Probabilistic reaction norm reveals family-related variation in the association between size, condition, and sexual maturation onset in Atlantic salmon (Salmo salar).

This study investigated the relationship between the size, condition, year class, family, and sexual maturity of Atlantic salmon (Salmo salar) using data collected in an aquaculture selective breeding programme. Males that were sexually mature at 2 years of age (maiden spawn) have, on average, greater fork length and condition factor (K) at 1 year of age than their immature counterparts. For every 10-mm increase in fork length or 0.1 increase in K at 1 year of age, the odds of sexual maturity at 2 years of age increased by 1.48 or 1.22 times, respectively. Females that were sexually mature at 3 years of age (maiden spawn) have, on average, greater fork length and K at 2 years of age than their immature counterparts. For every 10-mm increase in fork length or 0.1 increase in K at 2 years of age, the odds of sexual maturity at 3 years of age increased by 1.06 or 1.44 times, respectively. The family explained 34.93% of the variation in sexual maturity among 2-year-old males that was not attributable to the average effects of fork length and K at 1 year of age and year class. The proportion of variation in sexual maturity among 3-year-old females explained by the family could not be investigated. These findings suggest that the onset of sexual maturation in Atlantic salmon is conditional on performance (with respect to energy availability) surpassing a threshold, the magnitude of which can vary between families and is determined by a genetic component. This could support the application of genetic selection to promote or inhibit the onset of sexual maturation in farmed stocks.

Development and validation of a RAD-Seq target-capture based genotyping assay for routine application in advanced black tiger shrimp (Penaeus monodon) breeding programs.

BACKGROUND: The development of genome-wide genotyping resources has provided terrestrial livestock and crop industries with the unique ability to accurately assess genomic relationships between individuals, uncover the genetic architecture of commercial traits, as well as identify superior individuals for selection based on their specific genetic profile. Utilising recent advancements in de-novo genome-wide genotyping technologies, it is now possible to provide aquaculture industries with these same important genotyping resources, even in the absence of existing genome assemblies. Here, we present the development of a genome-wide SNP assay for the Black Tiger shrimp (Penaeus monodon) through utilisation of a reduced-representation whole-genome genotyping approach (DArTseq). RESULTS: Based on a single reduced-representation library, 31,262 polymorphic SNPs were identified across 650 individuals obtained from Australian wild stocks and commercial aquaculture populations. After filtering to remove SNPs with low read depth, low MAF, low call rate, deviation from HWE, and non-Mendelian inheritance, 7542 high-quality SNPs were retained. From these, 4236 high-quality genome-wide loci were selected for baits-probe development and 4194 SNPs were included within a finalized target-capture genotype-by-sequence assay (DArTcap). This assay was designed for routine and cost effective commercial application in large scale breeding programs, and demonstrates higher confidence in genotype calls through increased call rate (from 80.2 ± 14.7 to 93.0% ± 3.5%), increased read depth (from 20.4 ± 15.6 to 80.0 ± 88.7), as well as a 3-fold reduction in cost over traditional genotype-by-sequencing approaches. CONCLUSION: Importantly, this assay equips the P. monodon industry with the ability to simultaneously assign parentage of communally reared animals, undertake genomic relationship analysis, manage mate pairings between cryptic family lines, as well as undertake advance studies of genome and trait architecture. Critically this assay can be cost effectively applied as P. monodon breeding programs transition to undertaking genomic selection.

Novel Allergen Discovery through Comprehensive De Novo Transcriptomic Analyses of Five Shrimp Species.

