The migration of anadromous fish in heterogenic environments unceasingly imposes a selective pressure that results in genetic variation for local adaptation. However, discrimination of anadromous fish populations by fine-scale local adaptation is challenging because of their high rate of gene flow, highly connected divergent population, and large population size. Recent advances in next-generation sequencing (NGS) have expanded the prospects of defining the weakly structured population of anadromous fish. Therefore, we used NGS-based restriction site-associated DNA (NextRAD) techniques on 300 individuals of an anadromous Hilsa shad (Tenualosa ilisha) species, collected from nine strategic habitats, across their diverse migratory habitats, which include sea, estuary, and different freshwater rivers. The NextRAD technique successfully identified 15,453 single nucleotide polymorphism (SNP) loci. Outlier tests using the F_ST OutFLANK and pcadapt approaches identified 74 and 449 SNPs (49 SNPs being common), respectively, as putative adaptive loci under a divergent selection process. Our results, based on the different cluster analyses of these putatively adaptive loci, suggested that local adaptation has divided the Hilsa shad population into two genetically structured clusters, in which marine and estuarine collection sites were dominated by individuals of one genetic cluster and different riverine collection sites were dominated by individuals of another genetic cluster. The phylogenetic analysis revealed that all the riverine populations of Hilsa shad were further subdivided into the north-western riverine (turbid freshwater) and the north-eastern riverine (clear freshwater) ecotypes. Among all of the putatively adaptive loci, only 36 loci were observed to be in the coding region, and the encoded genes might be associated with important biological functions related to the local adaptation of Hilsa shad. In summary, our study provides both neutral and adaptive contexts for the observed genetic divergence of Hilsa shad and, consequently, resolves the previous inconclusive findings on their population genetic structure across their diverse migratory habitats. Moreover, the study has clearly demonstrated that NextRAD sequencing is an innovative approach to explore how dispersal and local adaptation can shape genetic divergence of non-model anadromous fish that intersect diverse migratory habitats during their life-history stages.

The biological process of pearl formation is an ongoing research topic, and a number of genes associated with this process have been identified. However, the involvement of microRNAs (miRNAs) in biomineralization in the pearl oyster, Pinctada fucata, is not well understood. In order to investigate the divergence and function of miRNAs in P. fucata, we performed a transcriptome analysis of small RNA libraries prepared from adductor muscle, gill, ovary, and mantle tissues. We identified 186 known and 42 novel miRNAs in these tissues. Clustering analysis showed that the expression patterns of miRNAs were similar among the somatic tissues, but they differed significantly between the somatic and ovary tissues. To validate the existence of the identified miRNAs, nine known and three novel miRNAs were verified by stem-loop qRT-PCR using U6 snRNA as an internal reference. The expression abundance and target prediction between miRNAs and biomineralization-related genes indicated that miR-1990c-3p, miR-876, miR-9a-3p, and novel-3 may be key factors in the regulatory network that act by controlling the formation of matrix proteins or the differentiation of mineralogenic cells during shell formation in mantle tissue. Our findings serve to further clarify the processes underlying biomineralization in P. fucata.

The domestication of a wild-caught aquatic animal is an evolutionary process, which results in genetic discrimination at the genomic level in response to strong artificial selection. Although black tiger shrimp (Penaeus monodon) is one of the most commercially important aquaculture species, a systematic assessment of genetic divergence and structure of wild-caught and domesticated broodstock populations of the species is yet to be documented. Therefore, we used skim sequencing (SkimSeq) based genotyping approach to investigate the genetic structure of 50 broodstock individuals of P. monodon species, collected from five sampling sites (n = 10 in each site) across their distribution in Indo-Pacific regions. The wild-caught P. monodon broodstock population were collected from Malaysia (MS) and Japan (MJ), while domesticated broodstock populations were collected from Madagascar (MMD), Hawaii, HI, USA (MMO), and Thailand (MT). After various filtering process, a total of 194,259 single nucleotide polymorphism (SNP) loci were identified, in which 4983 SNP loci were identified as putatively adaptive by the pcadapt approach. In both datasets, pairwise F_ST estimates high genetic divergence between wild and domesticated broodstock populations. Consistently, different spatial clustering analyses in both datasets categorized divergent genetic structure into two clusters: (1) wild-caught populations (MS and MJ), and (2) domesticated populations (MMD, MMO and MT). Among 4983 putatively adaptive SNP loci, only 50 loci were observed to be in the coding region. The gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses suggested that non-synonymous mutated genes might be associated with the energy production, metabolic functions, respiration regulation and developmental rates, which likely act to promote adaptation to the strong artificial selection during the domestication process. This study has demonstrated the applicability of SkimSeq in a highly duplicated genome of P. monodon specifically, across a range of genetic backgrounds and geographical distributions, and would be useful for future genetic improvement program of this species in aquaculture.

