Comparative analysis of gill transcriptomes of two freshwater crayfish, Cherax cainii and C. destructor

We undertook deep sequencing of gill transcriptomes from two freshwater crayfish, Cherax cainii and Cherax destructor, in order to generate genomic resources for future genomics research. Over 83 and 100 million high quality (quality score (Q) ≥ 30) paired-end Illumina reads (150 bp) were assembled into 147,101 and 136,622 contigs in C. cainii and C. destructor, respectively. A total of 24,630 and 23,623 contigs received significant BLASTx hits and allowed the identification of multiple gill expressed candidate genes associated with pH and salinity balance. These functionally annotated transcripts will provide a resource to facilitate comparative genomic research in the genus Cherax, and in particular allow insights into respiratory and osmoregulatory physiology of this group of animals.

Molecular data extend Australian Cricotopus midge (Chironomidae) species diversity, and provide a phylogenetic hypothesis for biogeography and freshwater monitoring

Resolving species relationships and confirming diagnostic morphological characters for insect clades that are highly plastic, and/or include morphologically cryptic species, is crucial for both academic and applied reasons. Within the true fly (Diptera) family Chironomidae, a most ubiquitous freshwater insect group, the genera CricotopusWulp, 1874 and ParatrichocladiusSantos-Abreu, 1918 have long been taxonomically confusing. Indeed, until recently the Australian fauna had been examined in just two unpublished theses: most species were known by informal manuscript names only, with no concept of relationships. Understanding species limits, and the associated ecology and evolution, is essential to address taxonomic sufficiency in biomonitoring surveys. Immature stages are collected routinely, but tolerance is generalized at the genus level, despite marked variation among species. Here, we explored this issue using a multilocus molecular phylogenetic approach, including the standard mitochondrial barcode region, and tested explicitly for phylogenetic signal in ecological tolerance of species. Additionally, we addressed biogeographical patterns by conducting Bayesian divergence time estimation. We sampled all but one of the now recognized Australian Cricotopus species and tested monophyly using representatives from other austral and Asian locations. Cricotopus is revealed as paraphyletic by the inclusion of a nested monophyletic Paratrichocladius, with in-group diversification beginning in the Eocene. Previous morphological species concepts are largely corroborated, but some additional cryptic diversity is revealed. No significant relationship was observed between the phylogenetic position of a species and its ecology, implying either that tolerance to deleterious environmental impacts is a convergent trait among many Cricotopus species or that sensitive and restricted taxa have diversified into more narrow niches from a widely tolerant ancestor.

Identification of a putative cellulase gene in the giant freshwater prawn, Macrobrachium rosenbergii (De Man, 1879)

Nutrition plays an important role in the development of all organisms and in particular that of farmed aquatic species where costs associated with feed can often exceed 60% of total production costs. Crustacean species in addition, have the added metabolic requirement for regular moulting to allow normal growth and this requires large amounts of energy in the form of sugars (glucose). The current study explored the capacity of the giant freshwater prawn to produce endogenous cellulose-degrading enzymes capable of extracting nutrients (simple sugars) from plant sources in formulated feeds used in the prawn aquaculture industry. We identified a putative cellulase cDNA fragment in the target organism of 1576 base pairs in length of non-microbial origin that after protein modelling exhibited a TM-score of 0.916 with a described cellulase reported from another crustacean species. The functional role of cellulase enzymes is to hydrolyse cellulose to glucose and the fragment identified in GFP was highly expressed in the hepatopancreas, the site of primary food digestion and absorption in crustaceans. Hepatopancreatic tissue from Macrobrachium rosenbergii also showed active digestion of cellulose to glucose following an endoglucanase assay. Cellulase gene(s) are present in the genomes of many invertebrate taxa and play an active role in the conversion of cellulose to available energy. Identification and characterization of endogenous cellulase gene(s) in giant freshwater prawn can assist development of the culture industry because the findings confirm that potentially greater levels of low-cost plant-material could be included in artificial formulated diets in the future without necessarily compromising individual growth performance. Ultimately, this development may contribute to more efficient, cost-effective production systems for freshwater prawn culture stocks that meet the animal's basic nutritional requirements and that also support good individual growth rates.

A candidate gene association study for growth performance in an improved Giant Freshwater Prawn (Macrobrachium rosenbergii) culture line