Shellfish allergy affects 2% of the world's population and persists for life in most patients. The diagnosis of shellfish allergy, in particular shrimp, is challenging due to the similarity of allergenic proteins from other invertebrates. Despite the clinical importance of immunological cross-reactivity among shellfish species and between allergenic invertebrates such as dust mites, the underlying molecular basis is not well understood. Here we mine the complete transcriptome of five frequently consumed shrimp species to identify and compare allergens with all known allergen sources. The transcriptomes were assembled de novo, using Trinity, from raw RNA-Seq data of the whiteleg shrimp (Litopenaeus vannamei), black tiger shrimp (Penaeus monodon), banana shrimp (Fenneropenaeus merguiensis), king shrimp (Melicertus latisulcatus), and endeavour shrimp (Metapenaeus endeavouri). BLAST searching using the two major allergen databases, WHO/IUIS Allergen Nomenclature and AllergenOnline, successfully identified all seven known crustacean allergens. The analyses revealed up to 39 unreported allergens in the different shrimp species, including heat shock protein (HSP), alpha-tubulin, chymotrypsin, cyclophilin, beta-enolase, aldolase A, and glyceraldehyde-3-phosphate dehydrogenase (G3PD). Multiple sequence alignment (Clustal Omega) demonstrated high homology with allergens from other invertebrates including mites and cockroaches. This first transcriptomic analyses of allergens in a major food source provides a valuable resource for investigating shellfish allergens, comparing invertebrate allergens and future development of improved diagnostics for food allergy.

Adequate supply of dietary taurine stimulates expression of molecular markers of growth and protein turnover in juvenile barramundi (Lates calcarifer).

A trial was conducted to investigate the effect of dietary taurine (Tau) supply on the plasma amino acid composition and hepatic expression of several genes in juvenile barramundi (Lates calcarifer) after feeding. Triplicate tanks of fish (average weight, 89.3 g) were fed diets containing either a deficient (1 g kg-1), adequate (8 g kg-1) or excessive (19 g kg-1) level of dietary Tau. Liver tissues collected before feeding, and at 2- and 4-h post-feeding, were analysed for expression of genes involved in pathways of sulphur amino acid turnover, Tau biosynthesis and transport, target of rapamycin (TOR) signalling, the somatotropic axis and protein turnover. The treatment had no significant effect on the profiles of any amino acid in plasma collected over time after feeding, other than Tau and glycine. The expression profile of cystine and Tau synthetic genes suggested an effect of Tau excess on the metabolism of cystine. Markers of two pathways of Tau biosynthesis appear to be active in this species, providing proof that this species possesses the ability to synthesise Tau from SAA precursors. A marker for the regulation of Tau transport and homeostasis was shown to be directly regulated by Tau availability, whilst a link between adequate supply of Tau and TOR pathway-mediated growth stimulation was also apparent. An observed depression in expression of genes of the somatotropic axis, coupled with upregulation of the proteolytic and TOR-suppressing genes, in response to excessive Tau supply in the diet, signalled that excessive Tau may not be conducive to optimal growth in this species.

Impact of dietary starch on extrahepatic tissue lipid metabolism in farmed European (Dicentrarchus labrax) and Asian seabass (Lates calcarifer).

In aquaculture, there is high interest in substituting marine-derived with vegetable-based ingredients as energy source. Farmed carnivorous fish under high carbohydrate diets tend to increase adiposity but it remains unclear if this happens by increased lipid retention/accumulation, promotion of lipogenic pathways, or both. In order to determine the response of extrahepatic tissue to dietary starch, European (Dicentrarchus labrax) and Asian (Lates calcarifer) seabass were fed a control (low starch; LS) or experimental (high starch; HS) diet, for at least 21 days and then transferred for 6 days to saltwater enriched with deuterated water 2H2O. Incorporation of 2H-labelling follows well-defined metabolic steps, and analysis of triacylglycerols (TAG) 2H-enrichment by 2HNMR allowed evaluation of de novo lipogenesis (DNL) in muscle and visceral adipose tissue (VAT). Fractional synthetic rates for TAG-bound fatty acids and glycerol were quantified separately providing a detailed lipogenic profile. The FA profile differed substantially between muscle and VAT in both species, but their lipogenic fluxes revealed even greater differences. In European seabass, HS promoted DNL of TAG-bound FA, in muscle and VAT. High 2H-enrichment also found in muscle TAG-bound glycerol was indicative of its role on lipid cycling. In Asian seabass, HS had no effect on muscle FA composition and lipogenic flux, with no 2H-enriched TAG being detected. VAT on the other hand revealed a strong enhancement of DNL in HS-fed fish along with high TAG-bound glycerol cycling. This study consolidated the use of 2H2O as tracer for fish lipid metabolism in different tissues, under different dietary conditions and suitable to use in different fish models.

