Transcriptional landscape of small non-coding RNAs reveals diversity of categories and functions in molluscs.

Small non-coding RNAs (sncRNAs) are non-coding RNA molecules that play various roles in metazoans. Among the sncRNAs, microRNAs (miRNAs) guide post-translational gene regulation during cellular development, proliferation, apoptosis, and differentiation, while PIWI-interacting RNAs (piRNAs) suppress transposon activity to safeguard the genome from detrimental insertion mutagenesis. While an increasing number of piRNAs are being identified in the soma and germlines of various organisms, they are scarcely reported in molluscs. To unravel the small RNA (sRNA) expression patterns and genomic function in molluscs, we generated a comprehensive sRNA dataset by sRNA sequencing (sRNA-seq) of eight mollusc species. Abundant miRNAs were identified and characterized in all investigated molluscs, and ubiquitous piRNAs were discovered in both somatic and gonadal tissues in six of the investigated molluscs, which are more closely associated with transposon silencing. Tens of piRNA clusters were also identified based on the genomic mapping results, which varied among different tissues and species. Our dataset serves as important reference data for future genomic and genetic studies on sRNAs in these molluscs and related species, especially in elucidating the ancestral state of piRNAs in bilaterians.

The chromosome-level genome and key genes associated with mud-dwelling behavior and adaptations of hypoxia and noxious environments in loach (Misgurnus anguillicaudatus).

BACKGROUND: The loach (Misgurnus anguillicaudatus), the most widely distributed species of the family Cobitidae, displays a mud-dwelling behavior and intestinal air-breathing, inhabiting the muddy bottom of extensive freshwater habitats. However, lack of high-quality reference genome seriously limits the interpretation of the genetic basis of specialized adaptations of the loach to the adverse environments including but not limited to the extreme water temperature, hypoxic and noxious mud environment. RESULTS: This study generated a 1.10-Gb high-quality, chromosome-anchored genome assembly, with a contig N50 of 3.83 Mb. Multiple comparative genomic analyses found that proto-oncogene c-Fos (fos), a regulator of bone development, is positively selected in loach. Knockout of fos (ID: Mis0086400.1) led to severe osteopetrosis and movement difficulties, combined with the comparison results of bone mineral density, supporting the hypothesis that fos is associated with loach mud-dwelling behavior. Based on genomic and transcriptomic analysis, we identified two key elements involved in the intestinal air-breathing of loach: a novel gene (ID: mis0158000.1) and heat shock protein beta-1 (hspb1). The flavin-containing monooxygenase 5 (fmo5) genes, central to xenobiotic metabolism, undergone expansion in loach and were identified as differentially expressed genes in a drug stress trial. A fmo5-/- (ID: Mis0185930.1) loach displayed liver and intestine injury, indicating the importance of this gene to the adaptation of the loach to the noxious mud. CONCLUSIONS: Our work provides valuable insights into the genetic basis of biological adaptation to adverse environments.

Environmental DNA study on aquatic ecosystem monitoring and management: Recent advances and prospects.

Environmental DNA (eDNA) is organismal DNA that can be detected in the environment and is derived from cellular material of organisms shed into aquatic or terrestrial environments. It can be sampled and monitored using molecular methods, which is important for the early detection of invasive and native species as well as the discovery of rare and cryptic species. While few reviews have summarized the latest findings on eDNA for most aquatic animal categories in the aquatic ecosystem, especially for aquatic eDNA processing and application. In the present review, we first performed a bibliometric network analysis of eDNA studies on aquatic animals. Subsequently, we summarized the abiotic and biotic factors affecting aquatic eDNA occurrence. We also systematically discussed the relevant experiments and analyses of aquatic eDNA from various aquatic organisms, including fish, molluscans, crustaceans, amphibians, and reptiles. Subsequently, we discussed the major achievements of eDNA application in studies on the aquatic ecosystem and environment. The application of eDNA will provide an entirely new paradigm for biodiversity conservation, environment monitoring, and aquatic species management at a global scale.

Exosome-derived small non-coding RNAs reveal immune response upon grafting transplantation in Pinctada fucata (Mollusca).

