Farmed Species Research Articles

First insights into oxidative stress and theoretical environmental risk of Bronopol and Detarox® AP, two biocides claimed to be ecofriendly for a sustainable aquaculture

Bronopol and Detarox® AP are broad spectrum antimicrobial biocides of growing interest for the aquaculture sector. While their effectiveness against aquatic pathogens has been demonstrated, toxicity data on wild or farmed species are still lacking, as is information on their potential environmental risk for aquatic ecosystems. With this study, we assessed the acute and sublethal toxicity of Bronopol and Detarox® AP in the freshwater bivalve Sinanodonta woodiana and their theoretical risk for aquatic ecosystem. The 96-h median lethal concentration (LC50) was determined using the acute toxicity test, while for the sublethal toxicity test the bivalves were exposed to two concentrations for 14days of Bronopol (2.5 and 50mg/L) and Detarox® AP (1.11 and 22.26mg/L) followed by a 14-day withdrawal period. Biocide-mediated oxidative processes were investigated via a panel of oxidative stress biomarkers (malondialdehyde, superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase). Theoretical environmental risk assessment of both biocides, with predicted concentration of no effect (PNEC), expected theoretical concentration (TEC) in the environment, and risk quotient (RQ) was performed. TEC was calculated using a model based on the size of the aquaculture facility and the receiving basin, the estimated quantity of biocide dissolved in water, and published data on biocide stability in water. Although the LC50 was higher for Bronopol (2440mg/L) than for Detarox® AP (126mg/L), fluctuations in oxidative stress biomarkers levels indicated that both biocides exert a slight oxidative pressure on S. woodiana. Theoretical environmental risk assessment suggested a muted risk with Detarox® AP and greater eco-sustainability compared to Bronopol.

Lipids and fatty acid composition in the crustacean model organismArtemiasp. as influenced by poly‐β‐hydroxybutyrate (PHB) supplementation

The effects of dietary poly-beta-hydroxybutyrate (PHB) on the lipid and fatty acids (FA) in crustaceans were investigated usingArtemiaas model species. Supplying PHB either in crystalline or amorphous form significantly increased the whole-body lipid contents of starvedArtemia. Co-supplying dietary PHB with bacterial PHB degraderComamonas testosteroniat 10(6) cells ml(-1) for 24 hr also significantly increased this parameter. These findings have important implications on the lipid-saving effects of PHB in crustacean tissues. Dietary PHB, however, did not contribute to the increase in docosahexaenoic acid (DHA) of the naturally DHA-deprivedArtemia. Alternative strategy of co-supplying dietary PHB with highly unsaturated fatty acid (HUFA)-rich emulsion for 24 hr effectively improved the nutritional contents ofArtemiaand at the same time assured efficient delivery of PHB to the larval predator. According to a C-13 stable isotope tracer study, the significantly higher delta C-13 levels measured inArtemia2 hr postfeeding with C-13-labelledR. eutropha DSM545 indicate that PHB offered in natural matrix is rapidly assimilated. Overall, this study demonstrated the lipid-saving effects of PHB inArtemia. PHB assimilation following gastrointestinal degradation could attribute to its reported beneficial effects in various aquatic farmed species.

Francisella noatunensis subsp. noatunensis triggers calcium metabolism gene modulation in Eleginops maclovinus