A candidate gene approach using type I single nucleotide polymorphism (SNP) markers can provide an effective method for detecting genes and gene regions that underlie phenotypic variation in adaptively significant traits. In the absence of available genomic data resources, transcriptomes were recently generated in Macrobrachium rosenbergii to identify candidate genes and markers potentially associated with growth. The characterisation of 47 candidate loci by ABI re-sequencing of four cultured and eight wild samples revealed 342 putative SNPs. Among these, 28 SNPs were selected in 23 growth-related candidate genes to genotype in 200 animals selected for improved growth performance in an experimental GFP culture line in Vietnam. The associations between SNP markers and individual growth performance were then examined. For additive and dominant effects, a total of three exonic SNPs in glycogen phosphorylase (additive), heat shock protein 90 (additive and dominant) and peroxidasin (additive), and a total of six intronic SNPs in ankyrin repeats-like protein (additive and dominant), rolling pebbles (dominant), transforming growth factor-β induced precursor (dominant), and UTP-glucose-1-phosphate uridylyltransferase 2 (dominant) genes showed significant associations with the estimated breeding values in the experimental animals (P =0.001−0.031). Individually, they explained 2.6−4.8 % of the genetic variance (R2=0.026−0.048). This is the first large set of SNP markers reported for M. rosenbergii and will be useful for confirmation of associations in other samples or culture lines as well as having applications in marker-assisted selection in future breeding programs.

Optimizing de novo transcriptome assembly and extending genomic resources for striped catfish (Pangasianodon hypophthalmus)

Striped catfish (Pangasianodon hypophthalmus) is a commercially important freshwater fish used in inland aquaculture in the Mekong Delta, Vietnam. The culture industry is facing a significant challenge however from saltwater intrusion into many low topographical coastal provinces across the Mekong Delta as a result of predicted climate change impacts. Developing genomic resources for this species can facilitate the production of improved culture lines that can withstand raised salinity conditions, and so we have applied high-throughput Ion Torrent sequencing of transcriptome libraries from six target osmoregulatory organs from striped catfish as a genomic resource for use in future selection strategies. We obtained 12,177,770 reads after trimming and processing with an average length of 97 bp. De novo assemblies were generated using CLC Genomic Workbench, Trinity and Velvet/Oases with the best overall contig performance resulting from the CLC assembly. De novo assembly using CLC yielded 66,451 contigs with an average length of 478 bp and N50 length of 506 bp. A total of 37,969 contigs (57%) possessed significant similarity with proteins in the non-redundant database. Comparative analyses revealed that a significant number of contigs matched sequences reported in other teleost fishes, ranging in similarity from 45.2% with Atlantic cod to 52% with zebrafish. In addition, 28,879 simple sequence repeats (SSRs) and 55,721 single nucleotide polymorphisms (SNPs) were detected in the striped catfish transcriptome. The sequence collection generated in the current study represents the most comprehensive genomic resource for P. hypophthalmus available to date. Our results illustrate the utility of next-generation sequencing as an efficient tool for constructing a large genomic database for marker development in non-model species.

Shaping development through mechanical strain: The transcriptional basis of diet-induced phenotypic plasticity in a cichlid fish

Adaptive phenotypic plasticity, the ability of an organism to change its phenotype to match local environments, is increasingly recognized for its contribution to evolution. However, few empirical studies have explored the molecular basis of plastic traits. The East African cichlid fish Astatoreochromis alluaudi displays adaptive phenotypic plasticity in its pharyngeal jaw apparatus, a structure that is widely seen as an evolutionary key innovation that has contributed to the remarkable diversity of cichlid fishes. It has previously been shown that in response to different diets, the pharyngeal jaws change their size, shape and dentition: hard diets induce an adaptive robust molariform tooth phenotype with short jaws and strong internal bone structures, while soft diets induce a gracile papilliform tooth phenotype with elongated jaws and slender internal bone structures. To gain insight into the molecular underpinnings of these adaptations and enable future investigations of the role that phenotypic plasticity plays during the formation of adaptive radiations, the transcriptomes of the two divergent jaw phenotypes were examined. Our study identified a total of 187 genes whose expression differs in response to hard and soft diets, including immediate early genes, extracellular matrix genes and inflammatory factors. Transcriptome results are interpreted in light of expression of candidate genesmarkers for tooth size and shape, bone cells and mechanically sensitive pathways. This study opens up new avenues of research at new levels of biological organization into the roles of phenotypic plasticity during speciation and radiation of cichlid fishes.

Identification of bacterial community composition in freshwater aquaculture system farming of Litopenaeus vannamei reveals distinct temperature-driven patterns

Change in temperature is often a major environmental factor in triggering waterborne disease outbreaks. Previous research has revealed temporal and spatial patterns of bacterial population in several aquatic ecosystems. To date, very little information is available on aquaculture environment. Here, we assessed environmental temperature effects on bacterial community composition in freshwater aquaculture system farming of Litopenaeus vannamei (FASFL). Water samples were collected over a one-year period, and aquatic bacteria were characterized by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and 16S rDNA pyrosequencing. Resulting DGGE fingerprints revealed a specific and dynamic bacterial population structure with considerable variation over the seasonal change, suggesting that environmental temperature was a key driver of bacterial population in the FASFL. Pyrosequencing data further demonstrated substantial difference in bacterial community composition between the water at higher (WHT) and at lower (WLT) temperatures in the FASFL. Actinobacteria, Proteobacteria and Bacteroidetes were the highest abundant phyla in the FASFL, however, a large number of unclassified bacteria contributed the most to the observed variation in phylogenetic diversity. The WHT harbored remarkably higher diversity and richness in bacterial composition at genus and species levels when compared to the WLT. Some potential pathogenenic species were identified in both WHT and WLT, providing data in support of aquatic animal health management in the aquaculture industry.

