Genomic Basis of Freshwater Adaptation in the Palaemonid Prawn Genus Macrobrachium: Convergent Evolution Following Multiple Independent Colonization Events.

Adaptation to different salinity environments can enhance morphological and genomic divergence between related aquatic taxa. Species of prawns in the genus Macrobrachium naturally inhabit different osmotic niches and possess distinctive lifecycle traits associated with salinity tolerance. This study was conducted to investigate the patterns of adaptive genomic divergence during freshwater colonization in 34 Macrobrachium species collected from four continents; Australia, Asia, North and South America. Genotyping-by-sequencing (GBS) technique identified 5018 loci containing 82,636 single nucleotide polymorphisms (SNPs) that were used to reconstruct a phylogenomic tree. An additional phylogeny was reconstructed based on 43 candidate genes, previously identified as being potentially associated with freshwater adaptation. Comparison of the two phylogenetic trees revealed contrasting topologies. The GBS tree indicated multiple independent continent-specific invasions into freshwater by Macrobrachium lineages following common marine ancestry, as species with abbreviated larval development (ALD), i.e., species having a full freshwater life history, appeared reciprocally monophyletic within each continent. In contrast, the candidate gene tree showed convergent evolution for all ALD species worldwide, forming a single, well-supported clade. This latter pattern is likely the result of common evolutionary pressures selecting key mutations favored in continental freshwater habitats Results suggest that following multiple independent invasions into continental freshwaters at different evolutionary timescales, Macrobrachium taxa experienced adaptive genomic divergence, and in particular, convergence in the same genomic regions with parallel shifts in specific conserved phenotypic traits, such as evolution of larger eggs with abbreviated larval developmental.

Do plasticity in gene expression and physiological responses in Palaemonid prawns facilitate adaptive response to different osmotic challenges?

Integrating physiological and genomic approaches in a comparative framework offers excellent opportunity to investigate the underlying mechanisms for acclimation to specific challenges. The present study was conducted on three different prawn species (inhabitants of different salinity environments) of the genus Macrobrachium (M. australiense, M. tolmerum and M. novaehollandiae) to investigate the salinity induced changes in expression patterns of 10 candidate genes in the gill tissue (that previously had been inferred to play important functional roles in acclimation and adaptation to freshwater environments), and hemolymph osmolality. The prawn individuals were maintained in laboratory condition under three different salinity levels (0‰, 6‰ and 12‰) for 28 days using 6‰ as the control. All of the genes studied, showed salinity induced differential expression patterns. Genes with more important functional roles under low ionic conditions (i.e. Claudin, Na+/H+exchanger, V-type H+-ATPase and UNT2) showed 2.5 to 6 fold higher expression at 0‰ compared with at higher salinities (6‰ and 12‰) but no significant differences (p > 0.05) were observed between 6‰ and 12‰ for the same genes. In contrast, 1.5 to 4 fold higher expression levels were observed at 6‰ and 12‰ for genes that have important roles in mediating salinity tolerance (i.e., Na+/K+-ATPase, Na+/K+/2Cl-Co-transporter, Diuretic Hormone, Crustacean Hyperglycaemic Hormone and UNT1). The osmotic stress response gene, Calreticulin, showed significant differences (p < 0.05) in expression between different salinity comparisons. Hemolymph osmolality also was impacted in all three species with a strong correlation evident between hemolymph osmolality and expression of genes influencing this trait. Findings indicate an important role of plasticity that facilitates rapid acclimation to changing salinity levels.

Understanding the Genomic Basis of Adaptive Response to Variable Osmotic Niches in Freshwater Prawns: A Comparative Intraspecific RNA-Seq Analysis of Macrobrachium australiense.

