregulated compared with those fed C33L11. Meanwhile, the protein levels of microtubule-associated protein 1 A/1B-light chain 3 membrane type (LC3-II) in the C41L5 group were also significantly higher than those in the C33L11 group. Moreover, the clam in the C41L5 group exhibited increased phosphorylation levels of the eukaryotic translation initiation factor 4E-binding protein (4E-BP) and decreased phosphorylation levels of the ribosomal protein S6 kinase beta-1 (S6K). Our results revealed that the optimal dietary carbohydrate/lipid ratio for juvenile S. constricta was approximately 3.01. The high-carbohydrate/low-lipid diet may repress the growth of clam by inhibiting mammalian target of rapamycin complex 1 (mTORC1) and subsequently activating autophagy, while the poor growth performance in the low-carbohydrate/high-lipid diet group may be associated with high lipid peroxidation levels in juvenile S. constricta.

Exploring the newly emerging effects of native seagrasses on survival and growth of non-native juvenile clams

The Manila clam (Ruditapes philippinarum) has been introduced into the Atlantic European coast for commercial exploitation. In this region, the population dynamics of this clam species may be determined by the interaction with the native dwarf eelgrass (Zostera noltei). We performed an experiment in the tidal flats of an estuary located in northern Spain to analyse the interaction between Z. noltei and juvenile Manila clams. Based on existing knowledge, it was hypothesised that seagrasses act as a nursery protecting juveniles from predation, which is an important cause of natural mortality, and that seagrass density influences the survival, growth and condition of clams. The results indicate that the effects of seagrasses on juvenile clams depends on shoot density, which mainly determine the relative prevalence of positive versus negative interactions. Dense seagrass meadows protect juvenile clams from predation, likely decreasing their visibility and reducing the efficiency of predators. However, in these dense meadows, a decline in the condition of clams was also observed, although not in shell growth. This decline can be due to the fact that food supply in dense meadows is slow and does not fulfil the replenishment rates required by filter-feeding organisms or because competition for space with the seagrass causes stress. Clams are larger in populations where predator access is prevented, possibly indicating selective predation on larger organisms when this interaction is allowed. The knowledge gained from this experiment is of great interest for analysing possible future trends in clam populations and the derived social implications given its high economic value.

Characterization of Vibrio mediterranei Isolates as Causative Agents of Vibriosis in Marine Bivalves.

Marine bivalves include species important globally for aquaculture and estuary ecology. However, epizootics of variable etiologies often pose a threat to the marine fishery industry and ecosystem by causing significant mortalities in related species. One of such diseases is larval vibriosis caused by bacteria of the genus Vibrio, which frequently occurs and causes mass mortalities in bivalve larvae and juveniles in hatcheries. During a mass mortality of razor clam, Sinonovacula constricta, juveniles in a shellfish hatchery in 2019, Vibrio mediterranei was identified as a dominant bacterial species in diseased animals and their rearing water. In this study, we selected and characterized 11 V. mediterranei isolates and studied their pathogenicity to the larvae and juveniles of S. constricta and Crossostrea sikamea. We found that V. mediterranei isolates showed various degrees of pathogenicity to the experimental animals by immersion. Injection of the extracellular products (ECPs) of the strains into clam juveniles resulted in similar pathogenicity with strain immersion. Furthermore, the measurements of enzyme activity suggested the existence of virulence factors in the ECPs of disease-causing V. mediterranei strains. Additionally, proteomic analysis revealed that more than 700 differentially expressed proteins were detected in the ECPs among V. mediterranei strains with different levels of virulence, and the higher expressed proteins in the ECPs of highly virulent strains were involved mainly in the virulence-related pathways. This research represented the first characterization of the V. mediterranei strains as causative agents for larval bivalve vibriosis. The mechanisms underlying the pathogenicity and related strain variability are under further study. IMPORTANCE In the marine environment, Vibrio members have a significant impact on aquatic organisms. Larval vibriosis, caused by bacteria of the genus Vibrio, often poses a threat to the marine fishery industry and ecosystem by causing the mortality of bivalves. However, the emerging pathogens of larval vibriosis in bivalves have not been explored fully. Vibrio mediterranei, the dominant bacterium isolated from moribund clam juveniles in a mortality event, may be responsible for the massive mortality of bivalve juveniles and vibriosis occurrence. Thus, it is necessary to study the pathogenic mechanisms of V. mediterranei to bivalve larvae. We found that V. mediterranei was the pathogen of larval bivalve vibriosis, and its extracellular products contributed a critical role for virulence in juveniles. This research is the first report of V. mediterranei as a causative agent for vibriosis in bivalve juveniles. Our results provide valuable information for understanding the pathogenic mechanism of V. mediterranei to bivalve larvae.