Francisella noatunensis subsp. noatunensis is the responsible agent of Francisellosis, a bacterial disease that affects an important amount of aquatic farmed species. Eleginops maclovinus is a fish that cohabits with salmonids cages in Chile and can also act as a vector of this bacterial disease. In the present study, we evaluated calcium metabolism in the liver of E. maclovinus injected intraperitoneally with different doses of F. noatunensis subsp. noatunensis (low 1.5 × 101, medium 1.5 × 105 and high doses 1.5 × 1010 cells/μL). Fish were sampled at 1, 3, 7, 14, 21 and 28 days post injection (dpi). No mortalities nor clinical signs were observed. Plasma calcium levels were higher in the high doses group of F. noatunensis subsp. noatunensis at day 7 and 14 compared to the control group (fish injected with bacterial medium alone). Hypercalcemic factors increased at day 14 and 21 for the medium and low dose (parathyroid hormone-related protein precursor), while vitamin D3 receptor increased its expression at times 1, 3 and 7 for the low dose. On the other hand, hypocalcemic factors such as calcitonin receptor and stanniocalcin increased its expression at time 7 and 14, respectively. Calmodulin involved in calcium storage decreased its expression during all experimental days in fish subjected to high bacterial dose. Proteins involved in calcium transport, such as L-type voltage-gated calcium channel and trpv5 increased their transcription at day 1 and 14, compared to calcium sensing-receptor and plasma membrane Ca2 +- ATPase that showed peak expression at times 14 and 28. The results suggest a clear alteration of calcium metabolism, mainly in high bacterial doses. This study provides new knowledge about the calcium metabolism in fish infected with bacteria.

Use of biofloc technology in shrimp aquaculture: a comprehensive review, with emphasis on the last decade

AbstractIn face of the shortage of, and competition with, land and water, the sustainability of aquaculture will have to depend on vertical development, through improving production environments, increasing productivity and enhancing aquaculture technologies. Biofloc technology (BFT) has emerged as new alternative for sustainable aquaculture, which could contribute to FAO Sustainable Development Goals (SDGs) related to food security. Extensive research has been carried out on the development and application of BFT in aquaculture since early 1990s, with emphasis on shrimp culture. Over 40% of BFT publications in aquaculture were directed to shrimp farming. Therefore, I strongly believe that the accumulated knowledge on the applications of BFT in shrimp farming and the experience gained, especially during the last 10 years (2010–2020), are now more than worthy of critical review and analysis. This review summarizes the most update knowledge on the use of BFT in different marine shrimp and freshwater prawn aquaculture. Emphasis has been on factors affecting shrimp production in BFT systems, integration of biofloc‐based shrimp farming with other aquatic farmed species, nutritional value of bioflocs as a natural food or feed ingredient for farmed shrimp and prawn, the application of BFT in different rearing phases, the use of biofloc as a natural probiotics and their effects on shrimp health and physiological functions, economic considerations and commercial applications of BFT‐based shrimp aquaculture, and the major challenges facing shrimp farming in biofloc systems.

Transcriptomic response to ammonia-N stress in the hepatopancreas of swimming crab Portunus trituberculatus

The swimming crab Portunus trituberculatus is an important farmed species in China. Ammonia-N represents a major physiological challenge for crab culture and the hepatopancreas plays a major role in physiological adaptation processes. To better understand the molecular mechanism of the crab in response to ammonia-N stress, we performed a transcriptome analysis in the hepatopancreas of P. trituberculatus challenged with ammonia-N stress (5 mg/L), using the high-throughput sequencing technology. In total, 52,280 unigenes were obtained from the hepatopancreas of P. trituberculatus, with an average length of 678 bp. Functional categorization and pathways showed some differentially expressed genes were involved in amino acid and nucleobase metabolism, energy metabolism, antioxidation, immune response, reproduction, moulting and material transport. The differential expression patterns of eight randomly selected annotated genes were validated by quantitative real-time PCR (qPCR). Results revealed a substantial number of genes modified by ammonia-N stress and a few significant ammonia acclimation pathways, which will serve as an invaluable resource for revealing the molecular basis of physiological adaptation mechanism in P. trituberculatus.

