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)

Impact of salinity changes on growth, oxygen consumption and expression pattern of selected candidate genes in the orange mud crab (Scylla olivacea)

Change in environmental salinity level is a major limiting factor for the aquaculture productivity because it imposes severe stress on organisms that in turn retards growth. The orange mud crab (Scylla olivacea) is an important coastal aquaculture species (farming is practised in 10‰–20‰ salinity levels) in Bangladesh. The present study was conducted to investigate the changes in growth, O2 consumption and mRNA expression levels of five selected genes in the orange mud crab (S. olivacea) exposed to three different experimental salinity levels (0‰, 10‰ and 20‰) for three months. Crabs reared at 10‰ and 20‰, showed significantly higher (p < .05) growth performance and expression of growth regulatory genes (Actin and α-amylase). The highest levels (p < .05) of O2 consumption and expression of ion regulatory genes (Na+-K+-ATPase, V-type H+-ATPase and Diuretic Hormone) were obtained at 0‰. Moderate levels of growth and expression of selected candidate genes were observed at 10‰ treatment while the highest levels of growth and gene expression were obtained at 20‰ (control salinity). Strong interactions were observed between growth performance and expression of growth genes (R2 = 0.81–0.91), and rate of O2 consumption and expression of ion regulatory genes (R2 = 0.83–0.93), implying that the selected genes are important candidates for growth and ionic balance in S. olivacea. Growth performance was found to be very low at 0‰ initially, after 30 days crabs showed better growth performance at this salinity level. It is thus inferred that orange mud crab individuals might require 3–5 days for acclimation to salinity stress but it can take at least 30 days for acclimation to regular growth. Results indicate that with proper acclimation, the orange mud crab (Scylla olivacea) can be farmed at low salinity conditions and possibly in freshwater condition

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

Understanding the molecular basis of adaptation to freshwater environments by prawns in the genus Macrobrachium