Effects of Supplementation Substances on Survival Rate of Lutraria Rhynchaena During Seed Transportation

This study was conducted to evaluate the effects of Chaetoceros algae and other substances (glucose, active char coal and Oxygen balls) on the survival rate of otter clam Lutraria rhynchaena during transportation and after 20 stocking days. Juvenile clams were packed in nylon bags (2 L of seawater 30‰) with the density of 300 ind./L and preserved during 24 hours at stable temperature of 25oC. Results showed that total ammonium concentration increased abruptly after 24 hours in the treatments without supplements. Adding glucose and Oxygen tablets resulted in higher algal clearance rate of otter clams (70.4 – 75.0%), these levels were significantly higher than those from others (p<0.05). Survival rates of otter clams in active charcoal (83.3%) or Oxygen tablets (83.3%) were significantly higher than in only algae (74.4%) or glucose (71.7%). Although there was not significant difference but after 20 days of rearing otter clams which were preserved with glucose showing the higher results in survival rate (78.9%) and total weight (0.23g). Our findings indicated the ability to supply Chaetoceros algae together either glucose, active charcoal or Oxygen tablets during long transportation to improve the survival rate of otter clams.

Growth, survival and fatty acid profile of hard clam, Mercenaria mercenaria, juveniles fed live microalgae diets

AbstractChoice of microalgae diet affects production outcomes in bivalve hatcheries. This study aimed to identify an optimal microalgae diet to improve hard clam, Mercenaria mercenaria, juvenile production. Four microalgae species (Tisochrysis lutea, Pavlova lutheri, Chaetoceros gracilis and Cyclotella nana) in six dietary combinations were fed for 6 weeks; then, growth, survival and fatty acid profiles of juveniles were evaluated. The highest numerical growth and survival of juveniles were attained by feeding a tetra‐species microalgae diet, but no statistical difference was found by feeding bi‐ or tri‐species diets that consisted of a mix of flagellate and diatom microalgae. Significant differences in fatty acid profiles were found among clams fed different microalgae diets. A contrasting polyunsaturated fatty acid (PUFA) profile was found in juveniles fed flagellate‐ or diatom‐only diets. Clams fed flagellate‐only diets contained a higher percentage of docosahexaenoic acid (DHA) but a lower percentage of (n‐6) PUFAs, whereas those fed diatom‐only diets had a higher percentage of arachidonic acid (ARA) and eicosapentaenoic acid (EPA) but a lower percentage of DHA. In contrast, the percentage of EPA, DHA and ARA was neither very high nor very low in juveniles fed the tetra‐species diet. The complementary nutritional profile of the tetra‐species microalgae diet resulted in enhanced production and exhibited a more balanced fatty acid profile in clam tissue. The tetra‐species microalgae diet should be a preferable choice for feeding hard clam juveniles, but the cost of producing multiple microalgae species for hatchery/nursery operations should be taken into consideration as bi‐ and tri‐species diets provided statistically similar growth and survival. Results of this study can be used to improve productivity and profitability in M. mercenaria hatcheries.

