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The large hemi-discoid diatom Palmerina ostenfeldii was common in subtropical Moreton Bay, Australia, following a flood in January 2011. All diatom cells exhibited diagnostic subapical folds settled by loricate peritrich ciliates, but which readily abandoned stressed diatom cells. We characterized both diatoms and ciliates by morphological and molecular analyses, including careful video observations on non-preserved cells immediately after collection. The fold in the diatom cell wall comprises a narrow shelf upon which the ciliates attach (on average seven per fold, and similar for the two folds of a single diatom cell) but without penetrating the diatom wall itself. Folds were fully developed in newly formed internal valves, indicating that the ciliates play no role in their morphogenesis. SSU rRNA sequences of P. ostenfeldii from Moreton Bay (with ciliates) differed by 25 bp (1.5%) from those of P. hardmaniana from Texas (without ciliates), but surprisingly rbcL chloroplast sequences for both diatom species were indistinguishable. The ciliate species epiphytic on P. ostenfeldii, previously referred to as the cold-water tintinnid Amphorella borealis could not be assigned to any known species. We formally describe it here as Vaginicola collariforma sp. nov. within the crown clade of peritrichs which also includes the closely related genus Cothurnia (but distinguished as having an external stalk). Beating of the peritrich oral cilia was observed to drive rotational movement of the large discoid diatoms as in a ferris wheel. The observed diatom-ciliate symbiosis may represent a unique evolutionary adaptation in which the ciliate is host-specific and attaches in a specific locality on the host cell.

In shrimp aquaculture, manufactured diets that include various supplements and alternative fishmeal ingredients are increasingly being used and their effect on the gastrointestinal (GI) microbiota studied. However, dietary effects on different shrimp GI samples are not known. We investigated how a high (HFM) or low (LFM) fishmeal diet affects bacterial communities from different sample types collected from Penaeus monodon gastrointestinal tract. Bacterial communities of the stomach, intestine tissue and intestine digesta were assessed using 16s rRNA gene sequencing. The feed pellets were also assessed as a potential source of bacteria in the GI tract. Results showed substantial differences in bacterial communities between the two diets as well as between the different sample types. Within the shrimp GI samples, stomach and digesta communities were most impacted by diet, while the community observed in the intestine tissue was less affected. Proteobacteria, Firmicutes and Bacteroidetes were the main phyla observed in shrimp samples, with enrichment of Bacteroidetes and Firmicutes in the LFM fed shrimp. The feed pellets were dominated by Firmicutes and were largely dissimilar to the shrimp samples. Several key taxa were shared however between the feed pellets and shrimp GI samples, particularly in the LFM fed shrimp, indicating the pellets may be a significant source of bacteria observed in shrimp GI samples. In summary, both diet and sample type influenced the bacterial communities characterised from the shrimp GI tract. Thus, it is important to consider the sample type collected from the GI tract when investigating dietary impacts on gut bacterial communities in shrimp. KEY POINTS: • Shrimp gastrointestinal communities are influenced by diet and sample type. • The low fishmeal diet enriched bacteria that aid in polysaccharide metabolism. • Feed pellets can be a source of bacteria-detected gastrointestinal tract of shrimp.

Natural and traditional diets have been gradually replaced with formulated diets for the Chinese mitten crab Eriocheir sinensis culture, but it is unclear what effects different feeding modes have on digestive physiology and metabolism of E. sinensis. This study investigated the effects of three feeding modes, formulated diets (FD), natural diets (ND, trash fish and freshwater snails) and traditional diets (TD, soybean, wheat, broad bean and corn) on digestive enzyme activity, physiological indices and metabolomics of adult female E. sinensis. The ND had the highest activities of trypsin and lipase in the hepatopancreas, while the highest amylase activity was detected in TD. The highest content of hepatopancreas triglyceride was detected in FD, while the highest levels of lipoprotein lipase, hepatic lipase, lactic acid, malonaldehyde, acid phosphatase and alkaline phosphatase in the hepatopancreas, as well as the total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol and γ-glutamyltransferase in serum were detected in ND. The TD had the highest content of glucose and uric acid in the hepatopancreas as well as the free cholesterol, lactic acid, aspartate aminotransferase and alanine aminotransferase in the serum. The metabolomics showed that most differential metabolites involved in amino acid anabolism in serum and hepatopancreas were significantly up-regulated in FD. The metabolites involved in energy metabolism and gluconeogenesis in the hepatopancreas were significantly down-regulated in FD. These results suggested that feeding with formulated diets promotes amino acid synthesis and reduces energy metabolism, which leads to more protein and glucose deposition in tissue.

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.

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 (EVEs) 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 EVEs 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 EVEs. In this context, reported here is a PacBio Sequel-based draft genome assembly of an Australian black tiger shrimp (Penaeus monodon) inbred for one 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 EVEs identified an element comprised of a 9,045 bp stretch of repeated, inverted and jumbled genome fragments of Infectious hypodermal and hematopoietic necrosis virus (IHHNV) bounded by a repeated 591/590 bp host sequence. As only near complete linear ~4 kb IHHNV 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 EVE types. The existence of joined inverted IHHNV genome fragments also provides a means by which hairpin dsRNAs could be expressed and processed by the shrimp RNA interference (RNAi) machinery.

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.