Exosomes, a subset of small extracellular vesicles, carry various nucleic acids, proteins, lipids, amino acids and metabolites. They function as a mode of intercellular communication and molecular transfer. Exosome cargo molecules, including small non-coding RNAs (sncRNAs), are involved in the immune response in various organisms. However, the role of exosome-derived sncRNAs in immune responses in molluscs remains unclear. Here, we aimed to reveal the sncRNAs involved in the immune response during grafting transplantation by the pearl oyster Pinctada fucata. Exosomes were successfully extracted from the P. fucata haemolymph during graft transplantation. Abundant microRNAs (miRNAs) and PIWI-interacting RNAs (piRNAs) were simultaneously discovered in P. fucata exosomes by small RNA sequencing. The expression patterns of the miRNAs and piRNAs at the grafting and initial stages were not substantially different, but varied significantly between the initial and later stages. Target prediction and functional analysis indicate that these miRNAs and piRNAs are related to immune response upon grafting transplantation, whereas piRNAs may also be associated with transposon silencing by targeting with genome transposon elements. This work provides the basis for a functional understanding of exosome-derived sncRNAs and helps to gain further insight into the PIWI/piRNA pathway function outside of germline cells in molluscs.

Depletion of LOXL2 improves respiratory capacity: From air-breathing fish to mammal under hypoxia.

Air-breathing fish are fascinating because of their ability to survive under hypoxia for a long time by using air-breathing organs (ABOs). Fish ABOs are thought to resemble the mammal lung all along. However, the link between the two has not been studied in depth. Here, we reported a markedly improved respiratory capacity in mice under hypoxia by inhibiting lysyl oxidase-like 2 (LOXL2), inspired from the intestinal air-breathing of loach (Misgurnus anguillicaudatus). Moreover, a posterior intestine (an ABO) transcriptome analysis revealed that the deletion of Loxl2b obviously inhibited PI3K-AKT and TGF-ß signaling, meanwhile, induced VEGF signaling, which could cause vasodilation and angiogenesis to improve the air-breathing ability of loach. The same phenomenon was found in LOXL2-inhibition mice under hypoxia, which significantly prolonged their living period relative to wild-type (WT) mice. In addition, compared with WT loach, Loxl2b-/- loach presented enhanced anaerobic metabolism, which could also make itself to better survive in hypoxic environment. This should be the magic of air-breathing fish! Supplied from air-breathing fish, this study provides a novel means of improving respiratory capacity in mammal under hypoxia.

Endogenic upregulations of HIF/VEGF signaling pathway genes promote air breathing organ angiogenesis in bimodal respiration fish.

Air-breathing has evolved independently serval times with a variety of air-breathing organs (ABOs) in fish. The physiology of the air-breathing in bimodal respiration fish has been well understood, while studies on molecular mechanisms of the character are very limited. In the present study, we first determined the gill indexes of 110 fish species including 25 and 85 kinds of bimodal respiration fishes and non-air-breathing fishes, respectively. Then combined with histological observations of gills and ABOs/non-ABOs in three bimodal respiration fishes and two non-air breathing fishes, we found that the bimodal respiration fish was always of a degeneration gill and a well-vascularized ABO. Meanwhile, a comparative transcriptome analysis of posterior intestines, namely a well vascularized ABO in Misgurnus anguillicaudatus and a non-ABO in Leptobotia elongata, was performed to expound molecular variations of the air-breathing character. A total of 5,003 orthologous genes were identified. Among them, 1,189 orthologous genes were differentially expressed, which were enriched in 14 KEGG pathways. More specially, the expressions of hemoglobin genes and various HIF/VEGF signaling pathway genes were obviously upregulated in the ABO of M. anguillicaudatus. Moreover, we found that HIF-1α, VEGFAa, and MAP2K1 were co-expressed dramatically higher in ABOs of bimodal respiration fishes than those of non-ABOs of non-air-breathing fishes. These results indicated that the HIF/VEGF pathway played an important role in ABO angiogenesis/formation to promote fish to do aerial respiration. This study will contribute to our understanding of molecular mechanisms of air-breathing in fish.

Comparative Transcriptomic Analysis of Regenerated Skins Provides Insights into Cutaneous Air-Breathing Formation in Fish.

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.