Understanding the molecular basis of adaptive response to variable environmental conditions is a central goal of evolutionary biology. Here, we sought to identify potential outlier single nucleotide polymorphisms (SNPs) in 3 wild populations of a freshwater prawn (Macrobrachium australiense) that are exposed to differing osmotic niches by using a comparative transcriptomics approach. De novo assembly of approximately 542 million (75 nt) pair end reads collected from 10 individuals revealed 123396 longer contigs/transcripts of variable length, that showed 97.38% transcriptome assembly completeness. Differential gene expression analysis of major osmoregulatory genes revealed that calreticulin, Na+/H+ exchanger, and V-type (H+) ATPase showed the highest expression levels in the Blunder Creek (low ionic) population, while Crustacean cardiovascular peptide (CCP), Na+/K+-ATPase, Na+/K+/2Cl- co-transporter (NKCC) and Na+/HCO3- exchanger showed the highest expression levels in the Bulimba Creek (higher ionic) population. In total, 16 gene ontology term categories were functionally enriched among the 3 studied populations. We identified 4144 raw and 835 high quality filtered SNPs in the 3 M. australiense populations, of which 84 SNPs were identified as outliers. Outliers were detected in 4 important osmoregulatory genes that include: calreticulin, Na+/H+ exchanger, Na+/K+-ATPase, and V-type-(H+)-ATPase. All outliers in the osmoregulatory genes were located in noncoding regulatory regions (untranslated regions) of the gene. We hypothesize that the outlier SNPs identified here in M. australiense populations exposed naturally to different osmotic conditions influence specific gene expression patterns that allow individuals to respond to local environmental conditions.

Integrated transcriptome catalog of Tenualosa ilisha as a resource for gene discovery and expression profiling.

The silver pride of Bangladesh, migratory shad, Tenualosa ilisha (Hilsa), makes the highest contribution to the total fish production of Bangladesh. Despite its noteworthy contribution, a well-annotated transcriptome data is not available. Here we report a transcriptomic catalog of Hilsa, constructed by assembling RNA-Seq reads from different tissues of the fish including brain, gill, kidney, liver, and muscle. Hilsa fish were collected from different aquatic habitats (fresh, brackish, and sea water) and the sequencing was performed in the next generation sequencing (NGS) platform. De novo assembly of the sequences obtained from 46 cDNA libraries revealed 462,085 transcript isoforms that were subsequently annotated using the Universal Protein Resource Knowledgebase (UniPortKB) as a reference. Starting from the sampling to final annotation, all the steps along with the workflow are reported here. This study will provide a significant resource for ongoing and future research on Hilsa for transcriptome based expression profiling and identification of candidate genes.

Effects of Salinity on Physiological, Biochemical and Gene Expression Parameters of Black Tiger Shrimp (Penaeus monodon): Potential for Farming in Low-Salinity Environments.

Salinity is one of the most important abiotic factors affecting growth, metabolism, immunity and survival of aquatic species in farming environments. As a euryhaline species, the black tiger shrimp (Penaeus monodon) can tolerate a wide range of salinity levels and is farmed between brackish to marine water conditions. The current study tested the effects of six different salinity levels (0‱, 2.5‱, 5‱, 10‱, 20‱ and 30‱) on the selected physiological, biochemical and genetic markers (individual changes in the expression pattern of selected candidate genes) in the black tiger shrimp. Experimental salinity levels significantly affected growth and survival performance (p < 0.05); the highest levels of growth and survival performance were observed at the control (20‱) salinity. Salinity reductions significantly increased free fatty acid (FFA), but reduced free amino acid (FAA) levels. Lower salinity treatments (0-10‱) significantly reduced hemolymph osmolality levels while 30‱ significantly increased osmolality levels. The five different salinity treatments increased the expression of osmoregulatory and hemolymph regulatory genes by 1.2-8-fold. In contrast, 1.2-1.6-fold lower expression levels were observed at the five salinity treatments for growth (alpha amylase) and immunity (toll-like receptor) genes. O2 consumption, glucose and serotonin levels, and expression of osmoregulatory genes showed rapid increase initially with salinity change, followed by reducing trend and stable patterns from the 5th day to the end. Hemocyte counts, expression of growth and immunity related genes showed initial decreasing trends, followed by an increasing trend and finally stability from 20th day to the end. Results indicate the farming potential of P. monodon at low salinity environments (possibly at freshwater) by proper acclimation prior to stocking with minimal effects on production performance.