Environmental issues in Chilean salmon farming: a review

AbstractThe growth of Chilean salmon production has not been free of important sanitary and environmental shortcomings. To ensure sustainability, it is necessary to understand the environmental impacts of salmon production on the Patagonian ecosystems. Currently, there is limited regulation or monitoring of impacts in the freshwater phase compared to the marine fattening stage, and there is some evidence of local eutrophication impact and diversity changes downstream the farms. Eutrophication of Patagonian channels and fjords from marine farms has been recognized as crucial environmental risk, although most scientific evidence comes from local effects below and around farms. So far, there are no regulations based on carrying capacity estimates to limit maximum fish biomass per area or water body. There is controversy regarding the potential role of nutrients derived from farming in triggering harmful algal blooms, yet current environmental monitoring and available information does not allow establishing or rejecting a cause–effect relationship. Pesticides used to control sea lice infestation have been shown to be deleterious to some non‐target species. There is evidence that the use of high quantities of antibiotics has allowed the development of antibiotic‐resistant bacteria in sediments and there is concern that salmon aquaculture has the potential to increase the proportion of antimicrobial‐resistant bacteria to antibiotics that are used in human medicine. There is an urgent need for more comprehensive ecosystem (beyond farm) studies on the impacts of antibiotics. Escapes of salmon (exotic species) from farms are a relevant environmental risk, although the most farmed species, Salmo salar, has shown little success in establishing wild populations. The review identifies critical knowledge gaps whose fulfilment is essential to advance towards an ecosystem approach to aquaculture and to protect Patagonian ecosystems.

Does Addition of Perch Larvae as Prey Affect the Growth, Development and Cannibalism Rate of Pikeperch Larvae?

Cannibalism occurs in many cultured fish species, especially at the larval and juvenile stages of piscivorous taxa. In farmed percid species, such as pikeperch (Sander lucioperca), intra-cohort cannibalism is a major issue inducing significant losses of the initial stocking density during the first weeks of rearing. Therefore, the present study was designed to investigate the effects of perch larvae (Perca fluviatilis) as live prey on growth, survival and cannibalism in pikeperch larvae under experimental conditions. Additionally, zootechnical and behavioural variables linked to aggressiveness (S postures, attacks, bites and ingestion), and group structures were considered. The survival rate was not different between the two groups (72% with prey vs. 69% without prey), but the cannibalism rate was higher in the group with the prey (28% vs. 10%). The means of final weight and length of pikeperch larvae were higher in the group fed with perch larvae, but size heterogeneity measured by the coefficients of variation for these two parameters did not differ. The specific growth rate was higher in the group fed with perch larvae, but there was no difference between the two groups concerning Fulton’s condition factor. Among all the behavioural variables (aggressiveness, group structure), none differed between the two groups.

Effect of exogenous hydrogen peroxide on ROS balance and antioxidant response in Chinese soft-shelled turtle Pelodiscus sinensis

Chinese soft-shelled turtle, Pelodiscus sinensis, is an important farmed species in the Southeast Asia and often encounters adverse environmental stresses during cultivation. Knowledge about the stress response, especially antioxidant response, in P. sinensis would help to understand the effect of adverse stress on turtles and to reduce the production loss. In the present study, intraperitoneal injection of H2O2 (0%, 0.3%, 0.6%, and 2.4%) was employed as an exogenous oxidative stressor to directly challenge the antioxidant defense in Chinese soft-shelled turtle, Pelodiscus sinensis, and evaluate the antioxidant capacity and characteristics of antioxidant defense in P. sinensis. Level of reactive oxygen species (ROS), total antioxidant capacity (TAC), antioxidant enzyme activities, ascorbic acid (AA) content and malondialdehyde (MDA) level were measured in the brain, liver, kidney and spleen of P. sinensis at 2 h, 6 h and 24 h post-injection. In general, all cerebral and hepatic parameters did not change, although there were some variations in the liver after 2 h, which recovered after 6 h. In the kidney, only glutathione peroxidase (GPx) activity and AA level increased by 24 h post-injection. Splenic ROS level decreased after 6 h; TAC level and catalase (CAT) activity increased, whereas other antioxidant enzyme activities and AA decreased following by H2O2 injection. Results for MDA level and histology indicated transitory oxidative damage in the liver, but longstanding damage in the spleen. In summary, turtle antioxidant defense work effectively to decrease ROS level even when tissues are faced with intense oxidative stress. However, spleen should be paid more attention because it seemed to be more fragile to intense or long-term oxidative stress than other tissues. Specifically, AA level was important in antioxidant defense of P. sinensis.

