The Australian redclaw crayfish (Cherax quadricarinatus) has recently received attention as an emerging candidate for sustainable aquaculture production in Australia and worldwide. More importantly, C. quadricarinatus serves as a good model organism for the commercially important group of decapod crustaceans as it is distributed worldwide, easy to maintain in the laboratory and its reproductive cycle has been well documented. In order to better understand the key reproduction and development regulating mechanisms in decapod crustaceans, the molecular toolkit available for model organisms such as C. quadricarinatus must be expanded. However, there has been no study undertaken to establish the C. quadricarinatus neuropeptidome. Here we report a comprehensive study of the neuropeptide genes expressed in the eyestalk in the Australian crayfish C. quadricarinatus. We characterised 53 putative neuropeptide-encoding transcripts based on key features of neuropeptides as characterised in other species. Of those, 14 neuropeptides implicated in reproduction regulation were chosen for assessment of their tissue distribution using RT-PCR. Further insights are discussed in relation to current knowledge of neuropeptides in other species and potential follow up studies. Overall, the resulting data lays the foundation for future gene-based neuroendocrinology studies in C. quadricarinatus.

Multiple biological processes across development and reproduction are modulated by neuropeptides that are predominantly produced and secreted from an animal's central nervous system. In the past few years, advancement of next-generation sequencing technologies has enabled large-scale prediction of putative neuropeptide genes in multiple non-model species, including commercially important decapod crustaceans. In contrast, knowledge of the G protein-coupled receptors (GPCRs), through which neuropeptides act on target cells, is still very limited. In the current study, we have used in silico transcriptome analysis to elucidate genes encoding neuropeptides and GPCRs in the Norway lobster (Nephrops norvegicus), which is one of the most valuable crustaceans in Europe. Fifty-seven neuropeptide precursor-encoding transcripts were detected, including phoenixin, a vertebrate neurohormone that has not been detected in any invertebrate species prior to this study. Neuropeptide gene expression analysis of immature and mature female N. norvegicus, revealed that some reproduction-related neuropeptides are almost exclusively expressed in immature females. In addition, a total of 223 GPCR-encoding transcripts were identified, of which 116 encode GPCR-A (Rhodopsin), 44 encode GPCR-B (Secretin) and 63 encode other GPCRs. Our findings increase the molecular toolbox of neural signaling components in N. norvegicus, allowing for further advances in the fisheries/larvae culture of this species.

BackgroundUnderstanding the genomic basis of osmoregulation (candidate genes and/or molecular mechanisms controlling the phenotype) addresses one of the fundamental questions in evolutionary ecology. Species distributions and adaptive radiations are thought to be controlled by environmental salinity levels, and efficient osmoregulatory (ionic balance) ability is the main mechanism to overcome the problems related to environmental salinity gradients.MethodsTo better understand how osmoregulatory performance in freshwater (FW) crustaceans allow individuals to acclimate and adapt to raised salinity conditions, here we (i), reviewed the literature on genes that have been identified to be associated with osmoregulation in FW crustaceans, and (ii), performed a transcriptomic analysis using cDNA libraries developed from mRNA isolated from three important osmoregulatory tissues (gill, antennal gland, hepatopancreas) and total mRNA from post larvae taken from the freshwater prawn, Macrobrachium australiense using Illumina deep sequencing technology. This species was targeted because it can complete its life cycle totally in freshwater but, like many Macrobrachium sp., can also tolerate brackish water conditions and hence should have genes associated with tolerance of both FW and saline conditions.ResultsWe obtained between 55.4 and 65.2 million Illumina read pairs from four cDNA libraries. Overall, paired end sequences assembled into a total of 125,196 non-redundant contigs (≥200 bp) with an N50 length of 2,282 bp and an average contig length of 968 bp. Transcriptomic analysis of M. australiense identified 32 different gene families that were potentially involved with osmoregulatory capacity. A total of 32,597 transcripts were specified with gene ontology (GO) terms identified on the basis of GO categories. Abundance estimation of expressed genes based on TPM (transcript per million) ≥20 showed 1625 transcripts commonly expressed in all four libraries. Among the top 10 genes expressed in four tissue libraries associated with osmoregulation, arginine kinase and Na+/K+- ATPase showed the highest transcript copy number with 7098 and 660, respectively in gill which is considered to be the most important organ involved in osmoregulation.DiscussionThe current study provides the first broad transcriptome from M. australiense using next generation sequencing and identifies potential candidate genes involved in salinity tolerance and osmoregulation that can provide a foundation for investigating osmoregulatory capacity in a wide variety of freshwater crustaceans.

Sexual development involves the successive and overlapping processes of sex determination, sexual differentiation, and ultimately sexual maturation, enabling animals to reproduce. This provides a mechanism for enriched genetic variation which enables populations to withstand ever-changing environments, selecting for adapted individuals and driving speciation. The molecular mechanisms of sexual development display a bewildering diversity, even in closely related taxa. Many sex determination mechanisms across animals include the key family of “doublesex- and male abnormal3-related transcription factors” (Dmrts). In a few exceptional species, a single Dmrt residing on a sex chromosome acts as the master sex regulator. In this study, we provide compelling evidence for this model of sex determination in the ornate spiny lobster Panulius ornatus, concurrent with recent reports in the eastern spiny lobster Sagmariasus verreauxi. Using a multi-tissue transcriptomic database established for P. ornatus, we screened for the key factors associated with sexual development (by homology search and using previous knowledge of these factors from related species), providing an in-depth understanding of sexual development in decapods. Further research has the potential to close significant gaps in our understanding of reproductive development in this ecologically and commercially significant order.

Next Generation Sequencing (NGS) has dramatically changed the way biological research is being conducted in the post-genomic era and they have only been utilized widely over the recent decade for studies of non-model decapod crustacean species, predominantly by sequencing the transcriptome of various tissues across different life stages. NGS can now provide a rapid, costeffective solution for discovery of genetic markers crucial in many applications that would previously have otherwise taken years to develop. Sequencing of the entire transcriptome (referred to as RNA sequencing; RNA-seq) is one of the most popular NGS tools. RNA-seq studies of non-model species in crustacean taxa however, have faced some problems, including a lack of "good" experimental study design, a relative paucity of gene annotations, combined with limited knowledge of genomic technologies and analyses. The aim of the current review is to assist crustacean biologists to develop a better appreciation of the applications and scope of RNA-seq analysis, understand the basic requirements for optimal RNA-seq studies and provide an overview of each step from RNA-seq experimental design to bioinformatics approaches to data analysis. Insights that have resulted from RNA-seq studies across a wide range of non-model decapod species are also summarized.