The effects of dietary methionine (Met) supply on the postprandial pattern of plasma free amino acids and the differential expression of several genes associated with a number of sulfur amino acid and protein turnover pathways in the liver of juvenile barramundi (Lates calcarifer) was investigated. At the conclusion of a 49-day growth trial assessing the requirement for dietary Met, three treatments were selected (with deficient (DEF; 8.6 g kg−1), adequate (ADQ; 14.9 g kg−1) and excessive (EXC; 21.4 g kg−1)) levels of dietary Met, based on their respective growth responses. A peak occurred in plasma free Met at 2 h post-feeding in fish fed the DEF and ADQ diets and at 4 h post-feeding in fish in the EXC treatment. Liver samples collected at these timepoints, as well as those taken as a pre-feeding control, were analyzed for expression of genes involved in Met turnover (CGL, MAT-1, MAT-2a) and taurine biosynthetic pathways (CSAD, ADO, CDO), target of rapamycin inhibition (Redd-1), the somatotropic axis (GHR-II, IGF I, IGF-II) and protein turnover pathways (MUL-1, ZFAND-5). Markers of sulfur amino acid turnover were more significantly affected by time after feeding than by dietary Met level, suggesting production of these enzymes may be primarily regulated by the consumption of feed or protein, rather than by the dietary composition. Further, metabolised Met appeared likely to have been directed through S-Adenosylmethionine (SAM) dependent pathways, rather than converted to Cys, which may have contributed to the observed growth response. Both genes influencing the conversion of Met to SAM appear to be active at this lifestage in barramundi. Previously described markers of proteolytic pathways appear to be conserved in this species and we have confirmed that ZFAND-5 is a reliable biomarker of this process in barramundi. A number of important genes were investigated for the first time in this species and shown to be nutritionally regulated.

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.

Juvenile western rock lobsters modify their shell colour from deep red (herein referred to as Red) to a pale pink during a mass migration off the coast of Western Australia. This colour change and subsequent migration coincides with the landing of a large proportion of the annual quota. Termed the ‘white’ phase by the industry (herein referred to as the White phase), these lobsters have a decreased market value and results in a multi-million dollar loss annually. Shell colour in decapod crustaceans is primarily determined by the carotenoid astaxanthin binding to carotenoid binding protein (CBP) to form a macromolecular protein complex known as crustacyanin. This data presented here comprehensively investigates P. cygnus CBP and the relationship of this protein with the White phase of the western rock lobster. Furthermore, this research demonstrates the use of this species as a model for investigating the regulation of shell colour formation in crustaceans. Firstly, carotenoid analysis of various tissues showed, as predicted, there is significantly less astaxanthin in White western rock lobsters in comparison to Reds. More importantly however, an esterified astaxanthin derivative within the hypodermal tissue appears to be involved in the regulation of shell colour. Secondly, environmental variables such as diet and background substrate colour are known to affect crustacean shell colour. These variables were thoroughly investigated and shown not to be involved in the onset of the White phase, suggesting this phenomenon is under genetic control. Thirdly, CBP was identified within the shell of P. cygnus, and the CBP gene isolated and characterised from this species. An antibody raised against CBP detected a distinct 20 kDa band that formed a large multimeric complex similar to the crustacyanin complex identified in other lobsters. This protein was detected along the outer margin of the shell in significantly lower levels in Whites compared to Reds. Nevertheless, quantitative analysis of CBP gene expression showed no difference between these two groups, suggesting that CBP abundance in the shell was due to protein accumulation not differential gene expression. Differential protein prevalence of CBP in White lobsters further supports the proposition that this colour difference is chiefly related to genetics and not diet. Lastly, a number of nuclear receptor genes were isolated from P. cygnus and their tissue expression profiles investigated. Many of these genes have been shown to be involved in regulating gene expression during the moult cycle in arthropods and may potentially play a role in morphological colour adaptation in crustaceans. Combined, these data suggest that lobsters held in the White phase provide a model for investigating the mechanisms of crustacean shell colour formation in all crustacean species. The results provide insight into the regulation of colour production in decapod crustaceans and the ability to modify crustacean shell colour in aquaculture. This research provides further implications for the molecular basis of the bathochromic colour shift produced by CBP upon binding to astaxanthin. Finally, these findings contribute to our understanding of the evolution of the lipocalin gene family and the evolution of cryptic and adaptive colouration in crustaceans.

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.

Shellfish allergy affects up to 2% of the world’s population and persists for life in most patients. The diagnosis of a shellfish allergy, in particular shrimp, is however often challenging due to the similarity of allergenic proteins in other invertebrates. Despite the clinical importance, the complete allergen repertoire of allergy-causing shrimps remains unclear. 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 assembledde novofrom 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). Trinity was used to assemble the transcriptome, and Transrate and BUSCO applied to verify the assembly. Blast search with the two major allergen databases, WHO/IUIS Allergen Nomenclature and AllergenOnline, successfully identified all seven known crustacean allergens. Salmon was utilised to measure their relative abundance, demonstrating sarcoplasmic calcium-binding protein, arginine kinase and myosin light chain as highly abundant allergens. In addition, the analyses revealed up to 40 unreported allergens in 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, conducted in Jalview 2.1 with 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 genomic resource for investigating shellfish allergens, comparing invertebrate allergens and developing improved diagnostics and novel immunotherapeutics for food allergy.

The natural flight response in shrimp is powered by rapid contractions of the abdominal muscle fibres to propel themselves backwards away from perceived danger. This muscle contraction is dependent on repetitive depolarization of muscle plasma membrane, triggering tightly spaced cytoplasmic [Ca2+] transients and rapidly rising tetanic force responses. To achieve such high amplitude and high frequency of Ca2+ transients requires a high abundance of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) to rapidly clear cytoplasmic Ca2+ between each transient and an efficient Ca2+ release system consisting of the Ryanodine Receptor (RyR), and voltage gated Ca2+ channels (CaVs). With the aim to expand our knowledge of muscle gene function and identify orthologous genes regulating muscle excitation-contraction (EC) coupling, this study assembled nine Penaeid shrimp muscle transcriptomes. On average, the nine transcriptomes contained 27,000 contigs, with an annotation rate of 36% and a BUSCO completeness of 70%. Despite maintaining their function, the crustacean RyR and CaV proteins showed evidence of significant diversification from mammalian orthologs, while SERCA remained more conserved. Several key components of protein interaction were conserved, while others showed distinct crustacean specific evolutionary adaptations. Lastly, this study revealed approximately 1,000 orthologous genes involved in muscle specific processes present across all nine species.