Acidification and high-temperature impacts on energetics and shell production of the edible clam Ameghinomya antiqua

Warming and ocean acidification are currently critical global change drivers for marine ecosystems due to their complex and irreversible effects on the ecology and evolution of marine communities. Changes in the chemistry and the temperature of the ocean impact the biological performance of marine resources by affecting their energy budget and thus imposing energetic restrictions and trade-offs on their survival, growth, and reproduction. In this study, we evaluated the interplaying effects of increased pCO2 levels and temperature on the economically relevant clam Ameghinomya antiqua, an infaunal bivalve inhabiting a wide distributional range along the coast of Chile. Juvenile clams collected from southern Chile were exposed to a 90-day experimental set-up emulating the current and a future scenario projeced to the end of the current century for both high pCO2/low-pH and temperature (10 and 15°C) projected for the Chilean coast. Clams showed physiological plasticity to different projected environmental scenarios without mortality. In addition, our results showed that the specimens under low-pH conditions were not able to meet the energetic requirements when increased temperature imposed high maintenance costs, consequently showing metabolic depression. Indeed, although the calcification rate was negative in the high-pCO2 scenario, it was the temperature that determined the amount of shell loss. These results indicate that the studied clam can face environmental changes for short-term periods modifying energetic allocation on maintenance and growth processes, but with possible long-term population costs, endangering the sustainability of an important benthic artisanal fisheries resource.

Acclimatory gene expression of primed clams enhances robustness to elevated pCO2.

Sublethal exposure to environmental challenges may enhance ability to cope with chronic or repeated change, a process known as priming. In a previous study, pre-exposure to seawater enriched with pCO2 improved growth and reduced antioxidant capacity of juvenile Pacific geoduck Panopea generosa clams, suggesting that transcriptional shifts may drive phenotypic modifications post-priming. To this end, juvenile clams were sampled and TagSeq gene expression data were analysed after (i) a 110-day acclimation under ambient (921 μatm, naïve) and moderately elevated pCO2 (2870 μatm, pre-exposed); then following (ii) a second 7-day exposure to three pCO2 treatments (ambient: 754 μatm; moderately elevated: 2750μatm; severely elevated: 4940 μatm), a 7-day return to ambient pCO2 and a third 7-day exposure to two pCO2 treatments (ambient: 967 μatm; moderately elevated: 3030 μatm). Pre-exposed geoducks frontloaded genes for stress and apoptosis/innate immune response, homeostatic processes, protein degradation and transcriptional modifiers. Pre-exposed geoducks were also responsive to subsequent encounters, with gene sets enriched for mitochondrial recycling and immune defence under elevated pCO2 and energy metabolism and biosynthesis under ambient recovery. In contrast, gene sets with higher expression in naïve clams were enriched for fatty-acid degradation and glutathione components, suggesting naïve clams could be depleting endogenous fuels, with unsustainable energetic requirements if changes in carbonate chemistry persist. Collectively, our transcriptomic data indicate that pCO2 priming during post-larval periods could, via gene expression regulation, enhance robustness in bivalves to environmental change. Such priming approaches may be beneficial for aquaculture, as seafood demand intensifies concurrent with increasing climate change in marine systems.

Deep-sea infauna with calcified exoskeletons imaged in situ using a new 3D acoustic coring system (A-core-2000)

The deep ocean is Earth’s largest habitable space inhabited by diverse benthic organisms. Infauna play crucial roles in shaping sedimentary structures, relocating organic matter, porewater chemistry, and hence biogeochemical cycles. However, the visualization and quantification of infauna in situ inside deep-sea sediment has been challenging, due to their sparse distribution and that deep-sea cameras do not visualize animals living below the sediment surface. Here, we newly developed a 3D acoustic “coring” system and applied it to visualize and detect burrowing bivalves in deep-sea sediments. The in situ acoustic observation was conducted at a dense colony of vesicomyid clams in a hydrocarbon seep in Sagami Bay, Japan, focusing on a patch of juvenile clams with a completely infaunal life style. We clearly observed strong backscatters from the top and lower edges of animals in our 3D acoustic data. At least 17 reflectors were identified in the survey area (625 cm2), interpreted to correspond to living clams. The estimated depths of the lower edge of clams ranged between 41 and 98 mm. The acoustic system presented here is effective for detecting and monitoring infauna with calcified exoskeletons. This novel tool will help us better assess and understand the distribution of deep-sea infauna, particularly those groups with hard exoskeletons, as well as biogeochemical cycles.