Effects of an unprecedented summer heatwave on the growth performance, flesh colour and plasma biochemistry of marine cage-farmed Atlantic salmon (Salmo salar).

Global seawater temperatures are increasing and becoming more variable, with consequences for all marine animals including those in food production systems. In several countries around the world,arming of Atlantic salmon (Salmo salar) occurs towards the upper end of the thermal tolerance window for this species, and marked effects on salmon production during summers have been experienced but never empirically investigated. This project tracked the effects of an extreme summer heatwave on two different cohorts of fish stocked into farm cages either during early winter (EW) or late winter (LW). The farm site experienced an unprecedented high water temperature event, with a peak water temperature of 22.9 °C and 117 days above 18 °C. Fish in both EW and LW cohorts experienced a temperature-induced cessation of voluntary feed intake as well as inefficient osmoregulatory, liver and renal function during high temperature periods. Flesh colour declined primarily in the dorsal and ventral regions of the fillet and secondarily along the midline, with over 20% of fish demonstrated a complete loss of flesh colour during the months of March and April. A return to feeding in autumn occurred faster in some fish and caused a marked bimodal size distribution to appear within both the EW and LW cohorts as autumn progressed. However, the LW cohort returned to feeding at seawater temperatures of 20.2 °C, compared with 18.6 °C for the EW cohort. There was a strong positive relationship between fillet colour recovery and residual condition index (RCI). These findings identified alkaline phosphatase as a potential marker to non-destructively track individual fish for signs of recovery after a thermal stress event, and shed light on the physiological consequences of marine heatwaves on fishes. This study also identified that supporting feed intake or promoting a return to feeding may help mitigate the negative impacts of climate warming on cultured Atlantic salmon.

Characterization of lipid metabolism genes and the influence of fatty acid supplementation in the hepatic lipid metabolism of dusky grouper (Epinephelus marginatus).

Dusky grouper is an important commercial fish species in many countries, but some factors such as overfishing has significantly reduced their natural stocks. Aquaculture emerges as a unique way to conserve this species, but very little biological information is available, limiting the production of this endangered species. To understand and generate more knowledge about this species, liver transcriptome sequencing and de novo assembly was performed for E. marginatus by Next Generation Sequencing (NGS). Sequences obtained were used as a tool to validate the presence of key genes relevant to lipid metabolism, and their expression was quantified by qPCR. Moreover, we investigated the influence of supplementing different dietary fatty acids on hepatic lipid metabolism. The results showed that the different fatty acids added to the diet dramatically changed the gene expression of some key enzymes associated with lipid metabolism as well as hepatic fatty acid profiles. Elongase 5 gene expression was shown to influence intermediate hepatic fatty acid elongation in all experimental groups. Hepatic triglycerides reflected the diet composition more than hepatic phospholipids, and were characterized mainly by the high percentage of 18:3n3 in animals fed with a linseed oil rich diet. Results for the saturated and monounsaturated fatty acids suggest a self-regulatory potential for retention and oxidation processes in liver, since in general the tissues did not directly reflect these fatty acid diet compositions. These results indicated that genes involved in lipid metabolism pathways might be potential biomarkers to assess lipid requirements in the formulated diet for this species.

Rapid expansion of pigmentation genes in penaeid shrimp with absolute preservation of function.