The digestibility of a suite of raw materials was determined when fed to black tiger shrimp (Penaeus monodon) in a series of three experiments. A total of 29 commercial and research raw materials were evaluated using the diet replacement digestibility method. Each of the reference and test diets were fed to tanks of shrimp for one-week prior to commencing faecal collection. The collected faecal samples were kept separate from any feed residue through using a discrete feeding period, after which uneaten feed was removed before a separate faecal collecting period. The same reference diet and soy protein concentrate diet were used across each of the three experiments and demonstrated consistent digestibility using this method. Most raw materials demonstrated some utility for use in diets for shrimp, with digestible protein or energy values >0.800. However, there were some raw materials (e.G. camelina meal) that provided very little nutritive value for shrimp. This study presents data on the digestibility and digestible nutrient content of a wide variety of raw materials, providing a clear basis for progressing to formulating shrimp diets on a digestible protein and energy basis, thereby optimising dietary formulation, maximising ingredient utilisation and reducing impacts of uneaten feed.

In shrimp aquaculture, the ability to reliably test for the presence and load of pathogens is vital for managing disease. As pathogen infection loads are not distributed homogenously throughout different tissues, detection by PCR can vary based on what tissue type is tested. Moreover, there is potential for infection loads to vary within the same tissue type depending on infection severity and nature of the tissue. This study used reverse transcriptase real-time quantitative PCR (RT-qPCR) to examine the sensitivity and variability in gill-associated virus (GAV) detection levels within and between different tissue types sampled from individual black tiger (Penaeus monodon) shrimp. From the group of juvenile shrimp (Group 1 - 10.9 ± 1.4 g, n = 10) with low-level GAV infection, eight gill filaments and eight pleopods were tested from each individual, and from another group of adult shrimp (Group 2 - 40.4 ± 2.3 g, n = 12) with higher-level GAV infection, 10 gill filaments, 10 pleopods and both lymphoid organ lobes were tested from each individual. For Group 1, 69/80 gill and 67/80 pleopod samples tested PCR-positive, and for Group 2, all samples tested PCR-positive. In some shrimp from each group, GAV detection levels among gill filaments or pleopods varied by up to ~3000-fold, highlighting the variability that is present in infection load among these tissues within individual shrimp. Statistical analyses used to determine the most sensitive and consistent tissue type to test identified no significant differences between mean GAV infection loads detected, either in gill or pleopod tissue, within each group (Group 1, p = 0.852; Group 2, p = 0. 146). Similarly, variability in GAV detection levels among individual gill filaments and pleopods did not differ between either tissue for each group (Group 1, p = 0.922; Group 2, p = 0.151). The mean GAV infection load (GAV RNA copies μg−1 TNA) detected in lymphoid organ lobes tested from Group 2 adult shrimp (5.96 × 107 ± 8.67 × 107 copies), was significantly higher (p

A range of inert digestibility markers, such as yttrium(Y) and ytterbium (Yb) oxides, are used to indirectly calculate the digestibility of nutrients in aquafeeds. Gross over-estimations of nutrient digestibility can be encountered if several assumptions are not met, including that nutrients move through the digestive system at the same rate as the markers. The present study sought to assess the effect of particle size on apparent nutrient digestibility, and traced the movement of several inert metal markers (Y, Yb and gold) of various sizes (>3 μm, 400 nm, 100 nm, 50 nm and 5 nm) through the shrimp digestive tract. The first of two experiments assessed the effect of particle size on nutrient digestibility using traditional faecal collection methods, while the second traced particle movement within the stomach, hepatopancreas and intestine at various times (1, 2, 3, 5, 10 and 18 h) after a set ration. This work showed that particle size had a significant effect on nutrient digestibility, and that 50 nm and 5 nm gold particles had relative particle retention coefficients of 37.3 ± 2.7% and 48.9 ± 1.8%, respectively. Gold particles were detected in the hepatopancreas of animals fed 5 nm gold for >18 h, indicating that small particles remain within the animal for long periods of time. This is despite that the shrimp digestion was extremely rapid, with evacuation rate of the digestive tract calculated at 22.1% h−1. Large Y and Yb particles (>3 μm) found within the animal 1-hour after feeding showed that the amount of true feed intake was lower than measured feed intake by 15–25%, and provided an estimate of manipulative losses during feeding. Overall, this study showed that the shrimp filter press was highly efficient at excluding small indigestible particles, to below 400 nm, and that extremely high rates of nutrient digestion were maintained despite rapid movement through the digestive system. This work may in part account for the disparity between high levels of apparent nutrient digestibility and low nutrient utilisation efficiency measured in shrimp.