The Molecular Basis of Freshwater Adaptation in Prawns: Insights from Comparative Transcriptomics of Three Macrobrachium Species.

Elucidating the molecular basis of adaptation to different environmental conditions is important because adaptive ability of a species can shape its distribution, influence speciation, and also drive a variety of evolutionary processes. For crustaceans, colonization of freshwater habitats has significantly impacted diversity, but the molecular basis of this process is poorly understood. In the current study, we examined three prawn species from the genus Macrobrachium (M. australiense, M. tolmerum, and M. novaehollandiae) to better understand the molecular basis of freshwater adaptation using a comparative transcriptomics approach. Each of these species naturally inhabit environments with different salinity levels; here, we exposed them to the same experimental salinity conditions (0‰ and 15‰), to compare expression patterns of candidate genes that previously have been shown to influence phenotypic traits associated with freshwater adaptation (e.g., genes associated with osmoregulation). Differential gene expression analysis revealed 876, 861, and 925 differentially expressed transcripts under the two salinities for M. australiense, M. tolmerum, and M. novaehollandiae, respectively. Of these, 16 were found to be unannotated novel transcripts and may be taxonomically restricted or orphan genes. Functional enrichment and molecular pathway mapping revealed 13 functionally enriched categories and 11 enriched molecular pathways that were common to the three Macrobrachium species. Pattern of selection analysis revealed 26 genes with signatures of positive selection among pairwise species comparisons. Overall, our results indicate that the same key genes and similar molecular pathways are likely to be involved with freshwater adaptation widely across this decapod group; with nonoverlapping sets of genes showing differential expression (mainly osmoregulatory genes) and signatures of positive selection (genes involved with different life history traits).

Candidate genes that have facilitated freshwater adaptation by palaemonid prawns in the genus Macrobrachium: identification and expression validation in a model species (M. koombooloomba).

BACKGROUND: The endemic Australian freshwater prawn, Macrobrachium koombooloomba, provides a model for exploring genes involved with freshwater adaptation because it is one of the relatively few Macrobrachium species that can complete its entire life cycle in freshwater. METHODS: The present study was conducted to identify potential candidate genes that are likely to contribute to effective freshwater adaptation by M. koombooloomba using a transcriptomics approach. De novo assembly of 75 bp paired end 227,564,643 high quality Illumina raw reads from 6 different cDNA libraries revealed 125,917 contigs of variable lengths (200-18,050 bp) with an N50 value of 1597. RESULTS: In total, 31,272 (24.83%) of the assembled contigs received significant blast hits, of which 27,686 and 22,560 contigs were mapped and functionally annotated, respectively. CEGMA (Core Eukaryotic Genes Mapping Approach) based transcriptome quality assessment revealed 96.37% completeness. We identified 43 different potential genes that are likely to be involved with freshwater adaptation in M. koombooloomba. Identified candidate genes included: 25 genes for osmoregulation, five for cell volume regulation, seven for stress tolerance, three for body fluid (haemolymph) maintenance, eight for epithelial permeability and water channel regulation, nine for egg size control and three for larval development. RSEM (RNA-Seq Expectation Maximization) based abundance estimation revealed that 6,253, 5,753 and 3,795 transcripts were expressed (at TPM value ≥10) in post larvae, juveniles and adults, respectively. Differential gene expression (DGE) analysis showed that 15 genes were expressed differentially in different individuals but these genes apparently were not involved with freshwater adaptation but rather were involved in growth, development and reproductive maturation. DISCUSSION: The genomic resources developed here will be useful for better understanding the molecular basis of freshwater adaptation in Macrobrachium prawns and other crustaceans more broadly.

A transcriptomic scan for potential candidate genes involved in osmoregulation in an obligate freshwater palaemonid prawn (Macrobrachium australiense).

BACKGROUND: Understanding 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. METHODS: To 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. RESULTS: We 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. DISCUSSION: The 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.