Understanding the processes that drive adaptive change in response to environmental variation and their consequences for speciation have long been key questions in evolutionary biology. Following origins in seawater, a number of animal groups invaded and colonized freshwater successfully over various evolutionary timeframes. Crustaceans represent a group of relatively recent colonizers of freshwater that now show extensive diversity with representative taxa found in virtually all aquatic environments. Macrobrachium (Family: Palaemonidae) are one of the most speciose and diversified of all crustacean lineages. Taxa in the genus Macrobrachium occupy a wide range of aquatic habitats, possess relatively large body size, and many are highly abundant. Macrobrachium species, as relatively recent freshwater colonizers, therefore provide excellent models for deciphering mechanisms that have facilitated freshwater adaptation. Modern genomic technologies now allow identification of genomic regions influencing adaptation and adaptive diversification (or speciation) at a finer scale. The current study employed a comparative genomics approach to investigate the molecular basis of freshwater adaptation in this decapod crustacean group. In the first study, a transcriptomic scan was performed to identify potential candidate genes involved in freshwater adaptation using an obligate freshwater species, M. koombooloomba as a model. M. koombooloomba was used essentially as the „control‟ because this species completes its entire life in freshwater; thus, all of the important genes affecting freshwater adaptation should be highly expressed in this species. We identified 43 candidate genes (based on BLAST matching with other species) that are likely to be important genes for adapting to a freshwater lifestyle in this species. Identified genes fell under seven broad biological categories including: osmoregulation, cell volume regulation, hemolymph regulation, water channel regulation, osmotic stress response, egg size control and control of larval developmental stage number. We used this gene list as the foundation for future studies. In the second study, we performed a comparative transcriptomics analysis of three Macrobrachium species (M. australiense, M. novaehollandiae and M. tolmerum) representing a range of salinity tolerances at various stages of the life cycle. The three species were maintained under two experimental salinity levels (0‰ and 15‰) over a period of six weeks. The study identified 59 candidate genes (all 43 identified in study 1) including 16 novel „lineage specific orphan transcripts/genes‟. A number of candidate genes (associated with osmoregulation, osmotic stress response, cell volume regulation, water channel and hemolymph regulation) showed different expression patterns between experimental salinities, while expression of others (associated with egg size control and larval development number) remained stable between salinities. Novel genes/transcripts also showed salinity induced differential gene expression patterns. Neutrality tests on all 59 genes revealed that differentially expressed genes showed signatures of purifying selection, but other genes (those that were not differentially expressed) showed patterns consistent with strong positive selection. A few genes (osmotic stress response, cell volume and hemolymph regulatory) showed both differential expression patterns and signatures of positive selection, depending on whether the comparison was between species with similar or dissimilar life history traits. Sequences were highly conserved across species for genes that were differentially expressed between salinities. Results suggest that both plasticity of gene expression and sequence divergence in coding regions (functional mutations), act in a co-ordinated way to promote adaptation. We argue that changes to gene expression pattern play a vital role in the initial adaptive response, while efficient adaptation via mutation/s act over prolonged evolutionary time. In the third study, we conducted a physiological genomic study of the same three species used in the previous study to investigate how regulation of gene expression and body fluid (hemolymph) change with salinity level over time. Individuals from each of the three species were maintained at three experimental salinity levels (0‰, 6‰ and 12‰) for 28 days after an initial acclimation phase to a common condition (6‰) for 14 days. In total, 12 genes were investigated in this study that are involved with different biological functions (based on study 2). For the majority of genes studied (10 out of 12), expression patterns were found to be significantly different among salinity treatments. Differentially expressed genes followed a common pattern; an initial rise in expression level up to 48 hours, followed by a fall in expression up to 96 hours after which expression levels stabilized until the end of the experimental period. Changes to hemolymph osmolality showed a similar pattern to gene expression, with significant differences in hemolymph osmolality evident among salinity treatments. Results demonstrate that salinity level has a strong influence on both hemolymph osmolality and gene expression pattern in the target Macrobrachium species. We conclude that rapid changes to physiological and genomic responses likely shape initial adaptive response to variable environmental salinities. In the final experiment, we employed a comparative genomics analysis using genotyping-by-sequencing (GBS) that screened sequences in 34 Macrobrachium species representing all life history character types from different continents (i.e., replicates of independent freshwater invasions). The study identified 5,018 single nucleotide polymorphisms (SNPs) from ≈310,000 aligned nucleotides in each species. Blasting of genotypes against both the Daphnia genome and Macrobrachium transcriptomes (sourced from studies 1 and 2) showed 65% sequence matching. Blast results revealed that the matched SNPs were located in 176 discrete genes. These genes are involved in an array of diversified functional roles including osmoregulation, hemolymph regulation, cell volume regulation, water channel regulation, egg size control, larval development pattern, energy budget, metabolism, and immune response. This suggests that many interacting genes and/or genomic regions are involved with adaptation to different environmental conditions. SNPs and aligned sequences were used to construct a maximum likelihood phylogenetic tree in addition to a „neutral‟ tree for the same 34 species using the mitochondrial (mtDNA) 16S gene. Topologies of the two trees were substantially different only at the within major clade level (distinct clade for each continent) and support an earlier hypothesis of multiple independent invasions in freshwater environments by ancestral Macrobrachium species. In the GBS tree, all continental freshwater species formed monophyletic groups indicating independent invasions of freshwater, following which Macrobrachium taxa underwent adaptive genomic divergence with respect to the environments they colonized. The GBS tree strongly supported the hypothesis that freshwater adaptation across the Macrobrachium genus likely involved convergent evolution of the same set of traits; so that all global freshwater Macrobrachium species evolved similar suites of phenotypic traits due to common selection pressures associated with a freshwater lifestyle. Overall, this study provides a comprehensive data set for resolving the genomic basis of freshwater adaptation by Palaemonid prawns in the genus Macrobrachium. We infer that response from the genome (rearrangement of the whole genome) is required for successful adaptation to a novel environment with major changes in phenotypic traits (morphology, physiology and overall organismal biology

Distributed under Creative Commons CC-BY 4.0 Candidate genes that have facilitated freshwater adaptation by palaemonid prawns in the genus Macrobrachium: identification and expression validation in a model species (M. koombooloomba)

ABSTRACT 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

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

Osmoregulation in decapod crustaceans: physiological and genomic perspectives

A central question in evolutionary biology represents understanding the molecular basis of adaptive response to differing environmental salinity gradients. The capacity to osmoregulate is considered to be the principal function through which adaptation occurs in different or fluctuating osmotic niches. Decapod crustaceans represent an interesting research group for exploring the underlying genetic/genomic mechanisms involved with this process. The genomic basis of osmoregulation involves modifying expression patterns of candidate genes for ionic balance for short-/long-term acclimation to salinity change while long-term persistence in the altered salinity conditions can facilitate adaptation via novel mutations over an evolutionary time frame. So far, 32 candidate genes have been identified that have important functional roles in maintaining ionic balance across decapod crustacean lineages. Certain genes are considered to play principal/vital roles while others apparently have secondary or minor roles. This group of genes falls under several broad biological categories including: sensing osmotic stress, signal transduction, activating candidate genes, osmotic stress tolerance, ion transportation, active ion exchange, and regulation of cell volume. Most studies conducted to date have focused only on a few principal genes to better understand osmoregulatory processes, while minor role-playing genes remained largely unexplored. The information available currently reviewed here can provide important clues to decipher the molecular mechanisms involved with osmoregulation broadly across crustacean lineages

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