Modeling meat yield based on measurements of body traits in genetically improved giant freshwater prawn (GFP) Macrobrachium rosenbergii

A single-generation dataset consisting of 1,730 records from a selection program for high growth rate in giant freshwater prawn (GFP, Macrobrachium rosenbergii) was used to derive prediction equations for meat weight and meat yield. Models were based on body traits [body weight, total length and abdominal width (AW)] and carcass measurements (tail weight and exoskeleton-off weight). Lengths and width were adjusted for the systematic effects of selection line, male morphotypes and female reproductive status, and for the covariables of age at slaughter within sex and body weight. Body and meat weights adjusted for the same effects (except body weight) were used to calculate meat yield (expressed as percentage of tail weight/body weight and exoskeleton-off weight/body weight). The edible meat weight and yield in this GFP population ranged from 12 to 15 g and 37 to 45 %, respectively. The simple (Pearson) correlation coefficients between body traits (body weight, total length and AW) and meat weight were moderate to very high and positive (0.75–0.94), but the correlations between body traits and meat yield were negative (−0.47 to −0.74). There were strong linear positive relationships between measurements of body traits and meat weight, whereas relationships of body traits with meat yield were moderate and negative. Step-wise multiple regression analysis showed that the best model to predict meat weight included all body traits, with a coefficient of determination (R 2) of 0.99 and a correlation between observed and predicted values of meat weight of 0.99. The corresponding figures for meat yield were 0.91 and 0.95, respectively. Body weight or length was the best predictor of meat weight, explaining 91–94 % of observed variance when it was fitted alone in the model. By contrast, tail width explained a lower proportion (69–82 %) of total variance in the single trait models. It is concluded that in practical breeding programs, improvement of meat weight can be easily made through indirect selection for body trait combinations. The improvement of meat yield, albeit being more difficult, is possible by genetic means, with 91 % of the variation in the trait explained by the body and carcass traits examined in this study.

Size-dependent natural mortality of juvenile banana prawns Penaeus merguiensis in the Gulf of Carpentaria, Australia

Natural mortality of marine invertebrates is often very high in the early life history stages and decreases in later stages. The possible size-dependent mortality of juvenile banana prawns, P. merguiensis (2-15 mm carapace length) in the Gulf of Carpentaria was investigated. The analysis was based on the data collected at 2-weekly intervals by beam trawls at four sites over a period of six years (between September 1986 and March 1992). It was assumed that mortality was a parametric function of size, rather than a constant. Another complication in estimating mortality for juvenile banana prawns is that a significant proportion of the population emigrates from the study area each year. This effect was accounted for by incorporating the size-frequency pattern of the emigrants in the analysis. Both the extra parameter in the model required to describe the size dependence of mortality, and that used to account for emigration were found to be significantly different from zero, and the instantaneous mortality rate declined from 0.89 week(-1) for 2 mm prawns to 0.02 week(-1) for 15 mm prawns.

Characterization of the carbonic anhydrase gene family and other key osmoregulatory genes in Australian freshwater crayfish (genus Cherax)

Cherax quadricarinatus (Redclaw), C. destructor (Yabby) and C. cainii (Marron) are a group of economically important freshwater crayfish and have been developed for aquaculture production in many countries. As crayfish are farmed in a wide range of culture conditions, optimisation of water quality parameters, are crucial for their maximum growth performance. Previous reports have shown that fluctuations in water quality can negatively impact on growth of crayfish. Therefore, this project aims to identify and characterize the major genes that enable freshwater crayfish to persist in different water chemistries and evaluate their patterns of expression under different water parameters. Overall, this project found a number of candidate genes in all three species and determined that water chemistry had a strong influence on the expression of candidate genes. This information is important in the optimization of water quality parameters in freshwater crayfish aquaculture production.

Transcriptome analysis and characterisation of gill-expressed carbonic anhydrase and other key osmoregulatory genes in freshwater crayfish Cherax quadricarinatus

The pH and salinity balance mechanisms of crayfish are controlled by a set of transport-related genes. We identified a set of the genes from the gill transcriptome from a freshwater crayfish Cherax quadricarinatus using the Illumina NGS-sequencing technology. We identified and characterized carbonic anhydrase (CA) genes and some other key genes involved in systematic acid-base balance and osmotic/ionic regulation. We also examined expression patterns of some of these genes across different sublethal pH levels [1]. A total of 72,382,710 paired-end Illumina reads were assembled into 36,128 contigs with an average length of 800 bp. About 37% of the contigs received significant BLAST hits and 22% were assigned gene ontology terms. These data will assist in further physiological-genomic studies in crayfish.