Increased disease tolerance is an important trait commonly incorporated into shrimp breeding programs. Sib-selection using experimental pathogen challenge tests are used to measure family based tolerance. It is critical for challenge tests to be standardised and repeatable for accurate genetic estimation. Accordingly, this study focused on establishing a method for uniformly challenging Black Tiger shrimp ( Penaeus monodon ) to an acute gill-associated virus (GAV) infection. A stock inoculum of GAV (10 5.27 GAV RNA copies μL − 1 inoculum) was prepared from infected shrimp and used as the source of infection for the challenge methodologies investigated. Three challenge approaches compared in groups of juvenile P. monodon (2–10 g) were; intramuscular injection, ingestion of fresh or frozen diced muscle tissue from GAV-infected shrimp and immersion in water containing the GAV inoculum. Intramuscular injection using a weight-standardised dose of the inoculum resulted in cumulative mortality surpassing 50% on Day 7 post-challenge. Immersion of shrimp for 2 h in water comprising a 1:1000 dilution of the inoculum resulted in no mortalities over the 11 day trial period. GAV RT-qPCR analysis also identified no evidence of any shrimp becoming infected by the immersion method. Feeding shrimp either fresh or frozen diced muscle tissue of moribund shrimp with an acute GAV infection generated cumulative mortality that reached/surpassed 50% on day 11 post-challenge. Compared to the survivors of the injection group (mean = 10 7.73 ± 0.49 GAV RNA copies/μg total RNA), RT-qPCR analysis identified GAV infection loads to be less uniform and 100- to 1000-fold lower among survivors of feeding either fresh or frozen muscle tissue (mean = 10 5.71 ± 1.97 or 10 4.77 ± 1.90 GAV RNA copies/μg total RNA, respectively). Coefficient of variation (CV) values for the GAV infection load data supported survivors of injection (CV = 0.06) having a more uniform infection load compared to the survivors of either fresh (CV = 0.41) or frozen (CV = 0.33) tissue ingestion. Of the three challenge approaches assessed for their suitability in measuring GAV tolerance, injection was clearly identified to provide the most uniform and reliable means of infecting shrimp with a defined dose of GAV and therefore is the preferred method of infection to use for GAV challenge tests.

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.

The serial replacement of fish meal (anchovetta) by canola meal (CM) (100, 200, 300 g kg − 1 as either solvent extracted (SE) CM or expeller extracted (EX) CM was undertaken to investigate the effects of increasing dietary CM levels on feed intake, growth, protein and energy retention, plasma biochemistry and the expression of a suite of hepatic genes in barramundi (Asian seabass; Lates calcarifer ) over an eight week feeding trial. An additional diet using lupin kernel meal (LM) to replace the fish meal was also included as a comparative reference. Eight iso-digestible nitrogenous (423 ± 29 g kg − 1 ) and iso-digestible energetic (14.6 ± 8 MJ kg − 1 DM) diets were formulated. Each diet was randomly allocated to triplicate groups of fish in seawater tanks (600 L), and each tank was stocked with 15 fish (53.4 ± 7.0 g). Fish were fed once daily (9:00–10:00) to apparent satiation, and uneaten feed was collected to determine feed consumption. The results showed that the survival, feed intake, growth, FCR, energy and protein retention of fish fed the diet containing SE CM were similar or even higher to those of fish fed the fish meal reference diet (FM) and the LM diet. Fish fed with the diet containing 300 g kg − 1 SE CM did not show any changes in biochemistry and gene expression in a suite of detoxification genes. However, the diet with 300 g kg − 1 EX CM depressed feed intake, growth performance and increased feed conversion ratio (FCR). Transcription of genes involving in fatty acid synthesis and the TCA cycle were not changed by different diets. The down regulation of gene expression in certain detoxification genes ( Lc CYP1A1 , Lc CYP3A , Lc CYP2N and Lc GST ) was observed in fish fed with the diet containing 300 g kg − 1 EX CM compared to the FM control diet and other experimental diets. In general, the SE CM can be used up to 300 g kg − 1 diet without negative performance effects or signs of clinical plasma biochemistry. By contrast the maximum acceptable level of the EX CM for barramundi was only 200 g kg − 1 . Higher inclusion level of the EX CM induced negative effects on growth performance, feed utilisation, plasma biochemistry and gene expression in relation to detoxification. Statement of relevance Previous research has demonstrated that canola meal is a potential plant protein source for fish meal replacement in diets for many fish species. The present work is designed to be the first to assess both two types of Australian canola meal in regard to different processing methods (solvent extraction and expeller extraction) in use for barramundi (Asian seabass) diets. The research not only assesses effects of canola meal on growth performance and feed utilisation but also investigate effects of canola meal use on fish health status using broad approach including plasma chemistry, histology and gene expression analysis. The present findings provide practical information for barramundi's diet formulation using canola meal as an additional plant ingredient. These also provide implications of potential health effects relating to using canola meal for barramundi.

For many fish species, dietary fish oil (FO) has been substituted with other oils such as poultry oil (PO) without affecting growth performance. However, in barramundi, the mechanisms by which fatty acid metabolism is regulated are poorly understood, and the effects of FO substitution are unknown. This study defined changes in the expression of genes controlling the metabolism of fatty acids in barramundi over a 24-h time period after a single meal. From one to 12 h after a single feeding event, the expression of fatty acid synthesis genes in the liver was upregulated, while genes involved in the β-oxidation showed minimal alteration. However, the expression of β-oxidation genes was significantly correlated with the expression of genes regulating fatty acid synthesis. In a second experiment, the changes in liver fatty acid composition and gene expression were defined after FO was substituted with PO. Liver fatty acid profile reflected the diet composition, with some subtle exceptions supporting the enrichment of certain long-chain polyunsaturated fatty acids in the liver. The fish from all experimental groups preferentially retained more docosahexaenoic acid than eicosapentaenoic acid in the liver, suggesting a bioconversion of this fatty acid to intermediate fatty acids. Replacement of FO with PO significantly regulated genes controlling both fatty acid synthesis and catabolism pathways, potentially related to a higher percentage of monounsaturated fatty acids, in the livers of fish fed these diets. The results demonstrated that diet composition significantly altered the lipid metabolism in barramundi and that there was a balance between direct dietary effects and endogenous synthetic capacity.

