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Loaches, Misgurnus anguillicaudatus, from Poyang Lake are a small economic fish, falling into three categories with typical blackspot characteristics, namely, big blackspot loaches (BBL), small blackspot loaches (SBL), and non-blackspot loaches (NBL), respectively. Pigment cell composition, pigment content, tyrosinase content and activity of three kinds of loaches were investigated. Our results showed that there were three types of skin pigment cells, namely, melanophores, xanthophores and iridophores. Melanophores in dorsum were more than in abdomen. Melanophore cytosomes in BBL were larger than those in SBL and NBL, and melanosomes were the largest in stage four. The melanophores in dorsal skin of SBL or NBL were small cell bodies, spindle-like, and in chain distribution. There was an extremely significant difference in melanin content in BBL between the dorsum and abdomen (P 0.05). In dorsal skin, tyrosinase content was the highest in BBL, and it was significantly lower in NBL than in BBL and SBL (P

Misgurnus anguillicaudatus (M. anguillicaudatus) is a widely cultivated fish. However, in M. anguillicaudatus breeding, the frequent cold stress during daily breeding could induce immune suppression and increase the risk of infection, causing serious economic loss. Based on existing findings, CpG Oligonucleotides (CpG-ODNs) may be an ideal protective agent for low temperature fish breeding, performing anti-infective when faced with cold stress with cold shock proteins Y box binding proteins (YBX). Although YBX has pleiotropic functions, its roles in CpG-ODNs-mediated immunity (especially under cold situations) remain largely unexplored. To clarify the relationship among them, we identified the YBX1/YBX2 in M. anguillicaudatus and analyzed using a series of bioinformatics methods. After that, we immunized the fish with 3 types of CpG-ODNs and challenged with Aeromonas hydrophila (A. hydrophila). Here we showed that the best anti-bacterial effect of CpG-B was accompanied by the significant upregulation of YBX1. And the detection of the YBX1 downstream effectors confirmed that CpG-B induced the YBX1-mediated Th1 oriented responses to A. hydrophila by regulation of the NLRP3 (Caspase-A/-B), IL-1β, IL-12 and IFN-γ. Afterwards, we found that under cold stress, CpG-B can activate the NLRP3 and NF-κB pathways through YBX1, a key mediator of anti-A. hydrophila in CpG-B immunization. In this study, we demonstrated CpG-B protection against infection in low temperature, and its interaction with YBX1, expanded the research of CpG-ODN under cold stress, and provided a new CpG-ODN application for low temperature fish farming.

The dojo loach Misgurnus anguillicaudatus is an endemic freshwater species in Asia. The effective conservation and molecular-aided selection of M. anguillicaudatus were limited without sufficient molecular markers. In this study, 112 novel single nucleotide polymorphism (SNP) markers were screened based on 2b-RAD sequencing database, and 56 SNPs of them were developed and characterized by genotyping 40 individuals using SNaPshot method. The observed heterozygosity (Ho) ranged from 0.025 to 0.675, while the expected heterozygosity (He) varied from 0.025 to 0.500. The minor allele frequency (MAF) ranged from 0.013 to 0.500. Among these SNPs, 18 loci were found to deviate significantly from the Hardy–Weinberg equilibrium after Bonferroni correction (P

This study represents a description of the diet composition of one of the largest European cobitids, the weatherfish Misgurnus fossilis. Specimens were collected in a drainage canal, representing a typical habitat for weatherfish, and with gut content analysis conducted with regard to individual total length and maturity stage. Overall, the weatherfish diet mainly consisted of Copepoda, Cladocera, Ostracoda, Oligochaeta, Asellus aquaticus, Chironomidae and Coleoptera larvae, Gastropoda, and detritus. To evaluate size‐related patterns of resource use, fish were assigned to two size classes, defined according to size at first maturation. ANOSIM analyses revealed major ontogenetic shifts in feeding strategy, which were related to size and maturity, with a significant ontogenetic shift in feeding pattern, marked by differences in the proportions of the main taxonomic groups of prey consumed. Copepoda and Cladocera dominated in the diet of small and immature individuals, while large weatherfish primarily fed on detritus. Similarly, cluster analysis of diet classified into these food types showed distinct two groups comprising juvenile and mature fish. The weatherfish is a food opportunist using all available resources, but spatially showed a change in feeding sites. Smaller and sexually immature individuals more often use prey caught in the water column and among macrophytes, while larger (sexually mature) individuals occupying the bottom, much more often use detritus as a food base.

Misgurnus anguillicaudatus and Paramisgurnus dabryanus hybrids were generated via full diallel crosses between the Meishan (M_M) and Fuyang (M_F) populations of M. anguillicaudatus and Meishan (P_M) and Ziyang (P_Z) populations of P. dabryanus. The purebred and hybrid offspring were co-cultured in a pool for 4 months, and 96.5% of the offspring could be assigned to a single parental pair based on nine potentially cross-species polymorphic microsatellites. The average body weight (BW) of the P_M × P_Z (female × male, in this order hereafter) hybrids was the highest (4.88 g), followed by that of the P_Z × P_M and P_M × P_M hybrids. The general combining abilities (GCA) of BW from both the sires and dams of P_M and P_Z were high, and that from M_M and M_F were low. The specific combining ability (SCA) of P_M × P_Z was the largest (16.61), followed by that of P_Z × P_M, M_F × M_M, and M_M × M_F (−15.89). The combination of M_F × P_M, M_F × P_Z, and P_M × M_F showed > 10% heterosis. This is the first study to use microsatellite-based parentage assignment in hybrid fish and lays foundation for loach crossbreeding.

Morphological signs (32 plastic and 7 meristic) and biological parameters (size, weight, age and sexual structure, fecundity) are considered for weather loaches of gen. Misgurnus inhabiting the Pad Bolshaya (Jewish Autonomous oblast), with the body length Ad 60.2-182.4 mm, body weigh 2.3-55.3 g, age 1-8 years and sex ratio about 1 : 1 (53 % males, 47 % females). Sexual dimorphism is found for antedorsal, anteventral and ventroanal distances, tail fin base length, base length and height of pectoral, pelvic and anal fins, and maximum body height. Ontogenetic variability of morphological indices in dependence on body length is detected for 26 of 32 plastic signs, whereas the dependence of anteanal, antedorsal, pectoventral, and anteventral distances, and maximum and minimum body height on the body length is not statistically significant. Males grow slower than females. Age of puberty is 4-6 years, mean individual absolute fecundity is 7409 eggs. The analysis concluded that the weather loaches Misgurnus in the Pad Bolshaya belonged to the species Misgurnus nikolskyi (Vasil’eva, 2001).