Fibronectin 1B Gene Plays an Important Role in Loach Barbel Air-Breathing.

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.

A Review of Discovery Profiling of PIWI-Interacting RNAs and Their Diverse Functions in Metazoans.

PIWI-interacting RNAs (piRNAs) are a class of small non-coding RNAs (sncRNAs) that perform crucial biological functions in metazoans and defend against transposable elements (TEs) in germ lines. Recently, ubiquitously expressed piRNAs were discovered in soma and germ lines using small RNA sequencing (sRNA-seq) in humans and animals, providing new insights into the diverse functions of piRNAs. However, the role of piRNAs has not yet been fully elucidated, and sRNA-seq studies continue to reveal different piRNA activities in the genome. In this review, we summarize a set of simplified processes for piRNA analysis in order to provide a useful guide for researchers to perform piRNA research suitable for their study objectives. These processes can help expand the functional research on piRNAs from previously reported sRNA-seq results in metazoans. Ubiquitously expressed piRNAs have been discovered in the soma and germ lines in Annelida, Cnidaria, Echinodermata, Crustacea, Arthropoda, and Mollusca, but they are limited to germ lines in Chordata. The roles of piRNAs in TE silencing, gene expression regulation, epigenetic regulation, embryonic development, immune response, and associated diseases will continue to be discovered via sRNA-seq.

Integrated proteomic and transcriptomic analysis reveals that polymorphic shell colors vary with melanin synthesis in Bellamya purificata snail.

The snail Bellamya purificata is an ecologically and economically important freshwater gastropod species. However, limited genomic resources are available for this snail. In this study, the transcriptome of mantle tissues and proteome of shells of B. purificata with two shell colors (namely light-cyan line (LC) and light-purple line (LP)) were deeply sequenced and characterized. A total of 5.72 million contigs were assembled into 157,015 unigenes, 21,455 (13.66%) of these unigenes were significantly matched to NR, Swiss-Prot, KOG, GO and KEGG database. 1807 differentially expressed genes (DEGs) were identified between the two different shell color lines. These DEGs were significantly enriched in five KEGG pathways including tyrosine metabolism, tryptophan metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, phenylalanine metabolism, and histidine metabolism, which suggested that the shell color polymorphism in B. purificata was a result of melanin synthesis variation. A total of 1521 proteins were identified in B. purificata here as well. The differentially expressed protein analysis showed that the tyrosinase content in LP was significantly decreased in comparison to LC, which agreed with the transcriptome analysis results. This study provides valuable genomic resources of B. purificata and improves our understanding of molecular mechanisms of biomineralization and shell color polymorphism in snail.

Discovery and functional understanding of MiRNAs in molluscs: a genome-wide profiling approach.

Small non-coding RNAs play a pivotal role in gene regulation, repression of transposable element and viral activity in various organisms. Among the various categories of these small non-coding RNAs, microRNAs (miRNAs) guide post-translational gene regulation in cellular development, proliferation, apoptosis, oncogenesis, and differentiation. Here, we performed a genome-wide computational prediction of miRNAs to improve the understanding of miRNA observation and function in molluscs. As an initial step, hundreds of conserved miRNAs were predicted in 35 species of molluscs through genome scanning. Afterwards, the miRNAs' population, isoforms, organization, and function were characterized in detail. Furthermore, the key miRNA biogenesis factors, including AGO2, DGCR8, DICER, DROSHA, TRABP2, RAN, and XPO5, were elucidated based on homologue sequence searching. We also summarized the miRNAs' function in biomineralization, immune and stress response, as well as growth and development in molluscs. Because miRNAs play a vital role in various lifeforms, this study will provide insight into miRNA biogenesis and function in molluscs, as well as other invertebrates.

Skim-Sequencing Based Genotyping Reveals Genetic Divergence of the Wild and Domesticated Population of Black Tiger Shrimp (Penaeus monodon) in the Indo-Pacific Region.

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 FST 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.

Characterisation of stem and proliferating cells on the retina and lens of loach Misgurnus anguillicaudatus.

