Meta-analysis to Unravel Core Transcriptomic Responses in Penaeus vannamei Exposed to Biotic and Abiotic Stresses.

Shrimp farming, a dominant economic activity in coastal areas, is affected by different abiotic and biotic stress factors. These stressors, under poor management conditions, could affect growth and health of farmed animals. Understanding the common gene expressions in response to stress, regardless of the specific stress factor, holds significant importance in the field of functional genomics. Scope of this study is to identify the core transcriptomic responses in the shrimp species Penaeus vannamei exposed to various abiotic and biotic stress conditions and to decipher their functional importance. To achieve our objective, we gathered and analyzed multiple RNA-seq datasets related to twelve abiotic and nine biotic stress conditions. Through the in silico meta-analysis, we predicted 961 differentially expressed genes (meta-DEGs) for abiotic stress conditions and 517 meta-DEGs for biotic stress conditions, respectively. These meta-DEGs represent genes that are commonly expressed across different stress factors and are indicative of the organism's general response to stress. The annotation of nineteen core up-regulated meta-DEGs revealed their diverse functions in detoxification, cell adhesion, metal ion binding, and oxidative phosphorylation. These genes play a crucial role in stress response and immune defense. For abiotic stress, significant pathways associated with the stress response include tryptophan metabolism, starch and sucrose metabolism, fatty acid degradation, carbohydrate digestion and absorption, phenylalanine metabolism, drug metabolism-other enzymes, arachidonic acid metabolism, and fatty acid elongation. Similarly, for biotic stress, metabolism of xenobiotics by cytochrome P450, pentose and glucuronate interconversions, steroid hormone biosynthesis, and drug metabolism-cytochrome P450 were found to be significant pathway associations. In addition, the study also predicted 17 stress regulatory motifs present in the identified meta-DEGs. These motifs have significance in identifying the stress responses of the organism. The metabolic pathways and regulatory motifs associated with abiotic and biotic stress factors identified through this study could be a valuable resource for developing stress management approaches in shrimp aquaculture.

MRF: a tool to overcome the barrier of inconsistent genome annotations and perform comparative genomics studies for the largest animal DNA virus.

BACKGROUND: The genome of the largest known animal virus, the white spot syndrome virus (WSSV) responsible for huge economic losses and loss of employment in aquaculture, suffers from inconsistent annotation nomenclature. Novel genome sequence, circular genome and variable genome length led to nomenclature inconsistencies. Since vast knowledge has already accumulated in the past two decades with inconsistent nomenclature, the insights gained on a genome could not be easily extendable to other genomes. Therefore, the present study aims to perform comparative genomics studies in WSSV on uniform nomenclature. METHODS: We have combined the standard mummer tool with custom scripts to develop missing regions finder (MRF) that documents the missing genome regions and coding sequences in virus genomes in comparison to a reference genome and in its annotation nomenclature. The procedure was implemented as web tool and in command-line interface. Using MRF, we have documented the missing coding sequences in WSSV and explored their role in virulence through application of phylogenomics, machine learning models and homologous genes. RESULTS: We have tabulated and depicted the missing genome regions, missing coding sequences and deletion hotspots in WSSV on a common annotation nomenclature and attempted to link them to virus virulence. It was observed that the ubiquitination, transcription regulation and nucleotide metabolism might be essentially required for WSSV pathogenesis; and the structural proteins, VP19, VP26 and VP28 are essential for virus assembly. Few minor structural proteins in WSSV would act as envelope glycoproteins. We have also demonstrated the advantage of MRF in providing detailed graphic/tabular output in less time and also in handling of low-complexity, repeat-rich and highly similar regions of the genomes using other virus cases. CONCLUSIONS: Pathogenic virus research benefits from tools that could directly indicate the missing genomic regions and coding sequences between isolates/strains. In virus research, the analyses performed in this study provides an advancement to find the differences between genomes and to quickly identify the important coding sequences/genomes that require early attention from researchers. To conclude, the approach implemented in MRF complements similarity-based tools in comparative genomics involving large, highly-similar, length-varying and/or inconsistently annotated viral genomes.

Transcriptomic variations associated with salinity stress in Penaeus indicus.