The combination of high-throughput sequencing technology and environmental DNA (eDNA) analysis has the potential to be a powerful tool for comprehensive, non-invasive monitoring of species in the environment. To understand the correlation between the abundance of eDNA and that of species in natural environments, we have to obtain quantitative eDNA data, usually via individual assays for each species. The recently developed quantitative sequencing (qSeq) technique enables simultaneous phylogenetic identification and quantification of individual species by counting random tags added to the 5′ end of the target sequence during the first DNA synthesis. Here, we applied qSeq to eDNA analysis to test its effectiveness in biodiversity monitoring. eDNA was extracted from water samples taken over 4 days from aquaria containing five fish species (Hemigrammocypris neglectus, Candidia temminckii, Oryzias latipes, Rhinogobius flumineus, and Misgurnus anguillicaudatus), and quantified by qSeq and microfluidic digital PCR (dPCR) using a TaqMan probe. The eDNA abundance quantified by qSeq was consistent with that quantified by dPCR for each fish species at each sampling time. The correlation coefficients between qSeq and dPCR were 0.643, 0.859, and 0.786 for H. neglectus, O. latipes, and M. anguillicaudatus, respectively, indicating that qSeq accurately quantifies fish eDNA.

Phenanthrene (PHE) as a tricyclic polycyclic aromatic hydrocarbon is one of the common pollutants in water and sediments, which can cause reproductive toxicity to aquatic organisms. In this study, enzyme-linked immunosorbent assay (ELISA) was used to detect the vitellogenin (VTG) of loach, and then to explore the estrogenic toxicity effect of PHE on loach. The results were as follows: (1) with the increase of PHE concentrations and the extension of exposure time, the gonadosomatic index (GSI) of males decreased significantly, while it increased in female loaches. In addition, males had more obvious changes than females and were more sensitive to PHE. (2) The increase of VTG contents in serum of males were stronger than that in females. Those results reveal that PHE has estrogenic effect, which can affect the generation of VTG, thus causing damage to the gonad development of loach.

The fecundity and sexual structure of weatherfish (Misgurnus fossilis) population, an inperilled and protected freshwater fish with a poorly known life history, was studied in three waterbodies: the River Ner, the Południowy canal and the Nowy Rów canal (Poland) differing in hydrological character. We compared reproductive traits; i.e. sex ratio, absolute and relative fecundity, oocyte size, gonado-somatic index and body condition. In all study sites, the sex ratio of weatherfish did not differ from parity (1: 1), though in the River Ner, the highest GSI values were recorded for females whilst male GSI did not differ among sites. The absolute (FA = 2860 ± 2065) and relative fecundity (FR = 120 ± 55 eggs per g of female body weight) in the River Ner were significantly lower than in the other two sites. In the River Ner the frequency distribution of oocyte diameter, decomposed using the Bhattacharya method, showed two distinct groups in equal numbers. Oocytes that were ready to spawn were larger in the River Ner than in the Południowy and Nowy Rów canals. Fish in the River Ner were also characterized by the lowest Fulton condition index (mean K = 0.36 ± 0.1). The trade-off between impaired fecundity and increased egg diameter may result from the different nature of the studied watercourses or levels of endocrine-disrupting chemicals (EDCs), such as steroid compounds.

In dojo loach (Misgurnus anguillicaudatus), although most wild types are gonochoristic diploids that are genetically differentiated into 2 groups, A and B, clonal lineages appear in certain localities. Clonal loaches have been considered to have hybrid origins between the 2 groups by a series of genetic studies. In this study, using FISH with a newly developed probe (ManDra-A), we identified 26 (1 pair of metacentric and 12 pairs of telocentric chromosomes) of 50 diploid chromosomes in contemporary wild-type group A loach. In contrast, ManDra-A signals were not detected on metacentric chromosomes derived from the ancestral group A of clonal loach. The FISH results clearly showed the presence of certain differentiations in metacentric chromosomes between ancestral and contemporary group A loach. Two-color FISH with ManDra-A and group B-specific ManDra (renamed ManDra-B) probes reconfirmed the hybrid origin of clones by identifying chromosomes from both groups A and B in metaphases. Our results showed the hybrid origin of clonally reproducing fish and the possibility that chromosomal differentiation between ancestral and contemporary fish can affect gametogenesis. In meiotic spermatocytes of sex-reversed clones, ManDra-A, and not ManDra-B, signals were detected in 12 out of 50 bivalents. Thus, the results further support the previous conclusion that clonal gametogenesis was assured by pairing between sister chromosomes duplicated from each ancestral chromosome from group A or B. Our study deepens the knowledge about the association between clonality and hybridity in unisexual vertebrates.

A noninvasive procedure for ultrasound imaging is developed to study the structure of fish embryos and measuring the speed of sound in their tissues. Loach (Misgurnus fossilis) eggs growing in the immersion cell of a device are examined using an acoustic microscope with a central frequency of 50 MHz. The speed of sound in the yolk and cell layer (blastula) of the embryo is measured at developmental stages 6–8.

The fish assemblage of the left Ross tributaries – the rivers Kamianka and Protoka is described. The total collected 2842 specimens. Studies were conducted in 2013 and 2020 in Kyiv and Zhytomyr regions. The current species composition includes 30 species for Kamianka and 20 species - Protoka. According to the research results, it has been established that the rivers are dominated by fish of the eurytopic ecological group, phytophils, zoobenthophages. It was found that the species composition in the studied parts of the Kamenka River is higher than in the Protoka River. This indicates the presence of a high level of biotopic diversity in comparison with other water bodies. In some areas studied there are species - from Resolution 6, adopted to create the Emerald Network. It’s such species as Rhodeus amarus (Bloch, 1782), Cobitis taenia (s.l.) Linnaeus, 1758, Misgurnus fossilis (Linnaeus, 1758). This allows us to take some reserved sites on rivers Kamianka and Protoka as objects of Emerald Network.

In the Naka-ikemi Wetland, a cryptic species of the dojo loach, Misgurnus sp. Type I (called Type I loach herein), inhabits along with the common nominal species, Misgurnus anguillicaudatus (called Type II loach herein). In this study, the distribution patterns of two loaches were investigated by environmental DNA (eDNA) analysis and previously suggested habitat segregation between two sympatric loaches was discussed. Type I loach tended to have a limited distribution, mainly inhabiting the northern part of the wetland; Type II loach tended to be widely distributed across the central to southern parts of the wetland. Both loaches occurred sympatrically in some areas throughout the sampling periods, indicating that they did not completely segregate their habitats.