Crustaceans form their distinct patterns and colours through the interaction of the carotenoid astaxanthin with a protein called crustacyanin (CRCN). Presently, the expression of just two CRCN genes is thought to provide the protein subunits that combine to form the crustacyanin complex and associated carotenoid colour change from red to blue. This study aimed to explore the genetic complexity underlying the production of pigmentation and camouflage in penaeid shrimp. We isolated 35 new CRCN genes from 12 species, and their sequence analysis indicated that this gene family has undergone significant expansion and diversification in this lineage. Despite this duplication and sequence divergence, the structure of the CRCN proteins and their functional role in shrimp colour production has been strictly conserved. Using CRCN isoforms from Penaeus monodon as an example, we showed that isoforms were differentially expressed, and that subtle phenotypes were produced by the specific downregulation of individual isoforms. These findings demonstrate that our knowledge of the molecular basis of pigmentation in shrimp was overly simplistic, and suggests that multiple copies of the CRCN genes within species may be advantageous for colour production. This result is of interest for the origin and evolution of pigmentation in crustaceans, and the mechanisms by which gene function is maintained, diversified or sub-functionalized.

An analysis of partial efficiencies of energy utilisation of different macronutrients by barramundi (Lates calcarifer) shows that starch restricts protein utilisation in carnivorous fish.

This study examined the effect of including different dietary proportions of starch, protein and lipid, in diets balanced for digestible energy, on the utilisation efficiencies of dietary energy by barramundi (Lates calcarifer). Each diet was fed at one of three ration levels (satiety, 80 % of initial satiety and 60 % of initial satiety) for a 42-d period. Fish performance measures (weight gain, feed intake and feed conversion ratio) were all affected by dietary energy source. The efficiency of energy utilisation was significantly reduced in fish fed the starch diet relative to the other diets, but there were no significant effects between the other macronutrients. This reduction in efficiency of utilisation was derived from a multifactorial change in both protein and lipid utilisation. The rate of protein utilisation deteriorated as the amount of starch included in the diet increased. Lipid utilisation was most dramatically affected by inclusion levels of lipid in the diet, with diets low in lipid producing component lipid utilisation rates well above 1·3, which indicates substantial lipid synthesis from other energy sources. However, the energetic cost of lipid gain was as low as 0·65 kJ per kJ of lipid deposited, indicating that barramundi very efficiently store energy in the form of lipid, particularly from dietary starch energy. This study defines how the utilisation efficiency of dietary digestible energy by barramundi is influenced by the macronutrient source providing that energy, and that the inclusion of starch causes problems with protein utilisation in this species.

Rapid effects of essential fatty acid deficiency on growth and development parameters and transcription of key fatty acid metabolism genes in juvenile barramundi (Lates calcarifer).

Barramundi (Lates calcarifer), a catadromous teleost of significant and growing commercial importance, are reported to have limited fatty acid bioconversion capability and therefore require preformed long-chain PUFA (LC-PUFA) as dietary essential fatty acid (EFA). In this study, the response of juvenile barramundi (47·0 g/fish initial weight) fed isolipidic and isoenergetic diets with 8·2% added oil was tested. The experimental test diets were either devoid of fish oil (FO), and thus with no n-3 LC-PUFA (FO FREE diet), or with a low inclusion of FO (FO LOW diet). These were compared against a control diet containing only FO (FO CTRL diet) as the added lipid source, over an 8-week period. Interim samples and measurements were taken fortnightly during the trial in order to define the aetiology of the onset and progression of EFA deficiency. After 2 weeks, the fish fed the FO FREE and FO LOW diets had significantly lower live-weights, and after 8 weeks significant differences were detected for all performance parameters. The fish fed the FO FREE diet also had a significantly higher incidence of external abnormalities. The transcription of several genes involved in fatty acid metabolism was affected after 2 weeks of feeding, showing a rapid nutritional regulation. This experiment documents the aetiology of the onset and the progression of EFA deficiency in juvenile barramundi and demonstrates that such deficiencies can be detected within 2 weeks in juvenile fish.

Postprandial molecular responses in the liver of the barramundi, Lates calcarifer.