BACKGROUND: The Indian white shrimp, Penaeus indicus a native species of India, is important brackishwater aquaculture species. Shrimps are euryhaline in nature and they regulate osmotic and ionic concentrations by osmoregulatory process. However, variations in abiotic factors such as salinity result in stress to the shrimps during culture period affecting their growth and immunity. METHODS AND RESULTS: To understand the adaptive mechanism to stress in low salinity conditions, RNA-seq was used to compare the transcriptomic response of P. indicus upto 3 weeks. De novo assembly using Trinity assembler generated a total of 173,582 transcripts. The assembly had a mean length of 854 bp, N50 value of 1243 bp and GC content of 42.33%. Differential gene expression analysis, resulted in identification of 2130, 3090, and 5351 DEGs in 7 days, 14 days and 21 days respectively of salinity stress period. The pathway prediction of the assembled trinity transcripts using KEGG database showed total number of 329 pathways linking 12,430 transcripts. KEGG pathway enrichment analyses led to the identification of several enriched pathways related to lipid metabolism, amino acid metabolism, glycolysis, signalling pathways etc. Selected genes involved in osmoregulatory process and immune response in shrimps were validated and analysed for the gene expression levels by quantitative real-time PCR (qPCR). CONCLUSION: This study on the adaptive transcriptomic response of P. indicus to low salinity, will further help in our understanding of the molecular mechanisms underlying osmoregulation mechanism in shrimps.

Exploration of the candidate beneficial bacteria for Penaeus vannamei culture by core microbiome analysis using amplicon sequencing.

Globally, Penaeus vannamei is the vital species in aquaculture production. Beneficial bacterial exploration of gut, sediment, and water were investigated in P. vannamei culture using Illumina Miseq sequencing of 16S RNA V3-V4 hypervariable regions. Predominant phyla identified were Proteobacteria, Tenericutes, Bacteroidetes in gut; Proteobacteria, Bacteroidetes, Planctomycetes in sediment and Cyanobacteria, Proteobacteria, and Planctomycetes in water. In total, 46 phyla, 509 families and 902 genera; 70 phyla, 735 families and 1255 genera; 55 phyla, 580 families and 996 genera were observed in gut, sediment and water, respectively. Diversity of microbial communities in respect of observed Operational Taxonomic Units, diversity indices (Shannon and Simpson), richness index (Chao1) were significantly high P (<0.05) in 60 DoC in gut and 30 DoC in sediment. Beta diversity indicated separate clusters for bacterial communities in gut, sediment and water samples and formation of distinct community profiles. Core microbiome in P. vannamei rearing ponds over a time consisted of 9, 21, and 20 OTUs in gut, rearing water and sediment, respectively. This study helps to intervene with suitable beneficial microbes to establish an aquaculture system thereby contributes to enhance the productivity, improve water quality and pond bottom condition, and control the pathogenic agents at each stage of the culture.

Correction: Chromosomal-Level Assembly of the Asian Seabass Genome Using Long Sequence Reads and Multi-layered Scaffolding.

[This corrects the article DOI: 10.1371/journal.pgen.1005954.].

Genome size estimation of brackishwater fishes and penaeid shrimps by flow cytometry.

Flow cytometry was used for estimating the genome size of five brackishwater finfish and four shrimp species. The genome size for Lutjanus argentimaculatus was 0.95 ± 0.10 and 0.79 ± 0.01 pg for Scatophagus argus. The genome sizes for Chanos chanos (0.72 ± 0.01 pg), Etroplus suratensis (1.71 ± 0.16 pg) and Liza macrolepis (0.87 ± 0.02 pg) which are important aquaculture species are reported for the first time in this study. The phylogenetic tree constructed using sixty-seven sequence accessions of cytochrome c oxidase subunit 1 (COI) gene of Lates calcarifer revealed two separate clades. The Indian Lates calcarifer species with estimated genome size of 0.44 ± 0.02 pg belonged to a clade different than that of South East Asia and Australia reported to have larger genome size. The genome size for the four major species of genus Penaeus (Penaeus monodon, Penaeus indicus, Penaeus vannamei and Penaeus japonicus) were found in similar range. The genome size of female shrimps ranged from 2.91 ± 0.03 pg (P. monodon) to 2.14 ± 0.02 pg (P. japonicus). In male shrimps, the genome size ranged from 2.86 ± 0.06 pg (P. monodon) to 2.19 ± 0.02 pg (P. indicus). Significant difference was observed in the genome size between male and female shrimp of all species except in P. monodon. The highest relative difference of 12.78% was observed in the genome size between the either sex in P. indicus. The interspecific relative difference of 30.59% in genome size was highest between the male shrimps of P. monodon and P. indicus and 35.98% between the female shrimps of P. monodon and P. japonicus. The stored gills and pleopod tissues could be successfully used up to 3 weeks to estimate the genome size in shrimps.