A two part experiment was conducted to assess the response of barramundi (Lates calcarifer; initial weight = 10.3 ± 0.03 g; mean ± S.D.) fed one of five diets with varying eicosapentaenoic acid (diets 1, 5, 10, 15 and 20 g/kg) or one of four diets with varying arachidonic acid (1, 6, 12, 18 g/kg) against a fish oil control diet. After 6 weeks of feeding, the addition of EPA or ARA did not impact on growth performance or feed utilisation. Analysis of the whole body fatty acids showed that these reflected those of the diets. The ARA retention demonstrated an inversely related curvilinear response to either EPA or ARA. The calculated marginal utilisation efficiencies of EPA and ARA were high (62.1 and 91.9 % respectively) and a dietary ARA requirement was defined (0.012 g/kg(0.796)/day). The partial cDNA sequences of genes regulating eicosanoid biosynthesis were identified in barramundi tissues, namely cyclooxygenase 1 (Lc COX1a, Lc COX1b), cyclooxygenase 2 (Lc COX2) and lipoxygenase (Lc ALOX-5). Both Lc COX2 and Lc ALOX-5 expression in the liver tissue were elevated in response to increasing dietary ARA, meanwhile expression levels of Lc COX2 and the mitochondrial fatty acid oxidation gene carnitine palmitoyltransferase 1 (Lc CPT1a) were elevated in the kidney. A low level of EPA increased the expression of Lc COX1b in the liver. Consideration should be given to the EPA to ARA balance for juvenile barramundi in light of nutritionally inducible nature of the cyclooxygenase and lipoxygenase enzymes.

An experiment was conducted to assess the response of juvenile barramundi ( Lates calcarifer ) to four diets containing either marine- or non-marine derived neutral lipid (NL) or polar lipid (PL) sources for eight weeks in a 2 × 2 factorial design. The four diets contained 8.2% added lipid composed of a 1% fish oil base with 7.2% test lipid (n − 3 NL: Fish oil, n − 3 PL: Krill oil, n − 6 NL: Soybean oil, n − 6 PL: Soybean lecithin). The results demonstrated that the different lipid sources (either n − 3 or n − 6 omega series from either NL or PL class) had significant effects on growth performance and feed utilisation with some interaction terms noted. Growth was negatively affected in the n − 6 NL fish and the feed conversion (FCR) was highest in the n − 6 PL fish. Digestibility of total lipid and some specific fatty acids (notably 18:2n − 6 and 18:3n − 3) were also negatively affected in the n − 6 PL fish. Analysis of the whole body neutral lipid fatty acid composition showed that these mirrored those of the diets and significant interaction terms were noted. However, the whole body polar lipid fatty acids appeared to be more tightly regulated in comparison. The blood plasma biochemistry and hepatic transcription of several fatty acid metabolism genes in the n − 6 PL fed and to a lesser extent in the n − 6 NL fed fish demonstrated a pattern consistent with modified metabolic function. These results support that there are potential advantages in using phospholipid-rich oils however there are clear differences in terms of their origin. Statement of relevance Juvenile barramundi may benefit from dietary phospholipid.

An experiment was conducted to examine the potential for the complete replacement of fishmeal (FM) and fish oil (FO) in diets for barramundi, Lates calcarifer . A series of diets were formulated to the same digestible protein and energy specifications, but which were designed with FM inclusion levels at 300, 200, 100 or 0 g/kg and FO at 100%, 30%, 15% or 0% of the added oil in the diets (4 × 4 factorial design). Ricebran oil was the alternative oil used in the growth study, while soybean meal and poultry meal were the main alternative protein sources used. For the growth study, fish of an initial weight of 154.4 ± 1.1 g were randomly allocated across 48 tanks (three replicates per treatment). After eight weeks, the average weight gain across all treatments was 187.7 ± 2.3 g/fish and feed conversion across all treatments averaged 1.04 ± 0.01 feed/gain. A significant effect of FM on both feed intake and weight gain was observed, and this was observed as early as within the first few weeks, but no similar such effect was observed with FO. No effects were observed on protein deposition efficiency, though both lipid and energy deposition efficiencies were affected by FM level. The reduction in FO had a notable effect on the fatty acid composition of the diets and subsequently the fish fatty acid composition. Expression of key LC-PUFA metabolism genes in the liver of the fish was influenced by both FM and FO levels, but was only significant at the extremes of the treatment ranges. The results from this study demonstrate that there is clear potential to replace almost all the FM content of barramundi diets without loss of fish performance, up to and including diets with as little as 100 g/kg fishmeal. Replacement of fish oil was more successful with the ability to completely replace all FO demonstrated at all but the lowest inclusion levels of FM. These results clearly demonstrate that the near complete replacement of both FM and FO in barramundi diets is a technical reality.

