Assessing mechanisms of potential local adaptation through a seascape genomic approach in a marine gastropod, Littoraria flava.

Understanding the combined effects of environmental heterogeneity and evolutionary processes on marine populations is a primary goal of seascape genomic approaches. Here we utilized genomic approaches to identify local adaptation signatures in Littoraria flava, a widely distributed marine gastropod in the tropical West Atlantic population. We also performed molecular evolution analyses to investigate potential selective signals across the genome. After obtaining 6,298 and 16,137 single nucleotide polymorphisms (SNPs) derived from genotyping-by-sequencing (GBS) and RNA sequencing (RNA-Seq), respectively, 69 from GBS (85 specimens) and four from RNA-Seq (40 specimens) candidate SNPs were selected and further evaluated. The correlation analyses support different evolutionary pressures over transcribed and non-transcribed regions. Thus, SNPs within transcribed regions could account for the genotypic and possibly phenotypic divergences in periwinkles. Our molecular evolution tests based on synonymous and non-synonymus ratio (kN/kS) showed that genotype divergences containing putative adaptive SNPs arose mainly from synonymous and/or UTR substitutions rather than polymorphic proteins. The distribution of genotypes across different localities seems to be influenced by marine currents, pH, and temperature variations, suggesting that these factors may impact the species dispersion. The combination of RNA-Seq and GBS derived datasets provides a deeper understanding of the molecular mechanisms underlying selective forces responses on distinct genomic regions and could guide further investigations on seascape genomics for non-model species.

Transcriptome Expression of Biomineralization Genes in Littoraria flava Gastropod in Brazilian Rocky Shore Reveals Evidence of Local Adaptation.

Understanding how selection shapes population differentiation and local adaptation in marine species remains one of the greatest challenges in the field of evolutionary biology. The selection of genes in response to environment-specific factors and microenvironmental variation often results in chaotic genetic patchiness, which is commonly observed in rocky shore organisms. To identify these genes, the expression profile of the marine gastropod Littoraria flava collected from four Southeast Brazilian locations in ten rocky shore sites was analyzed. In this first L. flava transcriptome, 250,641 unigenes were generated, and 24% returned hits after functional annotation. Independent paired comparisons between 1) transects, 2) sites within transects, and 3) sites from different transects were performed for differential expression, detecting 8,622 unique differentially expressed genes. Araçá (AR) and São João (SJ) transect comparisons showed the most divergent gene products. For local adaptation, fitness-related differentially expressed genes were chosen for selection tests. Nine and 24 genes under adaptative and purifying selection, respectively, were most related to biomineralization in AR and chaperones in SJ. The biomineralization-genes perlucin and gigasin-6 were positively selected exclusively in the site toward the open ocean in AR, with sequence variants leading to pronounced protein structure changes. Despite an intense gene flow among L. flava populations due to its planktonic larva, gene expression patterns within transects may be the result of selective pressures. Our findings represent the first step in understanding how microenvironmental genetic variation is maintained in rocky shore populations and the mechanisms underlying local adaptation in marine species.

Shedding the Light on Litopenaeus vannamei Differential Muscle and Hepatopancreas Immune Responses in White Spot Syndrome Virus (WSSV) Exposure.

White Spot Syndrome Virus (WSSV) is one of the main threats to farming Litopenaeus vannamei, the most important crustacean commercialized in aquaculture worldwide. Here, we performed RNA-seq analyses in hepatopancreas and muscle from WSSV-negative (healthy) and WSSV-positive (unhealthy) L. vannamei, previously exposed to the virus, to obtain new insights about the molecular basis of resistance to WSSV. We detected 71% of our reads mapped against the recently described L. vannamei genome. This is the first report mapping RNA-seq transcripts from shrimps exposed to WSSV against the species reference genome. Differentially expressed gene (DEG) analyses were performed for four independent comparisons, and 13,338 DEGs were identified. When the redundancies and isoforms were disregarded, we observed 8351 and 6514 DEGs, respectively. Interestingly, after crossing the data, we detected a common set of DEGs for hepatopancreas and healthy shrimps, as well as another one for muscle and unhealthy shrimps. Our findings indicate that genes related to apoptosis, melanization, and the Imd pathway are likely to be involved in response to WSSV, offering knowledge about WSSV defense in shrimps exposed to the virus but not infected. These data present potential to be applied in further genetic studies in penaeids and other farmed shrimp species.

Identification of SNPs potentially related to immune responses and growth performance in Litopenaeus vannamei by RNA-seq analyses.

Litopenaeus vannamei is one of the most important shrimp species for worldwide aquaculture. Despite this, little genomic information is available for this penaeid and other closely related taxonomic crustaceans. Consequently, genes, proteins and their respective polymorphisms are poorly known for these species. In this work, we used the RNA sequencing technology (RNA-seq) in L. vannamei shrimp evaluated for growth performance, and exposed to the White Spot Syndrome Virus (WSSV), in order to investigate the presence of Single Nucleotide Polymorphisms (SNPs) within genes related to innate immunity and growth, both features of great interest for aquaculture activity. We analyzed individuals with higher and lower growth rates; and infected (unhealthy) and non-infected (healthy), after exposure to WSSV. Approximately 7,000 SNPs were detected in the samples evaluated for growth, being 3,186 and 3,978 exclusive for individuals with higher and lower growth rates, respectively. In the animals exposed to WSSV we found about 16,300 unique SNPs, in which 9,338 were specific to non-infected shrimp, and 7,008 were exclusive to individuals infected with WSSV and symptomatic. In total, we describe 4,312 unigenes containing SNPs. About 60% of these unigenes returned GO blastX hits for Biological Process, Molecular Function and Cellular Component ontologies. We identified 512 KEGG unique KOs distributed among 275 pathways, elucidating the majority of metabolism roles related to high protein metabolism, growth and immunity. These polymorphisms are all located in coding regions, and certainly can be applied in further studies involving phenotype expression of complex traits, such as growth and immunity. Overall, the set of variants raised herein enriches the genomic databases available for shrimp, given that SNPs originated from nextgen are still rare for this relevant crustacean group, despite their huge potential of use in genomic selection approaches.

RNA-seq as a powerful tool for penaeid shrimp genetic progress.

The sequences of all different RNA transcripts present in a cell or tissue that are related to the gene expression and its functional control represent what it is called a transcriptome. The transcripts vary between cells, tissues, ontogenetic and environmental conditions, and the knowledge that can be gained through them is of a solid relevance for genetic applications in aquaculture. Some of the techniques used in transcriptome studies, such as microarrays, are being replaced for next-generation sequencing approaches. RNA-seq emerges as a new possibility for the transcriptome complexity analysis as well as for the candidate genes and polymorphisms identification of penaeid species. Thus, it may also help to understand the determination of complex traits mechanisms and genetic improvement of stocks. In this review, it is first introduced an overview of transcriptome analysis by RNA-seq, followed by a discussion of how this approach may be applied in genetic progress within penaeid stocks.