The regulation of gene expression by nutrients is an important mechanism governing energy storage and growth in most animals, including fish. At present, very few genes that regulate intermediary metabolism have been identified in barramundi, nor is there any understanding of their nutritional regulation. In this study, a partial barramundi liver transcriptome was assembled from next-generation sequencing data and published barramundi EST sequences. A large number of putative metabolism genes were identified in barramundi, and the changes in the expression of 24 key metabolic regulators of nutritional pathways were investigated in barramundi liver over a time series immediately after a meal of a nutritionally optimised diet for this species. Plasma glucose and free amino acid levels showed a mild postprandial elevation which peaked 2 h after feeding, and had returned to basal levels within 4 or 8 h, respectively. Significant activation or repression of metabolic nuclear receptor regulator genes were observed, in combination with activation of glycolytic and lipogenic pathways, repression of the final step of gluconeogenesis and activation of the Akt-mTOR pathway. Strong correlations were identified between a number of different metabolic genes, and the coordinated co-regulation of these genes may underlie the ability of this fish to utilise dietary nutrients. Overall, these data clearly demonstrate a number of unique postprandial responses in barramundi compared with other fish species and provide a critical step in defining the response to different dietary nutrient sources.

Post-prandial response in hepatopancreas and haemolymph of Penaeus monodon fed different diets. Omics insights into glycoconjugate metabolism, energy utilisation, chitin biosynthesis, immune function, and autophagy.

Raw materials or bioactive ingredients trigger mechanisms to assimilate nutrients and activate metabolic pathways that promote growth, immune function, or energy storage. Our understanding of these processes at a molecular level remains limited in aquaculture, especially in shrimp. Here, hepatopancreas proteomics and haemolymph metabolomics were used to investigate the post-prandial response of black tiger shrimps (Penaeus monodon) fed a conventional fishmeal diet (FM); a diet supplemented with the microbial biomass Novacq™ (NV); krill meal (KM); or, fasted (FS). Using FM as a control, a 2-fold change in abundance threshold was implemented to determine the significance of proteins and metabolites. NV fed shrimp showed preference for energy derived from carbohydrates indicated by a strong signature of glycoconjugate metabolism and activation of the amino- and nucleotide sugar metabolic pathway. KM activated the glyoxylate and dicarboxylate pathway that denoted shrimp preference for lipidic energy. KM also influenced energy generation by the TCA cycle inferred from higher abundance of the metabolites succinic semialdehyde, citric acid, isocitrate, alpha ketoglutarate and ATP and downregulation of the enzyme isocitrate dehydrogenase that catalyses oxidative decarboxylation of isocitrate. FS shrimp displayed down-regulation of oxidative phosphorylation and resorted to internal lipid reserves for energy homeostasis displaying a strong signature of autophagy. Pyrimidine metabolism was the preferred energy strategy in this group. Our study also provided evidence that during fasting or consumption of specific ingredients, shrimp share common pathways to meet their energy requirements, however, the intensity at which these pathways were impacted was diet dependent.

Mechanisms of colour adaptation in the prawn Penaeus monodon.

Exposure of prawns to dark- or light-coloured substrates is known to trigger a strong colour adaptation response through expansion or contraction of the colouration structures in the prawn hypodermis. Despite the difference in colour triggered by this adaptive response, total levels of the predominant carotenoid pigment, astaxanthin, are not modified, suggesting that another mechanism is regulating this phenomenon. Astaxanthin binds to a specific protein called crustacyanin (CRCN), and it is the interaction between the quantities of each of these compounds that produces the diverse range of colours seen in crustacean shells. In this study, we investigated the protein changes and genetic regulatory processes that occur in prawn hypodermal tissues during adaptation to black or white substrates. The amount of free astaxanthin was higher in animals adapted to dark substrate compared with those adapted to light substrate, and this difference was matched by a strong elevation of CRCN protein. However, there was no difference in the expression of CRCN genes either across the moult cycle or in response to background substrate colour. These results indicate that exposure to a dark-coloured substrate causes an accumulation of CRCN protein, bound with free astaxanthin, in the prawn hypodermis without modification of CRCN gene expression. On light-coloured substrates, levels of CRCN protein in the hypodermis are reduced, but the carotenoid is retained, undispersed in the hypodermal tissue, in an esterified form. Therefore, the abundance of CRCN protein affects the distribution of pigment in prawn hypodermal tissues, and is a crucial regulator of the colour adaptation response in prawns.