A 40-day reproductive performance trial was conducted to assess the effect of targeted supplementation of Penaeus monodon broodstock grow-out and maturation diets with microbial biomass (MB). Over a seven month grow-out period, shrimp were fed a typical pelleted grow-out diet with or without 10% MB. Broodstock were then transferred to a maturation facility and a subset of animals from each grow-out diet fed on a typical fresh-frozen maturation diet that included a pellet ration with or without 30% MB (5.5% of total diet fed). At nine months of age, female broodstock were unilaterally eyestalk-ablated and reproductive assessments commenced. No significant difference in ovary maturation, hepatosomatic index, spawning and egg and nauplii production parameters was found between diet treatments (p = > 0.05). However, females originating from control ponds displayed a higher gonadosomatic index at first spawn, whilst the percentage of eggs that hatched was lower in females fed a MB-inclusive maturation diet (p =

The colour of prawns, particularly the Giant Tiger Prawn Penaeus monodon, is highly desired and fetches premium market prices. Prawn pigmentation is influenced by the interaction of a range of factors, including the amount of dietary carotenoid, the distribution of hypodermal pigments, and genetics. These aspects have been studied in isolation, but there is limited knowledge on how these components interact to influence prawn pigmentation. This study tracked the colour of prawns that had been fed four different levels of dietary astaxanthin (Axn) over 6 weeks, and then transferred to either black or white coloured tanks. The dietary influence on colour was slow and had only developed after 6 weeks. Meanwhile the effect of background colour was rapid, within 15 min. Results showed that diet and background colour work in combination to affect prawn colour. The poorest colour was recorded in prawns fed without dietary Axn and transferred to white substrates, and this colour was improved by the addition of dietary Axn. Animals fed without dietary Axn and exposed to black substrates showed an intermediate colour, and this was further improved by the addition of dietary Axn. The best colour was recorded in prawns fed 100 mg/kg Axn and exposed to black substrates. The abundance of the epithelial pigment protein crustacyanin (CRCN) was not correlated with prawn colour, suggesting that this protein does not regulate the modifications in response to background colour. Finally, the effect of substrate exposure was assessed on farmed prawns, and indicated a small positive effect on colour during harvesting. These data demonstrate that while short term exposure to black substrates can have positive effects on prawn colour, dietary Axn supplementation can both improve pigmentation of animals exposed to black substrates, and prevent the negative effects of exposure to white substrates.

Selective breeding to improve performance of growth and increase disease resistance is becoming commonplace within shrimp aquaculture. Whilst estimated breeding values (EBV) for growth can be evaluated easily in on-farm trials, EBVs for disease resistance are usually obtained from controlled laboratory challenges and may not be indicative of natural infection and resistance in a commercial setting. If they are not the same trait, this will result in a low genetic correlation between the traits and application of EBVs derived from the laboratory challenge will lead to overall slow genetic progress for improved disease resistance in on-farm conditions. There is a need for alternative measures of predicting disease resistance based on performance in farm environments. Two such estimates may be viral infection status (presence/absence) and load of farm reared shrimp. This study presents genetic parameter estimates of Gill-Associated Virus (GAV) infection (viral infection presence and viral loading) and body weight traits from Penaeus monodon reared under commercial farm conditions. Body weight and GAV infection traits were assessed by sampling similar to 2000 shrimp from 80 full-sib families from two replicate commercial ponds. In addition, the Pearson correlation of EBVs of pond-reared shrimp with those of siblings derived from communal laboratory GAV challenge tests (intramuscular injection of standard doses of virus) were calculated to establish if prediction of genetic merit in controlled trials would be similar to disease resistance seen on-farm. Heritability under commercial farm conditions for GAV infection status was 0.06 +/- 0.03 (percent GAV positive 45.5%) and infection load 0.21 +/- 0.10 (mean GAV infection load 3.11 +/- 1.14 log(10) GAV copies mu g(-1) TNA) and for body weight h(2) = 0.38 +/- 0.07. A high genetic correlation was observed among the two on-farm GAV infection traits (r(g) = 0.90 +/- 0.24), whereas the genetic correlation between the two GAV infection traits and body weight on-farm were positive although weak (r(g) = 0.26 +/- 0.25; 0.36 +/- 0.26). No significant correlation was found for EBVs of GAV infection (infection status and load) on-farm and GAV-induced mortality or viral load under laboratory challenge testing (R-EBV ranged from -0.06 +/- 0.14 to -0.27 +/- 0.15). Additionally, there was no correlation of EBVs between body weight of shrimp on-farm and GAV-induced disease under controlled challenge. In conclusion, measures of GAV infection from shrimp on-farm and GAV induced mortality measured under controlled challenge conditions may represent different genetic traits, at least when mortality is measured using an 'unnatural' challenge method and when no apparent disease outbreak occurs on farm.

The raw carapace color of Eriocheir sinensis is normally greenblack, while a small percentage of wild crabs have a purple carapace that also attract a higher market price. This study was conducted to quantify color parameters, carotenoids composition, and nutritional composition between purple and greenblack crabs. The redness (a*) values, total astaxanthin and astaxanthin ester content (P

P. monodon juveniles were fed krill meal (KM), krill hydrolysate (KH), whole squid (SQ), and the microbial biomass, Novacq™ (NQ), at 10% inclusion, in practical fishmeal and soybean meal based diets. Diets were fed to apparent satiation as well as restrictively pair-fed (~60% of control diet satiation) for 6 weeks to examine the effects of the test ingredients and feed restriction on shrimp growth, feed efficiency, digestive enzyme activity, nutritional condition, and gut microbiota. All diets when fed to satiety significantly outperformed the control diet, with shrimp fed NQ 87%, SQ 73%, KH 41% and KM 38% larger than shrimp fed the control diet. NQ fed shrimp had the best feed conversion ratio (FCR) and significantly improved retention efficiency (RE) of dietary lipid (RETL), protein (RECP) and gross energy (REGE). SQ significantly promoted feed intake over control and KM fed shrimp, but neither KM, KH nor SQ improved RETL, RECP or REGE. While there was little change in the apparent digestibility (AD) of each diet, the apparent biological value (ABV) of lipid (ABVTL), protein (ABVCP) and gross energy (ABVGE) were significantly improved by inclusion of NQ; and ABVTL and ABVGE were significantly improved by addition of SQ. Feed restriction had a stronger effect on hepatopancreas digestive enzymes and gut microbiota than addition of any feed ingredient. Total protease activity was positively correlated with feed efficiency, including FCR, RETL and REGE. Shrimp fed to satiety tended to have high levels of Vibrio whereas those on the restricted ration tended to have higher levels of bacteria in Rhodobacteracaea, Flavobacteriales and Bacteroidales. We conclude that shrimp are more efficient in using digested dietary macronutrients for growth when whole squid and Novacq™ are provided, due to improvements in feed intake and post-absorptive processes. Feeding restriction was shown to be a useful strategy to improve P. monodon feeding efficiency, digestive capacity and modulate gut microbiota, however it reduced the scope for shrimp growth especially on high performing diets. Novacq™ was more effective than some of the known invertebrate meals and a more environmentally sustainable growth and nutrient utilisation promoter for shrimp.