Genomic Tools and Selective Breeding in Molluscs.

The production of most farmed molluscs, including mussels, oysters, scallops, abalone, and clams, is heavily dependent on natural seed from the plankton. Closing the lifecycle of species in hatcheries can secure independence from wild stocks and enables long-term genetic improvement of broodstock through selective breeding. Genomic techniques have the potential to revolutionize hatchery-based selective breeding by improving our understanding of the characteristics of mollusc genetics that can pose a challenge for intensive aquaculture and by providing a new suite of tools for genetic improvement. Here we review characteristics of the life history and genetics of molluscs including high fecundity, self-fertilization, high genetic diversity, genetic load, high incidence of deleterious mutations and segregation distortion, and critically assess their impact on the design and effectiveness of selective breeding strategies. A survey of the results of current breeding programs in the literature show that selective breeding with inbreeding control is likely the best strategy for genetic improvement of most molluscs, and on average growth rate can be improved by 10% per generation and disease resistance by 15% per generation across the major farmed species by implementing individual or family-based selection. Rapid advances in sequencing technology have resulted in a wealth of genomic resources for key species with the potential to greatly improve hatchery-based selective breeding of molluscs. In this review, we catalog the range of genomic resources currently available for molluscs of aquaculture interest and discuss the bottlenecks, including lack of high-quality reference genomes and the relatively high cost of genotyping, as well as opportunities for applying genomics-based selection.

Efficient Gene Transfer and Gene Editing in Sterlet (Acipenser ruthenus).

The sturgeon (Acipenseriformes) is an important farmed species because of its economical value. However, neither gene transfer nor gene editing techniques have been established in sturgeon for molecular breeding and gene functional study until now. In this study, we accomplished gene transfer and gene editing in sterlet (Acipenser ruthenus), which has the shortest sexual maturation period of sturgeons. The plasmid encoding enhanced green fluorescent protein (EGFP) was transferred into the embryos of sterlet at injection concentration of 100 ng/μL, under which condition high survival rate and gene transfer rate could be achieved. Subsequently, exogenous EGFP was efficiently disrupted by transcription activator-like effector nucleases (TALENs) or clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 nuclease/guide RNA (gRNA), with injection concentrations of 300 ng/μL TALENs, or 100 ng/μL Cas9 nuclease and 30 ng/μL gRNA, respectively, under which condition high survival rate and gene mutation rate could be achieved. Finally, the endogenous gene no tail in sterlet was successfully mutated by Cas9 nuclease/gRNA. We observed the CRISPR-induced no tail mutation, at a high efficiency with the mutant P0 embryos displaying the expected phenotype of bent spine and twisted tail.

Photosynthetic response of Bangia fuscopurpurea (Bangiales, Rhodophyta) towards dehydration and hyposalinity

Bangia fuscopurpurea, an important farmed species in China, inhabits upper intertidal zones where it suffers periodical desiccation and salinity stress. However, the physiological response and acclimation mechanism of Bangia to abiotic stress is unknown. Here, the photosynthetic response of B. fuscopurpurea to desiccation and hyposalinity was investigated by using chlorophyll fluorescence measurement. The optimum photosynthetic efficiency of photosystem II (Fv/Fm), photochemical quenching (qP) and the non-photochemical quenching (NPQ) of B. fuscopurpurea thalli maintained at basal level when the absolute water content (AWC) was 32%. As AWC decreased from 32% to 9%, Fv/Fm dropped from 0.62 to 0.1 and NPQ increased from 0.2 to 1.2. No significant change occurred in the mean qP but great standard deviation was present as AWC was 9%. Fv/Fm, qP and NPQ of the thalli with 9% AWC fully recovered after rehydration. That B. fuscopurpurea kept high photosystem II photochemical reactions even when AWC was mere 32% enabled this species to survive extreme air drying at low tide. Fv/Fm and qP dropped while NPQ increased with 1 h of varying hyposaline treatment and they regained the basal levels after 6–24 h treatment. Nine days later, Fv/Fm, qP and NPQ levels of the thalli in 100% freshwater was equal to the control level (0.62, 0.9, 0.1, respectively). The present finding suggested that this alga has high photosynthetic capacity to survive during low tide, even during heavy rainfall. We hope this study would facilitate further investigation on the stress acclimation mechanism of B. fuscopurpurea.