QTL for white spot syndrome virus resistance and the sex-determining locus in the Indian black tiger shrimp (Penaeus monodon).

BACKGROUND: Shrimp culture is a fast growing aquaculture sector, but in recent years there has been a shift away from tiger shrimp Penaeus monodon to other species. This is largely due to the susceptibility of P. monodon to white spot syndrome virus disease (Whispovirus sp.) which has impacted production around the world. As female penaeid shrimp grow more rapidly than males, mono-sex production would be advantageous, however little is known about genes controlling or markers associated with sex determination in shrimp. In this study, a mapped set of 3959 transcribed single nucleotide polymorphisms were used to scan the P. monodon genome for loci associated with resistance to white-spot syndrome virus and sex in seven full-sibling tiger shrimp families challenged with white spot syndrome virus. RESULTS: Linkage groups 2, 3, 5, 6, 17, 18, 19, 22, 27 and 43 were found to contain quantitative trait loci significantly associated with hours of survival after white spot syndrome virus infection (P < 0.05 after Bonferroni correction). Nine QTL were significantly associated with hours of survival. Of the SNPs mapping to these and other regions with suggestive associations, many were found to occur in transcripts showing homology to genes with putative immune functions of interest, including genes affecting the action of the ubiquitin-proteasome pathway, lymphocyte-cell function, heat shock proteins, the TOLL pathway, protein kinase signal transduction pathways, mRNA binding proteins, lectins and genes affecting the development and differentiation of the immune system (eg. RUNT protein 1A). Several SNPs significantly associated with sex were mapped to linkage group 30, the strongest associations (P < 0.001 after Bonferroni correction) for 3 SNPs located in a 0.8 cM stretch between positions 43.5 and 44.3 cM where the feminisation gene (FEM-1, affecting sexual differentiation in Caenorhabditis elegans) mapped. CONCLUSIONS: The markers for disease resistance and sexual differentiation identified by this study could be useful for marker assisted selection to improve resistance to WSSV and for identifying homogametic female individuals for mono-sex (all female) production. The genes with putative functions affecting immunity and sexual differentiation that were found to closely map to these loci provide leads about the mechanisms affecting these important economic traits in shrimp.

Genome assembly, Full-length transcriptome, and isoform diversity of Red Snapper, Lutjanus argentimaculatus.

The mangrove red snapper, Lutjanus argentimaculatus, is a marine food fish of economic and aquaculture importance. The application of genomic selection-based breeding programs for this species is limited by the absence of a reference genome and transcriptome profiles. The current study attempted to fill this void by generating genomic and transcriptomic resources for red snapper. Using PacBio long reads, and Arima Hi-C linked reads, a scaffold-level genome assembly was generated for L. argentimaculatus. The assembly is of 1.03 Gb comprising of 400 scaffolds with N50 of 33.8 Mb and was assessed to be 97.2% complete upon benchmarking with BUSCO. Full-length transcriptome generated with PacBio Iso-Sequencing strategy using six tissues (muscle, gills, liver, kidney, stomach, and gonad) contained 56,515 isoforms belonging to 18,108 unique genes with N50 length of 3,973 bp. The resources generated will have potential applications in the functional studies, conservation, broodstock management and selective breeding programmes of L. argentimaculatus.

In silico prediction of potential indigenous microbial biomarkers in Penaeus vannamei identified through meta-analysis and genome-scale metabolic modelling.

BACKGROUND: Understanding the microbiome is crucial as it contributes to the metabolic health of the host and, upon dysbiosis, may influence disease development. With the recent surge in high-throughput sequencing technology, the availability of microbial genomic data has increased dramatically. Amplicon sequence-based analyses majorly profile microbial abundance and determine taxonomic markers. Furthermore, the availability of genome sequences for various microbial organisms has prompted the integration of genome-scale metabolic modelling that provides insights into the metabolic interactions influencing host health. However, the analysis from a single study may not be consistent, necessitating a meta-analysis. RESULTS: We conducted a meta-analysis and integrated with constraint-based metabolic modelling approach, focusing on the microbiome of pacific white shrimp Penaeus vannamei, an extensively cultured marine candidate species. Meta-analysis revealed that Acinetobacter and Alteromonas are significant indicators of "health" and "disease" specific taxonomic biomarkers, respectively. Further, we enumerated metabolic interactions among the taxonomic biomarkers by applying a constraint-based approach to the community metabolic models (4416 pairs). Under different nutrient environments, a constraint-based flux simulation identified five beneficial species: Acinetobacter spWCHA55, Acinetobacter tandoii SE63, Bifidobacterium pseudolongum 49 D6, Brevundimonas pondensis LVF1, and Lutibacter profundi LP1 mediating parasitic interactions majorly under sucrose environment in the pairwise community. The study also reports the healthy biomarkers that can co-exist and have functionally dependent relationships to maintain a healthy state in the host. CONCLUSIONS: Toward this, we collected and re-analysed the amplicon sequence data of P. vannamei (encompassing 117 healthy and 142 disease datasets). By capturing the taxonomic biomarkers and modelling the metabolic interaction between them, our study provides a valuable resource, a first-of-its-kind analysis in aquaculture scenario toward a sustainable shrimp farming.