Diseases in shrimp aquaculture cause significant economic losses, particularly those caused by viruses. Genetic improvement of disease tolerance through selective breeding can be an important tool for managing the risk of disease in shrimp farming as long as the trait has a genetic basis (i.e. heritable) and is able to be accurately and reliably measured. This study presents the first estimates of genetic variation associated with tolerance to gill-associated virus (GAV) in Penaeus monodon using a unique controlled challenge test design. Shrimp families (n = 72) were communally spawned and reared prior to replicated communal challenge testing when they reached an average size of ~8 g. Pedigree determination was carried out using genotyping and shrimp were challenged by intramuscular injection with a weight standardised dose of GAV. Daily mortality data were collected over 15 days from 1717 individuals and GAV infection load was measured on the challenge test survivors (n = 963). Overall mortality after 15 days was 35.5% and ranged from 0 to 71% among families. Heritability estimates for mortality were 0.11 ± 0.03 using a binomial model and 0.14 (standard error not estimated) using Cox's proportional hazards model. The estimated heritability for GAV infection load was 0.21 ± 0.07. The genetic correlation between mortality and GAV infection load was not significantly different from zero, rg = 0.30 ± 0.23, suggesting that the two traits may be genetically independent, at least in the way they were measured in this study. Results from this study suggest a low but significant genetic basis for GAV tolerance in P. monodon and that selection may be a viable option to manage GAV-related disease. The study also demonstrated, for the first time in shrimp, communal-based spawning and rearing can be used for large-scale genetic analysis of disease tolerance, eliminating the need for single family rearing tanks and associated confounding environmental effects.

Crustaceans are exposed to a range of environmental factors that can impact their condition, physiological function and growth. Condition indices are broadly defined as the extent to which stored nutrient reserves allow normal physiological function and growth, but can also represent more than nutrition alone. There is currently no reliable indicator to non-destructively measure shrimp physiological or nutritional condition. In this study, haemolymph and biochemical indices were benchmarked against a range of physiological status condition indicators, including haemolymph biochemistry parameters and carcass traits, in fed and unfed sub-adult black tiger shrimp (Penaeus monodon) and across the moult cycle. Results based on fluorescent marker injection and analysis of recovered amounts showed that haemolymph volume was elevated immediately after the moult, but decreased significantly within 2 days and remained stable for the remainder of the inter-moult period. Brix index (Brix) strongly correlated with haemolymph biochemical, shrimp condition and carcass composition indices. These included haemolymph volume, total protein and triglycerides, as well as gross energy, hepatosomatic index (HSI) and body weight gain per moult. Overall, results demonstrated that Brix was the simplest, fastest and most cost-effective indicator to accurately and non-destructively estimate physiological condition in P. monodon. Given the correlations with a broad range of indices, Brix may represent a more holistic estimate of condition, incorporating multifactorial aspects of shrimp condition including moult cycle and nutrition. Additionally, the baseline measurements of metabolites across the moult and under starvation conditions improves our fundamental understanding of overall condition in P. monodon.

Made available in DSpace on 2018-11-26T17:49:15Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-05-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Marine Biology Center of University of Sao Paulo (CEBIMar) 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. Univ Sao Paulo, Dept Fisiol, Inst Biociencias, Rua Matao,Trav 14,321, BR-05508090 Sao Paulo, SP, Brazil Univ Sao Paulo CEBIMar USP, Ctr Biol Marinha, Rodovia Manoel Hipolito do Rego,Km 131,5, BR-11600000 Sao Sebastiao, SP, Brazil CSIRO Agr & Food, Queensland Biosci Precinct, 306 Carmody Rd, St Lucia, Qld 4067, Australia Fundacao Inst Pesca Estado Rio de Janeiro, Praca Fonseca Ramos S-N, BR-24030020 Niteroi, RJ, Brazil Univ Estadual Paulista, Ctr Aquicultura CAUNESP, Via Acesso Prof Paulo Donato Castelane S-N, BR-14884900 Jaboticabal, SP, Brazil Univ Estadual Paulista, Ctr Aquicultura CAUNESP, Via Acesso Prof Paulo Donato Castelane S-N, BR-14884900 Jaboticabal, SP, Brazil FAPESP: 2011/504074 FAPESP: 2011/50287-2