Genetic parameters of color phenotypes of black tiger shrimp (Penaeus monodon).

Black tiger shrimp (Penaeus monodon) is the second most important aquaculture species of shrimp in the world. In addition to growth traits, uncooked and cooked body color of shrimp are traits of significance for profitability and consumer acceptance. This study investigated for the first time, the phenotypic and genetic variances and relationships for body weight and body color traits, obtained from image analyses of 838 shrimp, representing the progeny from 55 sires and 52 dams. The color of uncooked shrimp was subjectively scored on a scale from 1 to 4, with "1" being the lightest/pale color and "4" being the darkest color. For cooked shrimp color, shrimp were graded firstly by subjective scoring using a commercial grading score card, where the score ranged from 1 to 12 representing light to deep coloration which was subsequently found to not be sufficiently reliable with poor repeatability of measurement (r = 0.68-0.78) Therefore, all images of cooked color were regraded on a three-point scale from brightest and lightest colored cooked shrimp, to darkest and most color-intense, with a high repeatability (r = 0.80-0.92). Objective color of both cooked and uncooked color was obtained by measurement of RGB intensities (values range from 0 to 255) for each pixel from each shrimp. Using the "convertColor" function in "R", the RGB values were converted to L*a*b* (CIE Lab) systems of color properties. This system of color space was established in 1976, by the International Commission of Illumination (CIE) where "L*" represents the measure of degree of lightness, values range from 0 to 100, where 0 = pure black and 100 = pure white. The value "a*" represents red to green coloration, where a positive value represents the color progression towards red and a negative value towards green. The value "b*" represents blue to yellow coloration, where a positive value refers to more yellowish and negative towards the blue coloration. In total, eight color-related traits were investigated. An ordinal mixed (threshold) model was adopted for manually (subjectively) scored color phenotypes, whereas all other traits were analyzed by linear mixed models using ASReml software to derive variance components and estimated breeding values (EBVs). Moderate to low heritability estimates (0.05-0.35) were obtained for body color traits. For subjectively scored cooked and uncooked color, EBV-based selection would result in substantial genetic improvement in these traits. The genetic correlations among cooked, uncooked and body weight traits were high and ranged from -0.88 to 0.81. These suggest for the first time that 1) cooked color can be improved indirectly by genetic selection based on color of uncooked/live shrimp, and 2) intensity of coloration is positively correlated with body weight traits and hence selection for body weight will also improve color traits in this population.

Genome assembly of the Australian black tiger shrimp (Penaeus monodon) reveals a novel fragmented IHHNV EVE sequence.

Shrimp are a valuable aquaculture species globally; however, disease remains a major hindrance to shrimp aquaculture sustainability and growth. Mechanisms mediated by endogenous viral elements have been proposed as a means by which shrimp that encounter a new virus start to accommodate rather than succumb to infection over time. However, evidence on the nature of such endogenous viral elements and how they mediate viral accommodation is limited. More extensive genomic data on Penaeid shrimp from different geographical locations should assist in exposing the diversity of endogenous viral elements. In this context, reported here is a PacBio Sequel-based draft genome assembly of an Australian black tiger shrimp (Penaeus monodon) inbred for 1 generation. The 1.89 Gbp draft genome is comprised of 31,922 scaffolds (N50: 496,398 bp) covering 85.9% of the projected genome size. The genome repeat content (61.8% with 30% representing simple sequence repeats) is almost the highest identified for any species. The functional annotation identified 35,517 gene models, of which 25,809 were protein-coding and 17,158 were annotated using interproscan. Scaffold scanning for specific endogenous viral elements identified an element comprised of a 9,045-bp stretch of repeated, inverted, and jumbled genome fragments of infectious hypodermal and hematopoietic necrosis virus bounded by a repeated 591/590 bp host sequence. As only near complete linear ∼4 kb infectious hypodermal and hematopoietic necrosis virus genomes have been found integrated in the genome of P. monodon previously, its discovery has implications regarding the validity of PCR tests designed to specifically detect such linear endogenous viral element types. The existence of joined inverted infectious hypodermal and hematopoietic necrosis virus genome fragments also provides a means by which hairpin double-stranded RNA could be expressed and processed by the shrimp RNA interference machinery.