The European weather loach (Misgurnus fossilis) is classified as highly endangered in several countries of Central Europe. Populations of M. fossilis are predominantly found in ditches with low water levels and thick sludge layers and are thus hard to detect using conventional fishing methods. Therefore, environmental DNA (eDNA) monitoring appears particularly relevant for this species. In previous studies, M. fossilis was surveyed following eDNA water sampling protocols, which were not optimized for this species. Therefore, we created two full factorial study designs to test six different eDNA workflows for sediment samples and twelve different workflows for water samples. We used qPCR to compare the threshold cycle (Ct) values of the different workflows, which indicate the target DNA amount in the sample, and spectrophotometry to quantify and compare the total DNA amount inside the samples. We analyzed 96 water samples and 48 sediment samples from a pond with a known population of M. fossilis. We tested several method combinations for long‐term sample preservation, DNA capture, and DNA extraction. Additionally, we analyzed the DNA yield of samples from a ditch with a natural M. fossilis population monthly over one year to determine the optimal sampling period. Our results showed that the long‐term water preservation method commonly used for eDNA surveys of M. fossilis did not lead to optimal DNA yields, and we present a valid long‐term sample preservation alternative. A cost‐efficient high salt DNA extraction led to the highest target DNA yields and can be used for sediment and water samples. Furthermore, we were able to show that in a natural habitat of M. fossilis, total and target eDNA were higher between June and September, which implies that this period is favorable for eDNA sampling. Our results will help to improve the reliability of future eDNA surveys of M. fossilis.

Artificial interspecific hybrids (3n) of tetraploid dojo loach Misgurnus anguillicaudatus (TTTT) and diploid large-scale loach Paramisgurnus dabryanus (PP) were viable. Allopentaploid loaches (TTTTP) were produced by cold-shocking the fertilized eggs of TTTT females fertilized with sperm of PP males. The TTTTP were viable and well developed. As there are currently no reports on the fertility and gamete ploidy of TTTTP, artificial propagation and ploidy analyses were conducted to determine them. The ovaries of TTTTP were well developed with primary oocytes and secondary oocytes; the testes were sterile with low-density sperm-like cells. By backcrossing with mature TTTT, TTTTP were found to be female fertile and male sterile. The fertility of female TTTTP was low; they produced few viable diploid eggs and a few inviable triploid and aneuploid eggs. Ploidy analysis of cold-shock-induced progenies of TTTTP × TTTT indicated that TTTTP could generate viable diploid eggs, along with diploid second polar bodies. The results of this study are helpful for further understanding of loach polyploidization and should encourage more investigations on hybridization and gamete formation in loaches.

Accessory respiratory is generally accepted to have evolved independently on numerous occasions in adaption to aquatic hypoxia in freshwater habitats. In general, the air-breathing organ in fish is believed to be structurally modified to supplement respiration. In this study, intuitive evidence for elaborate structural modifications of the intestine, an air-breathing organ in mud loach (Misgurnus anguillicaudatus), compared with two other obligate aquatic breathers, grass carp (Ctenopharyngodon idellus) and yellow catfish (Pelteobagrus fulvidraco), were directly provided by histological and morphometric methods. As a result, a sharply decreasing height of mucosal folds and thickness of muscularis were manifested in loach intestine from its anterior to posterior region. Compared with grass carp and yellow catfish, loach had the smallest ratios of mucosal fold height/muscularis thickness to intestinal lumen radius in the posterior intestine. These suggested that the posterior intestine is the air-breathing location for the loach. Furthermore, length density of capillary (0.46±0.05 μm−2) in the posterior intestine of the loach was significantly higher than those of grass carp and yellow catfish. Meanwhile, diffusion distance of air-blood barrier (1.34±0.04 μm) in the posterior intestine of the loach was significantly smaller than those of the other two fish species. In summary, the characteristics of highly vascularized, short diffusion distance of air-blood barrier, thinned and flattened made the posterior intestine a perfect air-breathing location for the loach.

To study the effect on amino acid and mineral content of the loach meat by adding Fe (II) chelating hairtail protein hydrolysates (Fe (II)‐HPH) to the feed. A total of 100 healthy loaches were selected. After 1 week's adaptive feeding, they were randomly divided into five groups and fed with feeds containing of Fe (II)‐HPH (0, 0.5, 1, 2, and 4 g/kg). On the 40th day, detection work of general nutrients (moisture, ash, crude protein, and crude fat), mineral elements (Fe, Mn, Cu, Zn, Na, K, and Ca), amino acid and amino acid score (AAS), Chemical Score (CS) and essential amino acid index (EAAI) indexes were done. The results show that crude protein has the highest content while crude fat has the lowest when amount of added Fe (II)‐HPH in feed is 2 g/kg. The Fe content is significantly improved while amount of added is 1, 2, 4 g/kg. The Ca content is significantly improved and the Zn content is significantly improved while amount of added was 2 g/kg. Mn contents are significantly lower than control while amount of added is 4 g/kg. Based on analysis of amino acids in each group, the nutritional value of loach meat with 2 g/kg Fe (II)‐HPH addition amount is relatively high, total amount of essential amino acids increases significantly, and EAA/TAA and EAA/NEAA improve significantly. In conclusion, adding 2 g/kg Fe (II)‐HPH to feed could improve the nutritional values of loach meat.

We examined the functional morphology of loach Misgurnus anguillicaudatus skin by using synchrotron X-ray micro-computed tomography (SR-μCT) and high-contrast staining using osmium tetroxide or phosphotungstic acid (PTA), which enhances the image contrast of soft tissues. The captured high-spatial resolution images revealed that the surface ornamentations were stuck in the basement membrane of the loach scales. The ornamentations consisting of grooves (radii) and ridges (circuli) that can move freely and bend flexibly. The cross-sectional lateral microstructures of flat, concave and convex loach skins were observed from a live image of loach skin obtained through dark-field optical coherence tomography (OCT) imaging. The thickness of loach skin was changed with varying empty space between the mucous-cell layer and the scales by bending motion of loach. In addition, through direct measurement of drag reduction of loach skin, the mucous layer was found to have a strong influence on the reduction of skin friction. The present results enhance the understanding of the functional morphologies of mucous layer of loach to secrete mucus for skin friction reduction.

Flufiprole is an insecticide used in the rice field and may pose a potential threat to aquatic organisms including loach. To investigate the transformation products of flufiprole in loach, the accumulation, elimination, and tissue distribution in vivo as well as the metabolism in vitro at the enantiomeric level were studied. Flufiprole enantiomers rapidly accumulated and were metabolized to flufiprole sulfone, fipronil, and flufiprole amide in the tissues. Enantiomeric fractions showed the preferential accumulation and degradation of S-flufiprole. The residue of the chiral metabolite flufiprole amide was also enantioselective. The individual enantiomer treatment indicated that S-flufiprole was preferentially metabolized to flufiprole sulfone and R-flufiprole to fipronil. The metabolites were more persistent than flufiprole with longer half-lives. The metabolism in liver microsomes also reached consistent conclusions. The dietary risk assessment indicated that flufiprole would not cause unacceptable threats to human health. However, the metabolites of flufiprole should be considered in the risk evaluation.