The black tiger shrimp (Penaeus monodon) remains the second most widely cultured shrimp species globally. However, issues with disease and domestication have seen production levels stagnate over the past two decades. To help identify innovative solutions needed to resolve bottlenecks hampering the culture of this species, it is important to generate genetic and genomic resources. Towards this aim, we have produced the most complete publicly available P. monodon transcriptome database to date. The assembly was carried out in multiple assemblers using 2×125 bp HiSeq data from PolyA selected, ribo-depleted RNA extracted from nine adult tissues and eight early life-history stages. In total, approximately 700 million high-quality sequence reads were obtained and assembled into 236,388 clusters. These were then further segregated into 99,203 adult tissue specific clusters, and 58,678 early life-history stage specific clusters. The final transcriptome had a high TransRate score of 0.37, with 88% of all reads successfully mapping back to the transcriptome. BUSCO statistics showed the assembly to be highly complete with low fragmentation, few genes missing, but higher redundancy or transcript duplication (Complete: 98.2% (Duplicated: 51.3%), Fragmented: 0.8%, Missing: 1.0%), and to greatly exceed the completeness of existing P. monodon transcriptomes. While annotation rates were low (approximately 30%), as is typical for a non-model organisms, annotated transcript clusters were successfully mapped to several hundred functional KEGG pathways. To help address the lack of annotation, transcripts were clustered into groups within tissues and early life-history stages, providing initial evidence for their roles in specific tissue functions, or developmental transitions. Additionally, transcripts of shrimp viruses previously not known to occur in Australia were also discovered. We expect the transcriptome to provide an essential resource to investigate the molecular basis of commercially relevant-significant traits in P. monodon and other shrimp species.

The carotenoid astaxanthin (Axn) plays a vital role in shrimp pigmentation, with direct influence on product quality, and forms a significant cost component of shrimp aquaculture feeds. However, the effects of dietary Axn on other measures of shrimp physiological performance are varied, and the efficiency of carotenoid uptake from the diet and deposition in shrimp tissues is poorly defined. This study fed juvenile shrimp (Penaeus monodon) diets that contained 0, 25, 50 or 100 mg kg−1 Axn for 6 weeks. Shrimp fed carotenoid-free diets had significantly reduced colour and growth than those fed carotenoids, but survival was unaffected. Carotenoid digestibility improved as dietary carotenoid levels increased, and was 98.5% in shrimp fed 100 mg kg−1 Axn. After 6 weeks, whole body carotenoid levels were significantly depleted in 0 or 25 mg kg−1 fed shrimp, compared with those fed 50 or 100 mg kg−1 or compared with initial shrimp. This study also showed that Axn monoesters were enriched with saturated fatty acids, whereas Axn diesters were enriched with monounsaturated and polyunsaturated fatty acids. Combined, these studies demonstrate that a total dietary carotenoid intake of between 25 and 50 mg kg−1 Axn is required for normal shrimp growth and health in P. monodon. Evidence suggests that there is a functional role for the accumulation of carotenoids and the formation of specific Axn fatty acid esters, and these may be linked to the metabolism, storage, mobilization or deposition of Axn within various tissues.

Crustaceans are cultured extensively around the world in intensive farming systems. High-performance formulated feeds have been developed for crustacean aquaculture, which are often supplemented with a number of natural and synthetic carotenoid sources. Studies over a number of years have consistently shown that dietary carotenoid supplementation is beneficial for crustacean aquaculture across a range of commercially relevant parameters. Most obvious is the effect on pigmentation, where carotenoid inclusion levels in feeds and duration of feeding diets with carotenoids have been optimised across many species to improve product colour, and subsequently quality and price. However, beneficial effects of carotenoid inclusion have increasingly been demonstrated on other parameters. This review updates the recent progress in our understanding of dietary carotenoid utilisation and storage, and the combined effects of diet, genetics and environment on crustacean pigmentation. In addition, the range of other physiological benefits this class of molecules brings to these animals is summarised. These include improvements in survival, growth, reproductive capacity, disease resistance and stress resistance.

A series of experiments were conducted to examine the nutrient and energy digestibility of a suite of diets and specific test raw materials when fed to juvenile (179 to 439 g) barramundi, Lates calcarifer. Each of the diets was prepared using a twin-screw extruder to mimic modern aquafeed manufacturing processes. Each of the diets was fed to juvenile barramundi for a minimum of a week to allow acclimation to the diet before the faeces were collected using stripping methods. A broad range of digestible nutrient and energy values among the different raw materials were observed, with protein digestibilities ranging from 36% to 106% and energy digestibilities ranging from 36% to 93%. This range in nutritional values of the different raw materials provides substantial utility in allowing the formulation of diets on a digestible nutrient and energy basis across the Asia Pacific region. These results also provide critical data to help underpin the replacement of both fishmeal and fish oil in barramundi diets.

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.

Cooked prawn colour is known to be a driver of market price and a visual indicator of product quality for the consumer. Although there is a general understanding that colour variation exists in farmed prawns, there has been no attempt to quantify this variation or identify where this variation is most prevalent. The objectives of this study were threefold: firstly to compare three different quantitative methods to measure prawn colour or pigmentation, two different colorimeters and colour quantification from digital images. Secondly, to quantify the amount of pigmentation variation that exists in farmed prawns within ponds, across ponds and across farms. Lastly, to assess the effects of ice storage or freeze-thawing of raw product prior to cooking. Each method was able to detect quantitative differences in prawn colour, although conversion of image based quantification of prawn colour from RGB to Lab was unreliable. Considerable colour variation was observed between prawns from different ponds and different farms, and this variation potentially affects product value. Different post-harvest methods prior to cooking were also shown to have a profound detrimental effect on prawn colour. Both long periods of ice storage and freeze thawing of raw product were detrimental to prawn colour. However, ice storage immediately after cooking was shown to be beneficial to prawn colour. Results demonstrated that darker prawn colour was preserved by holding harvested prawns alive in chilled seawater, limiting the time between harvesting and cooking, and avoiding long periods of ice storage or freeze thawing of uncooked product.