Applying Surfactin in the Removal of Blooms of Karlodinium veneficum Increases the Toxic Potential

Biosurfactant has potential application value in the removal of microalgal blooms, but the ecological risks require more research. In this paper, the effects of surfactin on the toxic dinoflagellate Karlodinium veneficum were studied. The coaction of surfactin and K. veneficum was also evaluated through toxicological experiments on Artemia and juvenile clams. The results showed that: (1) in the concentration range of 0–10 mg/L, surfactin significantly killed algal cells in a dose-dependent manner within 48 h; the 24 h EC50 was 3.065 mg/L; (2) K. veneficum had the ability to restore population growth after stress reduction and the restored proliferation was positively correlated with the initial surfactin concentration; (3) the ability to restore population growth was associated with protection afforded by the promotion of antioxidant enzymes, including catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD), whose increase was positively correlated with the surfactin concentration; (4) the toxicity of the coculture of surfactin and K. veneficum was significantly greater than that of the K. veneficum culture or surfactin alone and was dose and time dependent. The potential ecological risks should be considered when applying biosurfactants, such as surfactin, in the removal of harmful algal blooms.

Buffering muds with bivalve shell significantly increases the settlement, growth, survival, and burrowing of the early life stages of the Northern quahog, Mercenaria mercenaria, and other calcifying invertebrates

Acidic conditions in muddy sediments with high organic matter content can negatively impact the growth and survival of juvenile bivalves. Amending sediments with carbonate shell debris could be a strategy to buffer against low porewater pH and improve habitat suitability. To assess this approach, lab and field experiments were conducted to quantify the effects of shell amendments on sediment chemistry as well as growth, survival, recruitment, and burrowing behavior of larval and juvenile hard clams (Mercenaria mercenaria). Shell-amended treatments yielded statistically significant increases in porewater pH (+0.23–0.73) and aragonite saturation state, Ωar (+0.5–1.26). Clams transitioning from larval to juvenile stages showed significantly increased growth (4–5.7 μm day−1) and survival (33%) in buffered sediments, and settling larval clams benefited from sediment buffering more than recently settled juveniles. Juvenile hard clam (length 2–5 mm) burrowing was significantly increased (3–14%) in buffered sediments compared to unamended sediments. Consistent with laboratory findings, juvenile clams showed significantly increased growth rates (5.5–11.4 μm day−1) in carbonate-buffered sediment field experiments in Shinnecock Bay, NY, USA. In addition, native calcifying benthos (Gemma gemma and foraminifera) preferentially settled in buffered sediments, with increased settlement of 150% and 80% for Gemma gemma and foraminifera, respectively. These results substantially extend and generalize previous indications that the addition of shell to muddy sediments can buffer porewater carbonate chemistry and make sediments more hospitable for young hard clams. Amending muddy sediments with shell is a simple, effective strategy to promote settlement, growth, and survival of hard clams and likely other calcifying benthos.

Calibration of the Discrete Element Method Parameters in Living Juvenile Manila Clam (Ruditapes philippinarum) and Seeding Verification