Gram-negative Aeromonas bacteria is known to contaminate aquaculture products and retains the ability to infect wide range of host including fishes, shrimps and humans. This study is designed to evaluate the presence of Aeromonas in healthy loaches (Misgurnus anguillicaudatus) collected from the markets in Hainan and Guangdong provinces of China. Based on the molecular sequencing and phylogenetic analysis with single 16S rRNA and concatenated genes (gyrA and ropD), 104 isolates were identified as Aeromonas species followed by further classification to A. veronii (83.65%), A. jandaei (9.62%), A. hydrophila (3.85%) and A. allosaccharophila (2.88%). More than half of the isolates displayed hemolytic activity of 87.5% and were able to form moderate biofilm (78.26%). Fourteen antibiotics from ten representative classes were screened that demonstrated complete resistance to ampicillin, 89.4% and 68.3% to lincomycin and nalidixic acid. Moreover, a notable detection and prevalence was found in screening of ten virulence-related and nine resistance genes. To our knowledge, it is the first report of its kind demonstrating potential threat of the commercial loaches carrying Aeromonas to human health. These findings will assist professionals working in clinical settings to optimize their prescription accordingly and researchers to access the impacts of Aeromonas resistance on human health.ImportanceThis study demonstrates high infection rate of loaches by Aeromonas spp. in Hainan and Guangdong provinces of China.The most prevalent species was Aeromonas veronii.Most of the isolates displayed hemolytic activity and formed moderate biofilm.Multiple virulence genes, resistance genes and multi-antibiotic resistance were detected.Aeromonads risks should be considered in loach industry.

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.

Sexual and polyploidy size dimorphisms are widespread phenomena in fish, but the molecular mechanisms remain unclear. Loach (Misgurnus anguillicaudatus) displays both sexual and polyploid growth dimorphism phenomena, and are therefore ideal models to study these two phenomena. In this study, RNA-seq was used for the first time to explore the differentially expressed genes (DEGs) between both sexes of diploid and tetraploid loaches in four tissues (brain, gonad, liver, and muscle). Results showed that 21,003, 17, and 1 DEGs were identified in gonad, liver, and muscle tissues, respectively, between females and males in both diploids and tetraploids. Regarding the ploidy levels, 4956, 1496, 2187, and 1726 DEGs were identified in the brain, gonad, liver, and muscle tissues, respectively, between tetraploids and diploids of the same sex. When both sexual and polyploid size dimorphisms were considered simultaneously in the four tissues, only 424 DEGs were found in the gonads, indicating that these gonadal DEGs may play an important regulatory role in regulating sexual and polyploid size dimorphisms. Regardless of the sex or ploidy comparison, the significant DEGs involved in glycolysis/gluconeogenesis and oxidative phosphorylation pathways were upregulated in faster-growing individuals, while steroid hormone biosynthesis-related genes and fatty acid degradation and elongation-related genes were downregulated. This suggests that fast-growing loaches (tetraploids, females) have higher energy metabolism levels and lower steroid hormone synthesis and fatty acid degradation abilities than slow-growing loaches (diploids, males). Our findings provide an archive for future systematic research on fish sexual and polyploid dimorphisms.

The dojo loach Misgurnus anguillicaudatus is an important economic species in Asia because of its nutritional value and broad environmental adaptability. Despite its economic importance, genomic data from M. anguillicaudatus was unavailable. In the present study, we conducted a genome survey of M. anguillicaudatus using next-generation sequencing technology. Its genome size was estimated to be 1105.97 Mb by using K-mer analysis, and its heterozygosity ratio, repeat sequence content, GC content were 1.45%, 58.98%, and 38.03%, respectively. A total of 376,357 microsatellite motifs were identified and mononucleotides, with a frequency of 42.57%, were the most frequently repeated motifs, followed by 40.83% dinucleotide, 7.49% trinucleotide, 8.09% tetranucleotide, and 0.91% pentanucleotide motifs. The AC/GT, AAT/ATT, and ACAG/CTGT repeats were the most abundant motifs among dinucleotide, trinucleotide, and tetranucleotide motifs, respectively. Besides, a complete mitochondrial genome was sequenced. Based on the maximum likelihood and Bayesian inference analyses, M. anguillicaudatus in this study was the “introgressed” mitochondrial type. Furthermore, a total of 376,357 SSR motifs were detected from the genome survey assembly. Seventy microsatellite loci were randomly selected from these SSR loci to test polymorphic, of which, twenty microsatellite loci were assessed in 30 individuals from a wild population. The number of alleles (Na), observed heterozygosity (Ho), and expected heterozygosity (He) per locus ranged from 7 to 19, 0.400 to 0.933, and 0.752 to 0.938, respectively. All twenty loci were highly informative (PIC > 0.700). Eight loci deviated from Hardy–Weinberg equilibrium after Bonferroni correction (P M. anguillicaudatus, and genome information, mitochondrial genome, and microsatellite markers will be valuable for further studies on population genetic analysis, natural resource conservation, and molecular marker-assisted selective breeding.

Most wild types of dojo loach (Misgurnus anguillicaudatus) are gonochoristic diploids that are genetically diversified groups (A and B, further subdivided into B1 and B2), while clonal lineages inhabit certain localities in Japan. Through a series of genetic studies including DNA markers, the clonal loaches were deemed to originate from a hybridization event(s) between the A and B1 groups. However, combined analyses with other DNA markers are needed to identify each genetic group. In this study, we improved the PCR-restriction fragment length polymorphism (RFLP) analysis of the recombination activating gene 1 (RAG1) gene using digestion with two restriction enzymes, PvuII and StuI. The improved RAG1-RFLP analysis showed different fragment patterns for each group: two fragments (245 and 198 bp) for group A, three fragments (198, 147, and 98 bp) for group B1, and a single fragment (443 bp) for group B2. The clonal loaches exhibited four fragments (245, 198, 147, and 98 bp) derived from both groups A and B1. Moreover, the DNA markers were able to detect two different hybrid genotypes (A × B2 and B1 × B2). Thus, the improved RAG1-RFLP markers allowed for quick and accurate group identification of the dojo loaches.