Mitochondrial DNA based diversity studies reveal distinct and substructured populations of pearlspot, Etroplus suratensis (Bloch, 1790) in Indian waters.

Pearlspot (Etroplus suratensis) is one of the most commercially important brackish water fish species widely found along the coastal regions of peninsular India and Sri Lanka. Pearlspot is known for its tender flesh, delectable taste, culinary tourism and highyielding market value. Information on the genetic makeup of stocks/populations is extremely vital as it forms the basis for future genetic studies. For this, we utilized ATPase6/8 genes of mtDNA of pearlspot populations collected from nine different locations ranging from Ratnagiri in Maharashtra state on the west coast to Chilika in Odisha on the east coast. Sequence analyses of these genes revealed 33 polymorphic sites, which include 17 singleton and 16 parsimony informative sites. Pair-wise genetic differentiation study (FST = 0.75) indicated significant (P<0.001) differences among all the pairs of stocks except those from Chilika and Nagayalanka. The spatial analysis of molecular variance (SAMOVA) significantly delineated the population into four groups (FCT = 0.69, P = 0.0001), namely northwest (Ratnagiri and Goa); southwest (Mangalore and lakes at Vembanad, Ashtamudi and Vellayani in Kerala); southeast (Pulicat in Tamil Nadu) and northeast (Chilika in Odisha and Nagayalanka in Andhra Pradesh). The above delineation is supported by clades of the phylogenetic tree and also the clusters of median joining haplotype network. The high haplotype diversity (0.84), low nucleotide diversity (0.003), and negative values of Tajima's D (-1.47) and Fu's Fs statistic (-14.89) are characteristic of populations having recently undergone demographic expansion. Mantel test revealed significant isolation by distance. The study identifies highly delineated structured populations with restricted gene flow. If such a stock is overfished, it is highly unlikely that it would recover through migration. For any future breeding programme in this species, it would be desirable to form a base population which incorporates the genetic material from all the locations so that we get a wide gene pool to select from.

Flow cytometry analysis of apoptotic progression and expression analysis of four apoptosis-related genes in Penaeus vannamei in response to white spot syndrome virus infection.

Flow cytometry analysis was carried out to detect the progression of apoptosis in haemocytes of WSSV infected Penaeus vannamei at different time-points (1.5 hpi, 18 hpi and 56 hpi). Apoptosis in haemocytes was found to increase with time of infectivity from 5.06 to 69.63%. Quantitative real-time PCR (qPCR) was used for the expression analysis of four apoptosis-related genes such as Death-associated protein 1, caspase-5, translationally controlled tumor protein, and cathepsin D. The evidence of apoptosis in haemocytes of P. vannamei was established as shown by significant increase in the percentage of late apoptotic cells due to WSSV infection in shrimp. The present study gives an insight to the apoptosis rate in a WSSV infected shrimp during the course of infection and the role of apoptosis related genes.

In Silico Prediction of Novel Probiotic Species Limiting Pathogenic Vibrio Growth Using Constraint-Based Genome Scale Metabolic Modeling.

The prevalence of bacterial diseases and the application of probiotics to prevent them is a common practice in shrimp aquaculture. A wide range of bacterial species/strains is utilized in probiotic formulations, with proven beneficial effects. However, knowledge of their role in inhibiting the growth of a specific pathogen is restricted. In this study, we employed constraint-based genome-scale metabolic modeling approach to screen and identify the beneficial bacteria capable of limiting the growth of V. harveyi, a common pathogen in shrimp culture. Genome-scale models were built for 194 species (including strains from the genera Bacillus, Lactobacillus, and Lactococcus and the pathogenic strain V. harveyi) to explore the metabolic potential of these strains under different nutrient conditions in a consortium. In silico-based phenotypic analysis on 193 paired models predicted six candidate strains with growth enhancement and pathogen suppression. Growth simulations reveal that mannitol and glucoronate environments mediate parasitic interactions in a pairwise community. Furthermore, in a mannitol environment, the shortlisted six strains were purely metabolite consumers without donating metabolites to V. harveyi. The production of acetate by the screened species in a paired community suggests the natural metabolic end product's role in limiting pathogen survival. Our study employing in silico approach successfully predicted three novel candidate strains for probiotic applications, namely, Bacillus sp 1 (identified as B. licheniformis in this study), Bacillus weihaiensis Alg07, and Lactobacillus lindneri TMW 1.1993. The study is the first to apply genomic-scale metabolic models for aquaculture applications to detect bacterial species limiting Vibrio harveyi growth.