The Manila Clam is an important economic shellfish in China’s seafood industry. In order to improve the design of juvenile Manila Clam seeding equipment, a juvenile clam discrete element method (DEM) particle shape was established, which is based on 3D scanning and EDEM software. The DEM contact parameters of clam-stainless steel, and clam-acrylic were calibrated by combining direct measurements and test simulations (slope sliding and dropping). Then, clam DEM simulation and realistic seeding tests were carried out on a seeding wheel at different rotational speeds. The accuracy of the calibrated clam DEM model was evaluated in a clam seeding verification test by comparing the average error of the variation coefficient between the realistic and simulated seeding tests. The results showed that: (a) the static friction coefficients of clam-acrylic and clam-stainless steel were 0.31 and 0.23, respectively; (b) the restitution coefficients of clam-clam, clam-acrylic, and clam-stainless steel were 0.32, 0.48, and 0.32, respectively. Furthermore, the results of the static repose angle from response surface tests showed that when the contact wall was acrylic, the coefficient rolling friction and static friction of clam-clam were 0.17 and 1.12, respectively, and the coefficient rolling friction of clam-acrylic was 0.20. When the contact wall was formed of stainless steel, the coefficient rolling friction and static friction of clam-clam were 0.33 and 1.25, respectively, and the coefficient rolling friction of clam-stainless steel was 0.20. The results of the verification test showed that the average error between the realistic and simulated value was <5.00%. Following up from these results, the clam DEM model was applied in a clam seeding simulation.

Comparative assessment of the fate and toxicity of chemically and biologically synthesized silver nanoparticles to juvenile clams

Nanoparticles (NPs) can be produced via physical, chemical, or biological approaches. Yet, the impact of the synthesis approaches on the environmental fate and effects of NPs is poorly understood. Here, we synthesized AgNPs through chemical and biological approaches (cit-AgNPs and bio-AgNPs), characterized their properties, and toxicities relative to commercially available Ag nanopowder (np-AgNPs) to the clam Mercenaria mercenaria. The chemical synthesis is based on the reduction of ionic silver using sodium borohydride as a reducing agent and trisodium citrate as a capping agent. The biological synthesis is based on the reduction of ionic silver using biomolecules extracted from an atoxigenic strain of a filamentous fungus Aspergillus parasiticus. The properties of AgNPs were determined using UV–vis, dynamic light scattering, laser Doppler electrophoresis, (single particle)-inductively coupled plasma-mass spectroscopy, transmission electron microscopy, and asymmetric flow-field flow fractionation. Both chemical and biological synthesis approaches generated spherical AgNPs. The chemical synthesis produced AgNPs with narrower size distributions than those generated through biological synthesis. The polydispersity of bio-AgNPs decreased with increases in cell free extract (CFE):Ag ratios. The magnitude of the zeta potential of the cit-AgNPs was higher than those of bio-AgNPs. All AgNPs formed aggregates in the test media i.e., natural seawater. Based on the same total Ag concentrations, all AgNPs were less toxic than AgNO3. The toxicity of AgNPs toward the juvenile clam, Mercenaria mercenaria, decreased following the order np-AgNPs > cit-AgNPs > bio-AgNPs. Expressed as a function of dissolved Ag concentrations, the toxicity of Ag decreased following the order cit-AgNPs > bio-AgNPs > AgNO3 ~ np-AgNPs. Therefore, the toxicity of AgNP suspensions can be attributed to a combined effect of dissolved and particulate Ag forms. These results indicate that AgNP synthesis methods determine their environmental and biological behaviors and should be considered for a more comprehensive environmental risk assessment of AgNPs.

Particulated Microalgae Composite (PMC) As Substitute For Live Microalgae In Culture Of Juvenile Manila Clam Ruditapes Philippinarum (A. Adams And Reeve, 1850): A Feasibility Analysis

To evaluate the feasibility of particulated microalgae composite (PMC) as live microalgae substitute in artificial breeding of Manila clam Ruditapes philippinarum, effects of replacing Dicrateria inornata with partial or total PMC on the growth, digestive enzyme activities, nutritional ingredient composition and microbial composition in faeces of juvenile clams were investigated. The results showed that 50 and 100% PMC replacement did not affect the mean shell length, mean body weight and survival rate of juvenile clams (p>0.05). Juvenile clams fed with 100% PMC replacement had higher lipase and pepsin activities than those fed with 100% D. inornata (p<0.05), while 50% PMC replacement only increased lipase activity (p<0.05). Essential amino acid (EAA) and polyunsaturated fatty acid (PUFA) contents exhibited no obvious differences between clams fed with and without PMC replacement (p>0.05). Significant increase in delicious amino acid contents (p<0.05) was observed with 100% PMC replacement. PMC replacement did not affect the diversity of dominant phyla in faeces, but the abundance of each dominant phylum. Similar relative abundance of faecal bacteria was observed when D. inornata was replaced with 50 and 100% PMC. PMC appeared to be a potential substitute for microalgae in artificial Manila clam breeding. Keywords: Dicrateria inornata, Microalgae substitute, Particulated microalgae composite, Ruditapes philippinarum