Dojo loach (Misgurnus anguillicaudatus) that inhabit Japan are composed of two genetically divergent groups (A and B). Although most individual loach reproduce bisexually, clone lineages exist in certain populations that reproduce gynogenetically. To investigate the molecular phylogenetic relationships among the M. anguillicaudatus groups and clone lineages, complete mitogenomes of members from groups A and B and a clone lineage were sequenced using long range PCR and primer walking methods. The three groups of mitogenomes shared the same gene order and had similar base compositions and codon usage patterns. Phylogenetic analysis indicated group A and the clone lineage were genetically close with group B being genetically divergent.

Misgurnus anguillicaudatus is a highly valued, aquaculture-relevant food fish in East Asian countries. In this study, the complete mitochondrial genome of Poyang loach was obtained by PCR. The genome is 16,646 bp in length, including 2 ribosomal RNA genes. 13 protein-coding genes, 22 transfer RNA genes, and a non-coding control region, the gene composition and order of the species were similar to most reported from other vertebrates. The phylogenetic tree showed that Misgurnus family got together for one branch, which includes Poyang M. anguillicaudatus, and the other loaches had their own branches.

A monogenean flatworm Gyrodactylus jennyae Paetow, Cone, Huyse, McLaughlin & Marcogliese, 2009 was previously described as a pathogen on bullfrog Lithobates catesbeianus Shaw, 1802, in a Canadian captive population originating in Missouri, U.S.A. The ITS barcoding of G. jennyae showed relatedness to Asian Gyrodactylus macracanthus Hukuda 1940, a parasite of the Asian loach Misgurnus anguillicaudatus Cantor, 1842. The resulting suggestion that the globally invasive pet-trade of fish may be a mechanism for arrival of Gyrodactylus species to North America provided the framework for the current study. The present study was undertaken following the discovery of two other species of Gyrodactylus in a population of illegally introduced M. anguillicaudatus in New York State. Here the invasion hypothesis was tested via DNA sequencing of the ITS of the two Gyrodactylus species obtained from M. anguillicaudatus from New York, termed Gyrodactylus sp. A and Gyrodactylus sp. B. Both Gyrodactylus sp. A and Gyrodactylus sp. B were closely related to G. jennyae and G. macracanthus, and all belong to a molecularly well-supported monophyletic Asian freshwater group. In conclusion, this invasive fish has trafficked at least three parasite species to the U.S.A., one of them also found on frog. This route from the Asian wetlands to other continents is similar to that of amphibian chytrid fungi of genus Batrachochytrium Longcore, Pessier & Nichols, 1999.

Environmental DNA (eDNA) analysis has been used as a cost-efficient and non-invasive tool for monitoring invasive and threatened species. Previous studies typically involve two approaches; species-specific detection via PCR and multiple species detection via metabarcoding. However, the former could be costly when several species are targeted, and the latter could sometimes be insufficient to distinguish closely related species. Here, the simultaneous eDNA detection from multiple species via multiplex real-time PCR was applied to 99 ponds to evaluate the distribution of three exotic and three threatened native fish species over different seasons. We detected bluegill sunfish (Lepomis macrochirus) eDNA at 31 sites, largemouth bass (Micropterus salmoides) eDNA at 22 sites, smallmouth bass (Micropterus dolomieu) eDNA at one site, golden venus chub (Hemigrammocypris rasborella) eDNA at 11 sites, Japanese medaka (Oryzias latipes) eDNA at 26 sites, and weather loach (Misgurnus anguillicaudatus) eDNA at 41 sites. We found that eDNA detection rates were higher in early summer for all fish species. Moreover, exotic fish eDNA was detected more frequently in ponds which were easier to access by car and which have a larger surface area and higher pH. Furthermore, the detection rates of native fish eDNA were generally lower in the ponds where exotic fish eDNA was detected more frequently. Multiplex real-time PCR can help detect the distribution of exotic and threatened native species for conservation and ecosystem management. This method is expected to substantially contribute to the early detection of invasive species and the efficient protection of threatened species’ habitat.

Misgurnus anguillicaudatus, an important aquatic species, is mainly distributed in the Yangtze River basin. To reveal the population genetic structure of M. anguillicaudatus distributed in the Yangtze River basin, genotyping by sequencing (GBS) technique was employed to detect the genome wide genetic variations of M. anguillicaudatus. A total of 30.03 Gb raw data were yielded from 70 samples collected from 15 geographic sites located in the Yangtze River basin. Subsequently, 2092 high quality SNPs were genotyped across these samples and used for a series of genetic analysis. The results of genetic analysis showed that high levels of genetic diversity were observed and the populations from upper reaches (UR) were significantly differentiated from the middle and lower reaches (MLR) of Yangtze River basin. Meanwhile, no significant isolation by distance was detected among the populations. Ecological factors (e.g. complicated topography and climatic environment) and anthropogenic factors (e.g. aquaculture and agriculture cultivation) might account for the genetic disconnectivity between UR and MLR populations. This study provided valuable genetic data for the future breeding program and also for the conversation and scientific utilization of those abundant genetic resources stored in the Yangtze River basin.

Progesterone (P4) are aquatic contaminants that can impair fish reproduction even in low concentrations. The aim of this study was to investigate the effects of P4 on the sex differentiatio...

Identifying proteins that regulate fin injury is critical to our understanding of regeneration as it relates to both acute wound injury and tissue formation. We have cloned the full-length cDNA of the actinodin4 (and4) gene of Misgurnus anguillicaudatus (MaAnd4) by the RACE method (GenBank Accession No. MG385835). Quantitative RT-PCR analysis during fin regeneration indicated a sudden increase in MaAnd4 expression, with a peak at 3 days post amputation (dpa). In situ analysis showed that MaAnd4 is located in the distal blastema and cells lining the regions of actinotrichia formation at 3 dpa. The highest levels of MaAnd4 expression were observed in the adult testis as well as in the gastrulae during embryonic development. Southern blotting confirmed the existence of and4 in teleosts but not in tetrapods examined. The results show the expression of this gene in actinotrichia formation and its association with fin/limb regeneration ability in teleosts.