ITRAQ-based identification of differentially expressed proteins related to growth in the swimming crab, Portunus trituberculatus

The swimming crab, Portunus trituberculatus, is an important farmed species in China, and it has been studied extensively, which requires more information on its genetic background. To date, information on the proteomics of P. trituberculatus is scarce. In this study, we used isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomics to investigate growth regulation in P. trituberculatus. Total proteins were isolated from five tissues (eyestalk, gill, heart, hepatopancreas and muscle). Equal quantities of protein from each tissue were pooled at the proteome level using the iTRAQ method. A total of 961 proteins were identified using liquid chromatography coupled with tandem mass spectrometry and de novo sequencing data. Using a 1.2-fold change in expression as a physiologically significant benchmark, 30 differentially expressed proteins (DEPs) were found to undergo differential expression related to the growth of P. trituberculatus, and most of the growth-related proteins, including those involved in metabolism, immune responses, DNA duplication and protein synthesis, were upregulated, indicating that conservation of energy is an important strategy to cope with growth. There was a high consistency between the expression levels determined using iTRAQ and mRNA, highlighting the high reproducibility of our proteomic approach and its great value in elucidating the molecular mechanisms of P. trituberculatus.

Selection response in fish and shellfish: a review

AbstractThe aquaculture sector is significantly behind plant and farm animal production in applying selective breeding, in spite of the fact that it has been suggested that the world aquaculture production could be doubled in 13 years if breeding programmes were supplying stocks for the farmed species. It is estimated that as late as in 2010, only 8.2% of the world's total aquaculture production was based on material developed in selective breeding programmes. Reported estimates of genetic gain per generation for a key trait like growth rate average 13%, implying that the animal's potential for growth can be doubled in a time span of only six generations of selection, as demonstrated for major farmed species like Atlantic salmon and Nile tilapia. Likewise are reported genetic gains for improved disease resistance generally very high. This study offers an updated review of published estimates on genetic gains for a range of traits in aquaculture species. Results are highly encouraging and demonstrate a substantial potential for genetic improvement in aquatic productions, in particular for traits such as growth rate and resistance to diseases.

The supplementation of a microdiet with crystalline indispensable amino-acids affects muscle growth and the expression pattern of related genes in Senegalese sole (Solea senegalensis) larvae