Identification of SNPs associated with disease resistance in juveniles of Sinonovacula constricta using RNA-seq and high-resolution melting analysis

Sinonovacula constricta is one of the important economic bivalves in the world with high output value. However, vibriosis is a serious threat to the razor clam aquaculture industries, especially Vibrio parahaemolyticus. Molecular breeding such as marker-assisted selection or genomic selection is an efficient method for genetic improvement of disease resistance. In this study, the resistant and susceptible juvenile clams were evaluated according to the survival time after V. parahaemolyticus challenge and the two groups were genotyped by RNA sequencing technology to develop disease-resistant single nucleotide polymorphism (SNP) markers. The reference transcriptome was de novo assembled by Trinity, containing a total of 146,651 unigenes and 40,574 unigenes annotated. Almost 2 million SNPs were called from the two groups, of which, 23,970 SNPs showed significant allelic imbalance between groups (P valve <0.05). As shown by functional annotation, 396 SNPs were selected as significantly immune-related SNPs, which presented in immune-related genes participating in pathogen infection. It is interesting that significant SNPs in ABC transporters pathways were enriched, and some of SNPs located in ABC transporter genes were exclusive in the susceptible group indicating they maybe deleterious, which can be used as biomarker to distinguish disease-susceptible individuals. Six SNPs were further validated by High-resolution melting analysis in wild population, and five SNPs were verified successfully, which were correlated significantly with disease. These single nucleotide polymorphisms located in coding regions can be applied in the further studies elucidating the molecular mechanisms of immune responses. And the SNPs identified in this study could be used as molecular markers for molecular breeding of disease resistance in S. constricta.

Optimal sediment grain size and sorting for survival and growth of juvenile Manila clams, Venerupis philippinarum

The characteristics of the sedimentary environment are considered significant for increasing the aquaculture production of Manila clams, Venerupis philippinarum. Among these, the grain size and sorting of the sediment, which determine the amount of pore-water-dissolved oxygen and organic matter content, can affect the growth and survival of clams. To determine the optimal grain size and sediment sorting in relation to the conditions of Manila clams (e.g., survival, growth, burrowing behavior, and condition index), we conducted three experiments. First, juvenile clams were incubated in four different grain sizes (coarse sand, 0.335–710 mm; medium sand, 0.180–0.335 mm; fine sand, 0.090–0.180 mm; and very fine sediment, < 0.09 mm). Survival was significantly higher in medium sand than in very fine sediment. The condition index of the Manila clams was higher in the medium sand than in the coarse and very fine sediments. However, the sediment grain size did not influence the shell growth or clam emergence behavior. Second, juvenile clams were incubated in three different treatments of sediment sorting (well-sorted sediment: 0.58 mm; moderately-sorted sediment: 0.46 mm; and poorly-sorted sediment: 0.37 mm). The shell growth rate was significantly higher in the poorly-sorted sediment than in any other treatment, while sediment sorting did not influence the survivorship, condition index, or emergence behavior. Third, juvenile Manila clams were incubated for 30 days in the sediment taken from two locations on the clam culturing mudflat, where a structure was installed to facilitate the settling of clam larvae (which was more poorly-sorted) or without a structure (the control). Although the survival and emergence behavior were not significantly different between the two treatments, the shell growth rate in the sediment under the structure was higher than that in the sediment with no structure. Moreover, the condition index of the Manila clams in the sediment under the structure was significantly higher than that under no structure. The results suggest that poorly-sorted sediment with an average grain size of medium sand is optimal for the survival and growth of Manila clams.