Premature termination codons in PRPF31 cause retinitis pigmentosa via haploinsufficiency due to nonsense-mediated mRNA decay.

Dominant mutations in the gene encoding the mRNA splicing factor PRPF31 cause retinitis pigmentosa, a hereditary form of retinal degeneration. Most of these mutations are characterized by DNA changes that lead to premature termination codons. We investigated 6 different PRPF31 mutations, represented by single-base substitutions or microdeletions, in cell lines derived from 9 patients with dominant retinitis pigmentosa. Five of these mutations lead to premature termination codons, and 1 leads to the skipping of exon 2. Allele-specific measurement of PRPF31 transcripts revealed a strong reduction in the expression of mutant alleles. As a consequence, total PRPF31 protein abundance was decreased, and no truncated proteins were detected. Subnuclear localization of the full-length PRPF31 that was present remained unaffected. Blocking nonsense-mediated mRNA decay significantly restored the amount of mutant PRPF31 mRNA but did not restore the synthesis of mutant proteins, even in conjunction with inhibitors of protein degradation pathways. Our results indicate that most PRPF31 mutations ultimately result in null alleles through the activation of surveillance mechanisms that inactivate mutant mRNA and, possibly, proteins. Furthermore, these data provide compelling evidence that the pathogenic effect of PRPF31 mutations is likely due to haploinsufficiency rather than to gain of function.

Growing backwards: an inverted role for the shrimp ortholog of vertebrate myostatin and GDF11.

Myostatin (MSTN) and growth differentiation factor-11 (GDF11) are closely related proteins involved in muscle cell growth and differentiation as well as neurogenesis of vertebrates. Both MSTN and GDF11 negatively regulate their functions. Invertebrates possess a single ortholog of the MSTN/GDF11 family. In order to understand the role of MSTN/GDF11 in crustaceans, the gene ortholog was identified and characterized in the penaeid shrimp Penaeus monodon. The overall protein sequence and specific functional sites were highly conserved with other members of the MSTN/GDF11 family. Gene transcripts of pmMstn/Gdf11, assessed by real-time PCR, were detected in a variety of tissue types and were actively regulated in muscle across the moult cycle. To assess phenotypic function in shrimp, pmMstn/Gdf11 gene expression was downregulated by tail-muscle injection of sequence-specific double-stranded RNA. Shrimp with reduced levels of pmMstn/Gdf11 transcripts displayed a dramatic slowing in growth rate compared with control groups. Findings from this study place the MSTN/GDF11 gene at the centre of growth regulation in shrimp, but suggest that, compared with higher vertebrates, this gene has an opposite role in invertebrates such as shrimp, where levels of gene expression may positively regulate growth.

Penaeus monodon is protected against gill-associated virus by muscle injection but not oral delivery of bacterially expressed dsRNAs.