The full expression of growth potential in fish larvae largely depends on an efficient protein utilization, which requires that all the indispensable amino acids (IAAs) are provided at an optimum ratio. The effect of supplementing a practical microdiet with encapsulated crystalline-AA to correct possible IAA deficiencies was evaluated in Senegalese sole larvae. Two isonitrogenous and isoenergetic microdiets were formulated and processed to have approximately the same ingredients and proximate composition. The control diet (CTRL) was based on protein sources commonly used in the aquafeed industry. In the supplemented diet (SUP) 8% of an encapsulated fish protein hydrolysate was replaced by crystalline-AA in order to increase the dietary IAA levels. The microdiets were delivered from mouth-opening upon a co-feeding regime until 51days after hatching (DAH). The larvae capacity to utilize protein was evaluated using an in vivo method of controlled tube-feeding during relevant stages throughout development: pre-metamorphosis (13DAH); metamorphosis climax (19DAH) and metamorphosis completion (25DAH). Somatic growth was monitored during the whole trial. A possible effect on the regulation of muscle growth was evaluated through muscle cellularity and the expression of related genes (myf5, myod2, myogenin, mrf4, myhc and mstn1) at metamorphosis climax (19DAH) and at a juvenile stage (51DAH). The SUP diet had a negative impact on larvae somatic growth after the metamorphosis, even though it had no effect on the development of Senegalese sole larvae capacity to retain protein. Instead, changes in somatic growth may reflect alterations on muscle growth regulation, since muscle cellularity suggested delayed muscle development in the SUP group at 51DAH. Transcript levels of key genes regulating myogenesis changed between groups, during the metamorphosis climax and at the 51DAH. The group fed the SUP diet had lower dnmt3b mRNA levels compared to the CTRL group. Further studies are needed to ascertain whether this would possibly lead to an overall DNA hypomethylation in skeletal muscle. Statement of relevanceIn farmed fish species, there has been a great effort over the years to provide the best conditions for successful development of embryos and small larvae, as early environmental conditions can strongly affect muscle growth during early stages and influence the subsequent growth potential at later life stages. In spite of increased efforts to understand the regulation of myogenesis by intrinsic factors like genotype and extrinsic factors such as photoperiod and temperature, studies evaluating the impact of nutritional factors on fish larvae muscle development are still very scarce.This work will raise interest to the discussion on whether a nutritional cue during an early developmental stage can impact on the regulation of muscle growth and on further growth potential in a metamorphosing farmed fish species, such as Senegalese sole. Supplementing microdiets with crystalline-AA in order to correct dietary IAA did not improve Senegalese sole larvae somatic growth and led to changes on the regulation of muscle growth associated with changes in expression patterns of muscle growth markers during the trial (secondary MRFs myogenin and mrf4, myhc and mstn1). Dietary IAA affected the expression of a DNA methyltransferase essential for de novo methylation, dnmt3b, suggesting that an epigenetic effect at the transcriptional regulation level may explain differences found in somatic growth as a response to a nutritional cue.This work may contribute to lay down basis for future studies on nutritional programming of muscle growth in fish larvae of important farmed species.

Increasing pressure on freshwater resources due to terrestrial feed ingredients for aquaculture production

As aquaculture becomes more important for feeding the growing world population, so too do the required natural resources needed to produce aquaculture feed. While there is potential to replace fish meal and fish oil with terrestrial feed ingredients, it is important to understand both the positive and negative implications of such a development. The use of feed with a large proportion of terrestrial feed may reduce the pressure on fisheries to provide feed for fish, but at the same time it may significantly increase the pressure on freshwater resources, due to water consumption and pollution in crop production for aquafeed. Here the green, blue and gray water footprint of cultured fish and crustaceans related to the production of commercial feed for the year 2008 has been determined for the major farmed species, representing 88% of total fed production. The green, blue and gray production-weighted average feed water footprints of fish and crustaceans fed commercial aquafeed are estimated at 1629m3/t, 179m3/t and 166m3/t, respectively. The estimated global total water footprint of commercial aquafeed was 31–35km3 in 2008. The top five contributors to the total water footprint of commercial feed are Nile tilapia, Grass carp, Whiteleg shrimp, Common carp and Atlantic salmon, which together have a water footprint of 18.2km3. An analysis of alternative diets revealed that the replacement of fish meal and fish oil with terrestrial feed ingredients may further increase pressure on freshwater resources. At the same time economic consumptive water productivity may be reduced, especially for carnivorous species. The results of the present study show that, for the aquaculture sector to grow sustainably, freshwater consumption and pollution due to aquafeed need to be taken into account.