Loach (Misgurnus anguillicaudatus) is well known to perform air-breathing through the posterior intestine and skin. However, we find here for the first time a unique central vascular structure in the loach barbel, with a blood–gas diffusion distance as short as that of the posterior intestine. Under acute hypoxia, the distance of loach barbels became significantly shorter. Moreover, barbel removal significantly decreased air-breathing frequency of the loach. These findings imply that the barbel is another air-breathing organ of the loach. For further investigation of loach barbel air-breathing, a transcriptome analysis of barbels with air exposure treatment was performed. A total of 2546 differentially expressed genes (DEGs) between the T-XU (air exposure) and C-XU (control) group were identified, and 13 key DEGs related to barbel air-breathing were screened out. On this foundation, sequence, expression, and location analysis results indicated an important positive role of fibronectin 1b (fn1b) in loach barbel air-breathing. We further generated an fn1b-depletion loach (MT for short) using the CRISPR/Cas9 technique. It was indicated that depletion of fn1b could weaker barbel air-breathing ability. In conclusion, due to nonlethal and regenerative characteristics, the loach barbel, a newly discovered and fn1b-related fish air-breathing organ, can be a good model for fish air-breathing research.

Cutaneous air-breathing is one of the air-breathing patterns in bimodal respiration fishes, while little is known about its underlying formation mechanisms. Here, we first investigated the skin regeneration of loach (Misgurnus anguillicaudatus, a cutaneous air-breathing fish) and yellow catfish (Pelteobagrus fulvidraco, a water-breathing fish) through morphological and histological observations. Then, the original skins (OS: MOS, POS) and regenerated skins (RS: MRS, PRS) when their capillaries were the most abundant (the structural foundation of air-breathing in fish) during healing, of the two fish species were collected for high-throughput RNA-seq. A total of 56,054 unigenes and 53,731 unigenes were assembled in loach and yellow catfish, respectively. A total of 640 (460 up- and 180 down-regulated) and 4446 (2340 up- and 2106 down-regulated) differentially expressed genes (DEGs) were respectively observed in RS/OS of loach and yellow catfish. Subsequently, the two DEG datasets were clustered in GO, KOG and KEGG databases, and further analyzed by comparison and screening. Consequently, tens of genes and thirteen key pathways were targeted, indicating that these genes and pathways had strong ties to cutaneous skin air-breathing in loach. This study provides new insights into the formation mechanism of cutaneous air-breathing and also offers a substantial contribution to the gene expression profiles of skin regeneration in fish.

PIWI/piRNA suppress transposon activity in animals, thereby safeguarding the genome from detrimental insertion mutagenesis. Recently, evidence revealed additional piRNA targets and functions in various animals. Although piRNAs are ubiquitously expressed in somatic tissues of the pearl oyster Pinctada fucata , their role is not well-characterized. Here, we report a PIWI/piRNA pathway, including piRNA biogenesis and piRNA-mediated gene regulation in P. fucata . A locked-nucleic-acid modified oligonucleotide (LNA-antagonist) was used to silence a single piRNA (piRNA0001) expression in P. fucata , which resulted in the differential expression of hundreds of endogenous genes. Target prediction analysis revealed that, following silencing, tens of endogenous genes were targeted by piRNA0001, including twelve up-regulated and nine down-regulated genes. Bioinformatic analyses suggested that different piRNA populations participate in the ping-pong amplification loop in a tissue-specific manner. These findings have improved our knowledge of the role of piRNA in mollusks, and provided evidence to understand the regulatory function of the PIWI/piRNA pathway on protein-coding genes outside of germline cells.