Loaches are popular in aquaculture in China due to their flesh having a high concentration of nutrients important for human health, such as amino acids, vitamins, minerals, and taurine. As such, they are used in Chinese traditional medicine to restore health and slow aging. However, the quick expansion of intensive loach culture to meet consumer demand has been accompanied by the increased prevalence of disease outbreaks, most often caused by Aeromonas spp. bacteria. We collected and identified pathogenic bacteria causing hemorrhagic septicemia in pond loach sourced from two provinces of China (Hubei and Hunan), and found three Aeromonas species: hydrophila (the most prevalent), sobria and veronii. We performed a bacterial challenge on the pond loach (Misgurnus anguillicaudatus) using the different concentrations of A. hydrophila in order to determine the median lethal dose, and found that the LD50 value was 2.14 × 107 CFU/fish. Hepcidin protein encoded by hepcidin antimicrobial peptide (hamp) gene is a key factor for iron regulation, enhancement of the host's innate immune system and acts as antimicrobial peptide. To better understand the functions of this protein in pond loach, we cloned it, conducted in silico physicochemical characterization, and studied its expression at the mRNA level both in healthy loach, and after a challenge with A. hydrophila. We found two very similar paralogs (a and b) of the hamp 1 gene, the open reading frames of which were 276 bp, both encoding an identical 91 amino acid putative peptide. The purified recombinant hepcidin protein exhibited antibacterial activity against all six studied bacterial species at 100–3200 arbitrary units/ml and can decrease 50% of the cumulation mortality of loaches which were injected by A. hydrophila. Both hamp 1 paralogs were basally expressed in most tissues, and significantly up-regulated in the liver, spleen and kidney after the bacterial challenge. The results of this study strongly indicate that hepcidin plays a role in defense mechanisms against pathogenic bacteria and inflammation in loaches.

Gonochoristic wild-type dojo loaches (Misgurnus anguillicaudatus) are diploid (2n = 50) and reproduce bisexually. However, sympatric clonal diploids generate unreduced diploid isogenic eggs that develop gynogenetically. Clone-origin triploidy arises following the incorporation of a haploid wild-type sperm nucleus into the diploid egg. Triploid females produce fertile haploid eggs by meiotic hybridogenesis, while triploid males are sterile. Clonal loaches arose from past hybridization event(s) between genetically diverse groups, A and B. Artificial hybrid females between the 2 groups produce unreduced and/or aneuploid eggs, but the hybrid males are sterile. In this study using FISH, we analyzed chromosome pairing in meiotic cells of clone-origin triploid and inter-group hybrid males to clarify the cytogenetic mechanisms underlying the male-specific sterility. We used a repetitive sequence probe to identify group B-derived chromosomes and a 5.8S + 28S rDNA probe to identify pairs of homologous chromosomes. We found that asynapsis and irregular synapsis occur in triploid and hybrid males containing 2 different genomes and that this may cause the formation of sterile germ cells. These results will help us to understand hybrid sterility from the viewpoint of synapsis behavior.

Anthropogenic modifications of the environment have clear negative impacts on species. These effects may reach a higher magnitude in highly altered habitats, for example in wetlands transformed into rice paddies. This is the case for the amphibian species of the genus Hynobius in the Republic of Korea, which originally breed in slow streams and valleys. However, a comparatively high proportion of the natural breeding sites used by the species in the lowlands has been transformed into rice paddies. Here, we assessed whether anthropogenic modification of wetlands leads to an additional threat to breeding Hynobius spp. in the form of increased vulnerability of their egg clutches to loach predators (Misgurnus species) in such modified habitats. We conducted weekly occurrence surveys at 27 randomly selected sites in the Republic of Korea and recorded the following information: type of site (natural versus agricultural), air temperature, water conductivity, moon phase and predation by Misgurnus sp. Our results reveal, for the first time, cases of predation of Hynobius spp. eggs by Misgurnus loaches. We also show that the risk of predation was higher in agricultural sites in comparison to natural sites. In conclusion, we demonstrate the increased predation risk of Hynobius spp. eggs by Misgurnus sp. at anthropogenically disturbed sites, and thus a new type of threat to Hynobius populations. This new type of threat may, however, be due to expansion of the breeding habitats following human disruptions to landscapes. We therefore call for the development of mitigating measures to wetland modifications.

The dojo loach Misgurnus anguillicaudatus is an endemic freshwater species to Asia. The effective conservation and molecular-aided selection of M. anguillicaudatus have been limited without sufficient molecular markers. In this study, 112 novel single nucleotide polymorphisms (SNPs) were screened based on 2b-RAD sequencing database, and 57 SNP markers were developed and characterized by genotyping 40 individuals using SNaPshot method. The observed heterozygosity (Ho) ranged from 0.025 to 0.675, while the expected heterozygosity (He) varied from 0.025 to 0.500. The minor allele frequency (MAF) ranged from 0.013 to 0.500. Among these SNPs, 18 loci were found to deviate significantly from the Hardy–Weinberg equilibrium after Bonferroni correction (P M. anguillicaudatus will provide valuable information in further population genetic analysis and natural resource conservation.

C/EBP [CCAAT/enhancer-binding protein]-homologous protein gene (chop) which plays an important role in endoplasmic reticulum stress-induced apoptosis, was investigated here by RACE and qPCR in an aquaculture animal for the first time. The full-length cDNA sequences of loach (Misgurnus anguillicaudatus) chop were 2533bp, encoding 266 amino acids. The expression levels of loach chop changed obviously during different early life stages, and the highest appeared at the 8-cell stage. Among different tissues, loach chop predominantly expressed in gill, spleen and gonad. Then, we performed a hydrogen peroxide (H2O2, a common-used disinfectant) stress trial to explore the role of loach chop, with three different concentrations (0 μM, 50 μM and 100 μM) of H2O2. Half of the loaches from the 100 μM group were dead, while all loaches from the other two treatment groups were alive. The activities of CAT, SOD and GPX in loach gill, liver and spleen decreased with extended stress time and increased H2O2 concentration. The expression levels of gill chop in loaches from the 100 μM group were significantly higher than those from the other two treatment groups between 12 and 24 hours of exposure. atf4 and bax, two proapoptotic genes, were significantly up-regulated in gills of loaches from the 100 μM group compared to the other two groups 18 hours and 24 hours after treatment, while bcl2, an antiapoptotic gene, presented an opposite trend. These results indicated a close relationship between H2O2 stress and fish apoptosis, and loach chop played an important role in H2O2 stress response.

The hybrid loach of Misgurnus anguillicaudatus (female) and Paramisgurnus dabryanus ssp. (male) has the desirable trait of growth performance. Recently, farming scale of this hybrid has been gradually increased in Asia, suggesting a promise of a new variety for loach. In this study, the complete mitochondrial genome of the hybrid loach was obtained by PCR. The genome is 16,646 bp in length, including two ribosomal RNA genes. Thirteen proteins-coding genes, 22 transfer RNA genes, and a non-coding control region, the gene composition and order of were similar to most reported from other vertebrates. Sequence analysis showed that the overall base composition is 29.8% for A, 28.3% for T, 25.6% for C, and 16.3% for G. The phylogenetic tree showed that Misgurnus family got together for one branch, which includes this hybrid loach, and the other loaches had their own branches. Also the mitochondrial genome sequences of the hybrid loach were aligned by BLAST, compared with Cobitidae the sequence similarity could reach >95%, and the similarity to Misgurnus was >99%. The hybrid loach follows the matrilineal inheritance.