Transcriptomic response to low salinity stress in gills of the Pacific white shrimp, Litopenaeus vannamei

The Pacific white shrimp, Litopenaeus vannamei (L. vannamei), is one of the most farmed species. Salinity is an important environmental factor that affects its growth and distribution. However, the molecular mechanism of the shrimp in response to salinity stress remains largely unclear. High-throughput sequencing is a helpful tool to analyze the molecular response to salinity challenge in shrimp. In the present study, the transcriptomic responses of the gills in L. vannamei under low salinity stress were detected by Illumina's digital gene expression system. A total of 10,725,789 and 10,827,411 reads were generated from the non-changed and low salinity changed groups, respectively. 64,590 Unigenes with an average length of 764bp were generated. Compared with the control, 585 genes were differentially expressed under low salinity. GO functional analysis and KEGG pathway analysis indicated some vital genes in response to the challenge. Ten genes related to osmoregulation and ambient salinity adaption were selected to validate the DGE results by RT-qPCR. This work provides valuable information to study the mechanism of salinity adaption in L. vannamei. Genes and pathways from the results will be beneficial to reveal the molecular basis of osmoregulation. It also gives an insight into the response to the salinity challenge in L. vannamei.

Genomics in aquaculture to better understand species biology and accelerate genetic progress.

Genomics in aquaculture to better understand species biology and accelerate genetic progress.

Transcriptome analysis of the Portunus trituberculatus: de novo assembly, growth-related gene identification and marker discovery.

BackgroundThe swimming crab, Portunus trituberculatus, is an important farmed species in China, has been attracting extensive studies, which require more and more genome background knowledge. To date, the sequencing of its whole genome is unavailable and transcriptomic information is also scarce for this species. In the present study, we performed de novo transcriptome sequencing to produce a comprehensive transcript dataset for major tissues of Portunus trituberculatus by the Illumina paired-end sequencing technology.ResultsTotal RNA was isolated from eyestalk, gill, heart, hepatopancreas and muscle. Equal quantities of RNA from each tissue were pooled to construct a cDNA library. Using the Illumina paired-end sequencing technology, we generated a total of 120,137 transcripts with an average length of 1037 bp. Further assembly analysis showed that all contigs contributed to 87,100 unigenes, of these, 16,029 unigenes (18.40% of the total) can be matched in the GenBank non-redundant database. Potential genes and their functions were predicted by GO, KEGG pathway mapping and COG analysis. Based on our sequence analysis and published literature, many putative genes with fundamental roles in growth and muscle development, including actin, myosin, tropomyosin, troponin and other potentially important candidate genes were identified for the first time in this specie. Furthermore, 22,673 SSRs and 66,191 high-confidence SNPs were identified in this EST dataset.ConclusionThe transcriptome provides an invaluable new data for a functional genomics resource and future biological research in Portunus trituberculatus. The data will also instruct future functional studies to manipulate or select for genes influencing growth that should find practical applications in aquaculture breeding programs. The molecular markers identified in this study will provide a material basis for future genetic linkage and quantitative trait loci analyses, and will be essential for accelerating aquaculture breeding programs with this species.

Transcriptome Analysis of Portunus trituberculatus in Response to Salinity Stress Provides Insights into the Molecular Basis of Osmoregulation

BackgroundThe swimming crab, Portunus trituberculatus, which is naturally distributed in the coastal waters of Asia-Pacific countries, is an important farmed species in China. Salinity is one of the most important abiotic factors that influence not only the distribution and abundance of crustaceans, it is also an important factor for artificial propagation of the crab. To better understand the interaction between salinity stress and osmoregulation, we performed a transcriptome analysis in the gills of Portunus trituberculatus challenged with salinity stress, using the Illumina Deep Sequencing technology.ResultsWe obtained 27,696,835, 28,268,353 and 33,901,271 qualified Illumina read pairs from low salinity challenged (LC), non-challenged (NC), and high salinity challenged (HC) Portunus trituberculatus cDNA libraries, respectively. The overall de novo assembly of cDNA sequence data generated 94,511 unigenes, with an average length of 644 bp. Comparative genomic analysis revealed that 1,705 genes differentially expressed in salinity stress compared to the controls, including 615 and 1,516 unigenes in NC vs LC and NC vs HC respectively. GO functional enrichment analysis results showed some differentially expressed genes were involved in crucial processes related to osmoregulation, such as ion transport processes, amino acid metabolism and synthesis processes, proteolysis process and chitin metabolic process.ConclusionThis work represents the first report of the utilization of the next generation sequencing techniques for transcriptome analysis in Portunus trituberculatus and provides valuable information on salinity adaptation mechanism. Results reveal a substantial number of genes modified by salinity stress and a few important salinity acclimation pathways, which will serve as an invaluable resource for revealing the molecular basis of osmoregulation in Portunus trituberculatus. In addition, the most comprehensive sequences of transcripts reported in this study provide a rich source for identification of novel genes in the crab.