Gill-associated virus (GAV) is a nidovirus that commonly infects Penaeus monodon (black tiger shrimp) in eastern Australia, causing morbidity and mortalities in the acute stage of disease. Here we explored the possibility of inhibiting GAV replication and disease using double-stranded (ds)RNAs expressed in bacteria and delivered either orally or by muscle injection. To enhance potential RNA interference (RNAi) responses, 5 long dsRNAs were used that targeted open reading frame 1a/1b (ORF1a/b) gene regions and thus only the genomic length RNA. To examine oral delivery, P. monodon were fed pellets incorporating a pool of formalin-fixed bacteria containing the 5 GAV-specific dsRNAs before being injected with a minimal lethal GAV dose. Feeding with the pellets continued post-challenge but did not reduce mortality accumulation and elevation in GAV loads. In contrast, muscle injection of the dsRNAs purified from bacteria was highly effective at slowing GAV replication and protecting shrimp against acute disease and mortalities. In synergy with these data, dsRNA targeted to P. monodon beta-actin mRNA caused 100% mortality following injection, whilst its oral delivery caused no mortality. Findings confirm that injected dsRNA can mount effective RNAi responses in P. monodon to endogenous shrimp mRNA and exogenous viral RNAs, but when delivered orally in bacteria as a feed component, the same dsRNAs are ineffective. The efficacy of the RNAi response against GAV provided by injection of dsRNAs targeted to multiple genome sites suggests that this strategy might have general applicability in enhancing protection against other shrimp single-stranded (ss)RNA viruses, particularly in hatcheries or breeding programs where injection-based delivery systems are practical.

Evolution of a novel carotenoid-binding protein responsible for crustacean shell color.

Carotenoids are commonly used by disparate metazoans to produce external coloration, often in direct association with specific proteins. In one such example, crustacyanin (CRCN) and the carotenoid astaxanthin combine to form a multimeric protein complex that is critical for the array of external shell colors in clawed lobsters. Through a combined biochemical, molecular genetic, and bioinformatic survey of the distribution of CRCN across the animal kingdom, we have found that CRCNs are restricted to, but widespread among, malacostracan crustaceans. These crustacean-specific genes separate into two distinct clades within the lipocalin protein superfamily. We show that CRCN differentially localizes to colored shell territories and the underlying epithelium in panulirid lobsters. Given the paramount importance of CRCN in crustacean shell colors and patterns and the critical role these play in survival, reproduction, and communication, we submit that the origin of the CRCN gene family early in the evolution of malacostracan crustaceans significantly contributed to the success of this group of arthropods.

Resolving hemocyanin isoform complexity in haemolymph of black tiger shrimp Penaeus monodon - implications in aquaculture, medicine and food safety.

Hemocyanin (Hc) is a multifunctional macromolecule involved in oxygen transport and non-specific immunity in shrimp. Hc is crucial in physiology and nutrition linked with optimal performance in aquaculture production systems. In medicine, Hc has been approved for clinical use in humans as adjuvant and anticancer therapeutic. In contrast, Hc has also been identified as one of the proteins causing anaphylaxis following shrimp consumption. The role of individual Hc isoforms remains unknown due to a lack of resolved Hc isoforms. We successfully identified eleven different Penaeus monodon hemocyanin (PmoHc) γ isoforms including two truncated isoforms (50 and 20 kDa) and one PmoHc ß isoform in haemolymph using proteomics informed by transcriptomics. Amino acid sequence homology ranged from 24 to 97% between putative PmoHc gene isoforms. Hc isoforms showed specific patterns of transcript expression in shrimp larval stages and adult hepatopancreas. These findings enable isoform level investigations aiming to define molecular mechanisms underpinning Hc functionality in shrimp physiology and immunity, as well as their individual immunogenic role in human allergy. Our research demonstrates the power of proteomics informed by transcriptomics to resolve isoform complexity in non-model organisms and lay the foundations for improved performance within the aquaculture industry and advance allergenic applications in medicine. SIGNIFICANCE: The roles of hemocyanin (Hc) in shrimp homeostasis and immunity as well as in human allergy are not well understood because the complexity of Hc isoforms has remained unresolved. Our results have confirmed the existence of at least 12 individual Hc isoforms in shrimp haemolymph and validated putative Hc gene assemblies from transcriptomics. Our findings will enable monitoring the expression of specific Hc isoforms in shrimp haemolymph during different environmental, nutritional and pathogenic conditions, thus providing insights into isoform specific functional roles. In medicine, the potential allergenicity of each Hc isoform could be determined and advance allergenic applications. Lastly, since Hc comprises up to 95% of the total protein in haemolymph, these isoforms become ideal targets for prawn provenance, traceability and food contamination studies.