The eye of the fish has a lifelong persistent neurogenesis unlike eye of mammals, so it's highly interesting to study retinal neurogenesis and its genetic control to give complete knowledge about the cause of this property in fish in comparison to mammals. We performed fluorescent in situ hybridisation for loach Misgurnus anguillicaudatus bmi1, msi1 and sox2 genes, which are used as an indicator of the sites of multipotent stem cells. Proliferating cell nuclear antigen (PCNA), bromodeoxyuridine (BRDU) and KI67 markers were used as indicators of proliferating cells and glial fibrillary acidic protein (GFAP) immunofluorescence was used for detection of the glial property of cells, as well as, immunohistochemistry detected the role of peroxisome proliferator-activated receptor (PPAR)α and γ in retinal neurogenesis. Our results determined that the lens and the retina of loach M. anguillicaudatus contain proliferative and pluripotent stem cells that have both glial and neuroepithelial properties, which add new cells continuously throughout life even without injury-induced proliferation. The PPARα has an essential function in providing energy supply for retinal neurogenesis more than PPARγ.

Identification and Characterization of microRNAs and Their Predicted Functions in Biomineralization in the Pearl Oyster (Pinctada fucata).

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.

Piwi-interacting RNA (piRNA) expression patterns in pearl oyster (Pinctada fucata) somatic tissues.

Piwi-interacting RNAs (piRNAs) belong to a recently discovered class of small non-coding RNAs whose best-understood function is repressing transposable element activity. Most piRNA studies have been conducted on model organisms and little is known about piRNA expression and function in mollusks. We performed high-throughput sequencing of small RNAs extracted from the mantle, adductor muscle, gill, and ovary tissues of the pearl oyster, Pinctada fucata. RNA species with sequences of approximately 30 nt were widely expressed in all tissues. Uridine at the 5' terminal and protection from ß-elimination at the 3' terminal suggested that these were putative piRNAs. A total of 18.0 million putative piRNAs were assigned to 2.8 million unique piRNAs, and 35,848 piRNA clusters were identified. Mapping to the reference genome showed that 25% of the unique piRNAs mapped to multiple tandem loci on the scaffold. Expression patterns of the piRNA clusters were similar within the somatic tissues, but differed significantly between the somatic and gonadal tissues. These findings suggest that in pearl oysters piRNAs have important and novel functions beyond those in the germ line.

Population Genomics of an Anadromous Hilsa Shad Tenualosa ilisha Species across Its Diverse Migratory Habitats: Discrimination by Fine-Scale Local Adaptation.

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 FST 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.

Fertility and ploidy of gametes of allodiploid and allotriploid loaches produced by diploid Misgurnus anguillicaudatus females and Paramisgurnus dabryanus males.

Artificial and natural hybridization of dojo loach Misgurnus anguillicaudatus (2N = 50, DD for short) and large-scale loach Paramisgurnus dabryanus (2N = 48, PP for short) are well-grown. However, these hybrid loaches have not yet been examined for fertility and ploidy of gametes. Here, histological observations, artificial propagation, observations of embryonic development, larval morphology, and ploidy analyses were conducted to determine the fertility and ploidy of gametes of allodiploid (DP for short) and allotriploid (DDP for short) loaches, produced by DD females × PP males and induced from fertilized eggs of DD females × PP males by cold shock to prevent the second polar body release, respectively. The ovaries of DP and DDP included smaller number of eggs when compared with those of the control DD, while full-grown oocytes were observed. Testes of these two loaches were delayed-developed without spermatids or mature spermatozoa. Results obtained here showed that DP and DDP were fertility-weakened female and sterile male. Moreover, DP females and DDP females could, respectively, produce few viable haploid eggs and few viable haploid and diploid eggs. This study will provide valuable information for fish hybrid researches.

Molecular cloning, expression analysis and miRNA prediction of vascular endothelial growth factor A (VEGFAa and VEGFAb) in pond loach Misgurnus anguillicaudatus, an air-breathing fish.