Phylogenetic relations and mitogenome-wide similarity metrics reveal monophyly of Penaeus sensu lato.

Splitting of the genus Penaeus sensu lato into six new genera based on morphological features alone has been controversial in penaeid shrimp taxonomy. Several studies focused on building phylogenetic relations among the genera of Penaeus sensu lato. However, they lack in utilizing full mitochondrial DNA genome of shrimp representing all the six controversial genera. For the first time, the present study targeted the testing of all the six genera of Penaeus sensu lato for phylogenetic relations utilizing complete mitochondrial genome sequence. In addition, the study reports for the first time about the complete mitochondrial DNA genome sequence of Fenneropenaeus indicus, an important candidate species in aquaculture and fisheries, and utilized it for phylogenomics. The maximum likelihood and Bayesian approaches were deployed to generate and comprehend the phylogenetic relationship among the shrimp in the suborder, Dendrobranchiata. The phylogenetic relations established with limited taxon sampling considered in the study pointed to the monophyly of Penaeus sensu lato and suggested collapsing of the new genera to a single genus. Further, trends in mitogenome-wide estimates of average amino acid identity in the order Decapoda and the genus Penaeus sensu lato supported restoration of the old genus, Penaeus, rather promoting the creation of new genera.

Draft Genome Sequence of Vibrio parahaemolyticus Strain VP14, Isolated from a Penaeus vannamei Culture Farm.

Here, we report the draft genome sequence of an isolate of Vibrio parahaemolyticus, VP14, recovered from the gut of Penaeus vannamei shrimp farmed in southern India. The genome of VP14 comprised 5,224,046 bp with a GC content of 45.3% and contained 5,326 genes, including 4,972 coding sequences.

Draft Genome Sequence of the Luminescent Strain Vibrio campbellii LB102, Isolated from a Black Tiger Shrimp (Penaeus monodon) Broodstock Rearing System.

We report here the genome sequence of Vibrio campbellii LB102, isolated from the broodstock rearing system of a shrimp hatchery in India. Sequence analysis revealed the presence of effector toxins of the type III (YopT, sharing 39% identity with Yersinia pestis) and type VI (VgrG-3 and hemolysin coregulated protein of V. cholerae) secretion systems.

The development of a high density linkage map for black tiger shrimp (Penaeus monodon) based on cSNPs.

Transcriptome sequencing using Illumina RNA-seq was performed on populations of black tiger shrimp from India. Samples were collected from (i) four landing centres around the east coastline (EC) of India, (ii) survivors of a severe WSSV infection during pond culture (SUR) and (iii) the Andaman Islands (AI) in the Bay of Bengal. Equal quantities of purified total RNA from homogenates of hepatopancreas, muscle, nervous tissue, intestinal tract, heart, gonad, gills, pleopod and lymphoid organs were combined to create AI, EC and SUR pools for RNA sequencing. De novo transcriptome assembly resulted in 136,223 contigs (minimum size 100 base pairs, bp) with a total length 61 Mb, an average length of 446 bp and an average coverage of 163× across all pools. Approximately 16% of contigs were annotated with BLAST hit information and gene ontology annotations. A total of 473,620 putative SNPs/indels were identified. An Illumina iSelect genotyping array containing 6,000 SNPs was developed and used to genotype 1024 offspring belonging to seven full-sibling families. A total of 3959 SNPs were mapped to 44 linkage groups. The linkage groups consisted of between 16-129 and 13-130 markers, of length between 139-10.8 and 109.1-10.5 cM and with intervals averaging between 1.2 and 0.9 cM for the female and male maps respectively. The female map was 28% longer than the male map (4060 and 2917 cM respectively) with a 1.6 higher recombination rate observed for female compared to male meioses. This approach has substantially increased expressed sequence and DNA marker resources for tiger shrimp and is a useful resource for QTL mapping and association studies for evolutionarily and commercially important traits.