Background Loaches of Cobitinae, widely distributed in Eurasian continent, have high economic, ornamental and scientific value. However, the phylogeny of Cobitinae fishes within genera or family level remains complex and controversial. Up to now, about 60 Cobitinae mitogenomes had been deposited in GenBank, but their integrated characteristics were not elaborated. Results In this study, we sequenced and analyzed the complete mitogenomes of a female Cobits macrostigma. Then we conducted a comparative mitogenome analysis and revealed the conserved and unique characteristics of 58 Cobitinae mitogenomes, including C. macrostigma. Cobitinae mitogenomes display highly conserved tRNA secondary structure, overlaps and non-coding intergenic spacers. In addition, distinct base compositions were observed among different genus and significantly negative linear correlation between AT% and AT-skew were found among Cobitinae, genus Cobitis and Pangio mitogenomes, respectively. A specific 3 bp insertion (GCA) in the atp8-atp6 overlap was identified as a unique feature of loaches, compared to other Cypriniformes fish. Additionally, all protein coding genes underwent a strong purifying selection. Phylogenetic analysis strongly supported the paraphyly of Cobitis and polyphyly of Misgurnus. The strict molecular clock predicted that Cobitinae might have split into northern and southern lineages in the late Eocene (42.11 Ma), furthermore, mtDNA introgression might occur (14.40 Ma) between ancestral species of Cobitis and ancestral species of Misgurnus. Conclusions The current study represents the first comparative mitogenomic and phylogenetic analyses within Cobitinae and provides new insights into the mitogenome features and evolution of fishes belonging to the cobitinae family.

Tapeworms of the genus Paracaryophyllaeus Kulakovskaya, 1961 (Cestoda: Caryophyllidea) are specific parasites of loaches (Cypriniformes: Cobitoidea) and occur almost exclusively in the Palaearctic region. The only exception and example of vicariance over the borders of two zoogeographical regions is Paracaryophyllaeus lepidocephali (Kundu, 1985), an insufficiently known species described from the Indomalayan region, with uncertain generic allocation. In the present paper, the species is redescribed based on new material collected from the type host, Lepidocephalichthys guntea, in West Bengal, India. Molecular data reveal this species as a member of Paracaryophyllaeus, within which it is the most closely related to P. cf. gotoi from Misgurnus anguillicaudatus in China and Japan on the basis of large subunit of ribosomal nuclear DNA (28S rDNA). Generic position of P. lepidocephali examined herein is also confirmed by morphology including cross sections, in particular, by a small, cylindrical body, medullary testes with testicular fields crossing the anterior margin of the cirrus sac, medullary vitelline follicles, with some follicles paramuscular, a shallow common genital atrium, short vagina and uterus not extending anteriorly beyond the cirrus sac. It differs from all Palaearctic congeners but Paracaryophyllaeus vladkae Scholz, Oros and Aydogdu, 2014 by the testicular field crossing the anterior margin of the cirrus sac. It differs from P. vladkae by more anterior position of the first vitelline follicles compared to the first testes. This species is a unique example of a fish tapeworm crossing the borders of the Palaearctic and Indomalayan zoogeographical regions.

Maternal genes are important in directing early development and determining egg quality in fish. We here report the de novo transcriptome from four tissue libraries of the cyprinid loach, Misgurnus anguillicaudatus, and for the first time identified maternal gene transcripts in unfertilized eggs and suggest their immune system involvement. Expression profiles and functional enrichment revealed a total 24,116 transcripts were expressed as maternal transcripts in unfertilized eggs, which were involved in a wide range of biological functions and pathways. Comparison expression profiles and analysis of tissue specificity revealed that the large numbers of maternal transcripts were stored in unfertilized eggs near the late phase of ovarian maturation and before ovulation. Functional classification showed a total of 279 maternal immune-related transcripts classified with immune system process GO term and immune system KEGG pathway. qPCR analysis showed that transcript levels of identified maternal immune-related candidate genes were dynamically modulated during development and early ontogeny of M. anguillicaudatus. Taken together, this study could not only provide knowledge on the protective roles of maternal immune-related genes during early life stage of M. anguillicaudatus but could also be a valuable transcriptomic/genomic resource for further analysis of maternally provisioned genes in M. anguillicaudatus and other related teleost fishes.

The European weather loach (Misgurnus fossilis) is a cryptic and poorly known fish species of high conservation concern. The species is experiencing dramatic population collapses across its native range to the point of regional extinction. Although environmental DNA (eDNA)-based approaches offer clear advantages over conventional field methods for monitoring rare and endangered species, accurate detection and quantification remain difficult and quality assessment is often poorly incorporated. In this study, we developed and validated a novel digital droplet PCR (ddPCR) eDNA-based method for reliable detection and quantification, which allows accurate monitoring of M. fossilis across a number of habitat types. A dilution experiment under laboratory conditions allowed the definition of the limit of detection (LOD) and the limit of quantification (LOQ), which were set at concentrations of 0.07 and 0.14 copies μl-1 , respectively. A series of aquarium experiments revealed a significant and positive relationship between the number of individuals and the eDNA concentration measured. During a 3 year survey (2017-2019), we assessed 96 locations for the presence of M. fossilis in Flanders (Belgium). eDNA analyses on these samples highlighted 45% positive detections of the species. On the basis of the eDNA concentration per litre of water, only 12 sites appeared to harbour relatively dense populations. The other 31 sites gave a relatively weak positive signal that was typically situated below the LOQ. Combining sample-specific estimates of effective DNA quantity (Qe ) and conventional field sampling, we concluded that each of these weak positive sites still likely harboured the species and therefore they do not represent false positives. Further, only seven of the classified negative samples warrant additional sampling as our analyses identified a substantial risk of false-negative detections (i.e., type II errors) at these locations. Finally, we illustrated that ddPCR outcompetes conventional qPCR analyses, especially when target DNA concentrations are critically low, which could be attributed to a reduced sensitivity of ddPCR to inhibition effects, higher sample concentrations being accommodated and higher sensitivity obtained. ispartof: JOURNAL OF FISH BIOLOGY vol:98 issue:2 pages:399-414 ispartof: location:England status: published

Whey acidic protein domain (WAPD) occurs in a variety of proteins in animals and many of WAPD-containing proteins are involved in immunity. In the present study, a novel protein containing three WAPDs was identified from the weather loach, Misgurnus anguillicaudatus, designated as MaTWD. MaTWD share high identity with TWDs from fish but low identity with TWDs from other animal phyla. MaTWD transcripts mainly distributed in gills and head kidney responded to bacterial challenge with significant upregulation. In vitro assay with recombinant MaTWD protein revealed that MaTWD had antiprotease activity against bacterial proteases. Moreover, MaTWD exhibited bacterial binding capacity and antimicrobial activity. Most importantly, exogenous MaTWD protected loach against bacterial infection by reducing loach mortality. We infer that MaTWD participates in the antibacterial immunity of loach via its antiprotease and antimicrobial activities.