Vascular endothelial growth factor A (VEGFA) is the most studied and the best characterized member of the VEGF family and is a key regulator of angiogenesis via its ability to affect the proliferation, migration, and differentiation of endothelial cells. In this study, the full-length cDNAs encoding VEGFAa and VEGFAb from pond loach, Misgurnus anguillicaudatus, were isolated. The VEGFAa is constituted by an open reading frame (ORF) of 570bp encoding for a peptide of 189 amino acid residues, a 639bp 5'-untranslated region (UTR) and a 2383bp 3' UTR. The VEGFAb is constituted by an ORF of 687bp encoding for a peptide of 228 amino acid residues, a 560bp 5' UTR and a 1268bp 3' UTR. Phylogenetic analysis indicated that the VEGFAa and VEGFAb of pond loach were conserved in vertebrates. Expression levels of VEGFAa and VEGFAb were detected by RT-qPCR at different development stages of pond loach and in different tissues of 6-month-old, 12-month-old and 24-month-old pond loach. Moreover, eight predicted miRNAs (miR-200, miR-29, miR-218, miR-338, miR-103, miR-15, miR-17 and miR-223) targeting VEGFAa and VEGFAb were validated by an intestinal air-breathing inhibition experiment. This study will be of value for further studies into the function of VEGFA and its corresponding miRNAs, which will shed a light on the vascularization and accessory air-breathing process in pond loach.

Developmental transcriptome analysis and identification of genes involved in formation of intestinal air-breathing function of Dojo loach, Misgurnus anguillicaudatus.

Dojo loach, Misgurnus anguillicaudatus is a freshwater fish species of the loach family Cobitidae, using its posterior intestine as an accessory air-breathing organ. Little is known about the molecular regulatory mechanisms in the formation of intestinal air-breathing function of M. anguillicaudatus. Here high-throughput sequencing of mRNAs was performed from six developmental stages of posterior intestine of M. anguillicaudatus: 4-Dph (days post hatch) group, 8-Dph group, 12-Dph group, 20-Dph group, 40-Dph group and Oyd (one-year-old) group. These six libraries were assembled into 81300 unigenes. Totally 40757 unigenes were annotated. Subsequently, 35291 differentially expressed genes (DEGs) were scanned among different developmental stages and clustered into 20 gene expression profiles. Finally, 15 key pathways and 25 key genes were mined, providing potential targets for candidate gene selection involved in formation of intestinal air-breathing function in M. anguillicaudatus. This is the first report of developmental transcriptome of posterior intestine in M. anguillicaudatus, offering a substantial contribution to the sequence resources for this species and providing a deep insight into the formation mechanism of its intestinal air-breathing function. This report demonstrates that M. anguillicaudatus is a good model for studies to identify and characterize the molecular basis of accessory air-breathing organ development in fish.

Transcriptomic Analysis of Compromise Between Air-Breathing and Nutrient Uptake of Posterior Intestine in Loach (Misgurnus anguillicaudatus), an Air-Breathing Fish.

Dojo loach (Misgurnus anguillicaudatus) is an air-breathing fish species by using its posterior intestine to breathe on water surface. So far, the molecular mechanism about accessory air-breathing in fish is seldom addressed. Five cDNA libraries were constructed here for loach posterior intestines form T01 (the initial stage group), T02 (mid-stage of normal group), T03 (end stage of normal group), T04 (mid-stage of air-breathing inhibited group), and T05 (the end stage of air-breathing inhibited group) and subjected to perform RNA-seq to compare their transcriptomic profilings. A total of 92,962 unigenes were assembled, while 37,905 (40.77 %) unigenes were successfully annotated. 2298, 1091, and 3275 differentially expressed genes (fn1, ACE, EGFR, Pxdn, SDF, HIF, VEGF, SLC2A1, SLC5A8 etc.) were observed in T04/T02, T05/T03, and T05/T04, respectively. Expression levels of many genes associated with air-breathing and nutrient uptake varied significantly between normal and intestinal air-breathing inhibited group. Intraepithelial capillaries in posterior intestines of loaches from T05 were broken, while red blood cells were enriched at the surface of intestinal epithelial lining with 241 ± 39 cells per millimeter. There were periodic acid-schiff (PAS)-positive epithelial mucous cells in posterior intestines from both normal and air-breathing inhibited groups. Results obtained here suggested an overlap of air-breathing and nutrient uptake function of posterior intestine in loach. Intestinal air-breathing inhibition in loach would influence the posterior intestine's nutrient uptake ability and endothelial capillary structure stability. This study will contribute to our understanding on the molecular regulatory mechanisms of intestinal air-breathing in loach.