069 Advances in shellfish germplasm cryopreservation: Current status and future directions

Cryopreservation can be a powerful tool in shellfish aquaculture for selective breeding programmes and hatchery production. Most species that are commercially farmed are broadcast spawners, releasing sperm and eggs into the environment where they fertilize and develop. However, some species are brooders (e.g. flat oysters (Ostrea chilensis) ), which retain and rear embryos until they reach later developmental stages. Sperm cryopreservation has been successfully achieved for several farmed species including the Pacific oyster ( Crassostrea gigas ), greenshell™ mussel ( Perna canaliculus ) and abalone ( Haliotis iris ). In general, dimethyl sulphoxide and trehalose are the preferred cryoprotectants (CPAs) and simple methods to control cooling rate (e.g. floating rack on liquid nitrogen; methanol/dry ice bath) have worked well for our main species of interest (Pacific oyster, greenshell™ mussel and abalone). Interestingly, sperm perform better in CPAs that are prepared in Milli-Q water as opposed to seawater, indicating that a lower salt concentration during freezing is beneficial. Recent studies with flat oysters and geoducks ( Panopea zelandica ) have also resulted in successful sperm cryopreservation. Shellfish oocyte and larvae cryopreservation has proven more challenging, however, successful cryopreservation of Pacific oyster oocytes has been achieved. Pacific oyster oocytes are able to be fertilized and develop to normal spat post-thawing. However, for other species (e.g. greenshell™ mussel) and for larvae, cryo-injuries are not always immediately apparent post-thawing but manifest during development and are often lethal. greenshell™ mussel oocytes are irretrievably damaged when they reach -6 to -8°C and attempts to overcome this sub-zero chilling injury by vitrification have been unsuccessful. Towards understanding these injuries and their effects, we investigated the effect of cryopreservation on the meiotic spindle of greenshell™ mussel oocytes. Results show that the meiotic spindle undergoes major changes during both CPA addition and cooling. However, the spindle is able to re-form normally in most instances given sufficient time post thawing. For a proportion of oocytes, cryopreservation appears to trigger oocyte activation. The effect of cryopreservation on oxidative stress in greenshell™ mussel oocytes has also been investigated. Although, there are changes in oxidative stress parameters as a result of cryopreservation and strong correlations between fertilization, development and oxidative stress parameters (lipid hydroperoxides, superoxide dismutase, protein carbonyls, total glutathione and glutathione state), preliminary attempts to mitigate oxidative stress using antioxidants have so far failed to enable normal development following cooling, suggesting that oxidative stress may be a secondary rather than primary stress event. Research on shellfish larval cryopreservation utilizing SEM and confocal microscopy has also been carried out for greenshell™ mussel. Later larval stages (D-larvae) appear more resilient to cryopreservation than trochophore larvae, but abnormalities are still apparent (e.g.shell abnormalities and delayed organogenesis). Future studies are aimed toward elucidating mechanisms of injury using new tools such as proteomics and then targeting susceptible cellular processes for protection during cryopreservation. Source of funding: This research was funded by the New Zealand Ministry of Business Innovation and Employment (CAWX0802). Conflict of interest: None declared. serean.adams@cawthron.org.nz