Bacteriophages are increasingly being used as biological control agents against pathogenic bacteria. In the present study, we isolate and characterize bacteriophage Akh-2 from Geoje Island, South Korea, to evaluate its utility in controlling motile Aeromonas septicemia. Akh-2 lysed four of the seven Aeromonas hydrophila strains tested. Transmission electron microscopy analysis showed that Akh-2 belongs to the Siphoviridae family, with head and tail sizes of 50 &plusmn, 5 and 170 &plusmn, 5 nm, respectively. One-step growth curve analysis revealed that the phage has a latent period of 50 &plusmn, 5 min and a burst size of 139 &plusmn, 5 plaque-forming units per infected cell. The phage appeared stable in a pH range of 6&ndash, 8 and a temperature range of &minus, 80 to 46 &deg, C. Based on next-generation sequencing analysis, its genome is 114,901 bp in size, with a 44.22% G + C content and 254 open reading frames. During an artificial induction of the disease, loach (Misgurnus anguillicaudatus) treated with Akh-2 showed an increased survival rate and time compared with the non-treated control. Our results suggest that Akh-2 is a potential biological agent for the treatment of Aeromonas infections in fish.

Aeromonas veronii is a major pathogenic bacterium in humans and animals. When it causes outbreaks, there are enormous economic losses to the aquaculture industry. An effective live attenuated vaccine strain, ΔhisJ, was obtained in our previous studies by gene knockout in Aeromonas veronii TH0426 using the suicide vector pRE112. Here, we evaluated whether the live attenuated vaccine ΔhisJ was suitable for prevention of Aeromonas veronii infection by injection and immersion in loaches. Compared with that of the TH0426 wild-type strain, the virulence of the live vaccine was significantly weakened. Vaccine safety assessment results also indicated that 1 × 107 CFU/mL live vaccine was safe and did not induce clinical symptoms or obvious pathological changes. Additionally, after challenging loaches with Aeromonas veronii TH0426, the relative percent survival of the IN3 injection group was 65.66%, and that of the IM group was 50.78%. Our data show that the live attenuated vaccine ΔhisJ can improve the immune protection rate of loaches. Furthermore, increased enzyme activity parameters (SOD, LZM, ACP, and AKP) in the skin mucus, increased enzyme activity parameters (SOD, LZM, ACP, AKP, and GPx) in the serum, increased specific IgM antibodies and cytokine IL-1β contents in the serum, and increased cytokine (IL-15, pIgR, IL-1β, and TNF-α) expression in the liver and spleen were observed. These data are the first to indicate that the live attenuated vaccine ΔhisJ is suitable for the development of a safe and effective vaccine against Aeromonas veronii infection in loach aquaculture.

Faithful analysis of transcripts represents a critical component of the gene discovery, genomic characterization and annotation process in species without a reference genome. The present study generated 28,001 full-length transcripts of Misgurnus anguillicaudatus using the Pacific Bioscience (PacBio) single-molecule real-time (SMRT) sequencing system. A total of 77,346 consensus isoforms were identified, and 18,991 complete open reading frames (ORFs) detected. In total, 1345 lncRNAs were identified with high-confidence, with additional identification of a number of well-known transcription factors. The present study may facilitate additional exploration of the genomic signatures of M. anguillicaudatus.

Dojo loach (Misgurnus anguillicaudatus) (DD, 2n = 50) and large-scale loach (Paramisgurnus dabryanus) (PP, 2n = 48) are freshwater fish species widely distributed across East Asia. Hybrids of these two loach species have disrupted gonadal development, but few studies have reported the actual fertility and chromosome number in gametes of reciprocal hybrid loaches (DD × PP and PP × DD, assigned as DP and PD, 2n = 49). In this study, selfing and backcrossing experiments between M. anguilicaudatus and P. dabryanus were conducted. The fertilization, hatching, deformity, and survival rates were examined along with the embryonic development and growth performance to analyze the actual fertility of the hybrid loach. The karyotypes of viable backcross offspring were studied to indirectly assess the chromosome number in eggs of DP and PD hybrids. The failure in fertilization among crosses using male hybrid proved that the male hybrid was sterile. The moderate fertilization rates (11.49%‐–49.16%), high hatching rates (82.78%–86.31%), high survival rates (78.67%–81.33%), and the normal embryonic development among crosses using female hybrid revealed that the female hybrid was fertile, albeit with relatively high deformity rates (12.68%–27.45%). The karyotype analysis of viable backcross offspring showed 49 chromosomes for DP × DD (assigned as DPD) and PD × DD (assigned as PDD) hybrids, and 48 chromosomes for DP × PP (assigned as DPP) and PD × PP (assigned as PDP) hybrids. This indicated that fertile gametes produced by female hybrid loach contained 24 chromosomes. This study provides valuable information for loach hybrid breeding and in-depth research on the precise mechanism of meiosis in these hybrid loach.

SummaryPolyspermy was initiated by microinjecting a multiple number of sperm into the activated and dechorionated eggs of dojo loach Misgurnus anguillicaudatus (Teleostei: Cobitidae). A 10-nl sperm suspension from an albino (recessive trait) male (105, 106, 107 or 108 sperm ml −1) was microinjected into eggs from a wild-type female. Although the rates of embryos developing into the blastula stage in the injection group at the highest sperm concentration were similar to that of the control group, the hatching rates of the injection group were much lower. A large proportion of embryos that developed from the injected eggs was haploid and were mosaics containing haploid cells. Most of the haploid and mosaic embryos inherited only paternally derived alleles in the microsatellite markers (i.e. androgenesis was initiated by injecting multiple sperm). In contrast, some haploid embryos contained both paternal and maternal alleles despite haploidy, suggesting that they were mosaics consisting of cells with either paternal or maternal inheritance. The injected eggs displayed diploid, hypotriploid and triploid cells, all of which included both maternally and paternally derived alleles. One albino tetraploid with only paternal alleles was also observed from the injected eggs. These results suggested that part of the sperm microinjected into the ooplasm should form a male pronucleus/pronuclei, which could develop by androgenesis or could fuse with the female pronucleus/pronuclei. Therefore, microinjection of multiple sperm should be considered a potential technique to induce androgenesis and polyploidy.