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Collections of vertebrates and macroinvertebrates over two years from 26 lentic temporary water bodies each surveyed 1–4 times, in Ulster and Dutchess counties in the Hudson Valley of New York, USA, resulted in identifications of 166 species. These included species resident in temporary waters that have life stages resistant to drying and freezing, species that reproduce in temporary waters that need to mature before drying occurs, predatory species that visit temporary waters, and highly aquatic species that move overland to temporary waters. Temporary waters sampled had a range of hydroperiods from a few weeks to almost a year. Of the macroinvertebrates we identified to species, 75 were each collected in a single pool-survey (n = 62 pool-surveys). Pools with greater volume, more vegetation cover, lower pH and turbidity, and more sodium yielded greater species richness of macroinvertebrates, and species richness of certain taxa (eg Mollusca) were correlated with additional elements. We briefly discuss several unusual animal occurrences. [ABSTRACT FROM AUTHOR]

A new species of pond loaches is described from the systems of Chibisanskiye–Vavaiskiye lakes in the south of Sakhalin Island. This species is distinguished from all other Misgurnus species by having a moderately deep body (maximum body depth 7.3–8.6 times in standard length, SL); poorly developed adipose crests; elongated caudal peduncle with its length usually exceeding 19% SL; caudal peduncle depth 1.9–2.4 times in length; dorsal fin begins much closer to the tip of the snout than to the end of the caudal fin; short pelvic fins with their length 1.2–1.7 times in length between the pelvic and anal fins in males and 1.7 times in females; length from pelvic fin base to anal fin base 1.9–2.4 times in the length from anal fin base to caudal fin base; more than 140 transverse rows of scales in the side series; lamina circularis rounded and flat; no distinct black spot at the upper base of the caudal fin. Analysis of several genetic markers (mitochondrial cytb and fragments of nuclear genes Rag1 and IRBP) proved that the new species is an independent phylogenetic lineage within Misgurnus, the same rank as the M. nikolskyi lineage or the group including M. mohoity and M. anguillicaudatus. [ABSTRACT FROM AUTHOR]

The current research work was undertaken to compare and analyze the body index, nutrient composition, and digestive enzyme activity of Pond loach (Misgurnus anguillicaudatus), large-scale loach (Paramisgurnus dabryanus), and Taiwan loach (Paramisgurnus dabryanus ssp). Viscerosomatic ratio (VR), condition factor, (CF), W/L and H/L were highest in Taiwan loach (P < 0.05). Muscle protein content was highest, whereas lipid content was lowest in pond loach (P < 0.05). The content of total amino acids (TAA), total essential amino acids (EAA), and delicious amino acids (DAA) in the muscle of pond loach was highest (P < 0.05). The content of polyunsaturated fatty acids (PUFA), monounsaturated fatty acids (MUFA) and linoleic acid was highest in pond loach, Taiwan loach and large-scale loach, respectively (P < 0.05). The trypsin activities and amylase activities of the pond loach were significantly higher than those of the large-scale loach and Taiwan loach in the intestine and liver (P < 0.05). These results indicate that the three kinds of loaches are of high nutritional value and have breeding prospects, among which pond loach has higher nutritional value. [ABSTRACT FROM AUTHOR]

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 taxonomic revision of the Chinese loach fishes of the genera of Misgurnus and Paramisgurnus was made in this paper based on the morphology features and sequences of mitochondrial Cyt b and CO1 gene. The results indicated that the species of Misgurnus and Paramisgurnus can be distinguished from the size of the scale, the length of the beard, and the degree of healing of the end of the basioccipital. The genetic distance between Misgurnus and Paramisgurnus were 15.2%--17.5%, which was very close to those among species for Misgurnus (10.7%--18.4%). Molecular phylogenetic analysis showed that the genus Misgurnus was not monophyletic, and Paramisgurnus was nested within Misgurnus. Misgurnus bipartitus and M. anguillicaudatus clustered together, and then clustered with Paramisgurnus dabryanus, M. mohoity was located at the basal of the phylogenetic tree. Based on the results of the morphology features and molecular data, Paramisgurnus should be recognized as a junior synonym of Misgurnus; M. bipartitus is a valid species distinct from M. mohoity. [ABSTRACT FROM AUTHOR]

Tetraploid fish are important for mass production of triploids in polyploid breeding. In this study, we reported a novel protocol for artificial induction of tetraploidy by a combination of cold shock and hydrostatic pressure administered to gynogenetically developed eggs in Japanese flounder Paralichthys olivaceus. The induction was carried out by activating the eggs with UV-irradiated sperm of red sea bream Pagrus major, administering a cold shock (0°C, 5 to 45 min) 3 min after fertilization to inhibit second polar body exclusion, incubating the eggs for 60 min at 17°C, and treating them with a 650 kg/cm2 hydrostatics pressure shock for 6 min. We named the embryos gyno-tetraploids that developed from eggs after such treatments. The hatching rate of the gyno-tetraploids ranged from 20.99%±3.66% to 36.01%±2.79%, and the tetraploid rate ranged from 80.00% to 100.00%. All-maternal inheritance was verified using 6 high-recombination-rate microsatellite markers. This method successfully induced gyno-tetraploidy. The successful induction of gyno-tetraploidy lays the foundation for triploidization of new varieties with improved economic traits of interest that can benefit commercial culture. [ABSTRACT FROM AUTHOR]

Abstract Teleost fish are the most primitive bony vertebrates that contain immunoglobulin (Ig). Although teleost Ig is known to be important during tetrapod evolution and comparative immunology, little is known about the genomic organization of the immunoglobulin heavy-chain (IgH) locus. Here, three Ig isotype classes, IgM, IgD and IgT, were firstly identified in dojo loach (Misgurnus anguillicaudatus), and the IgH locus covering τ, μ and δ genes was also illustrated. Variable (V) gene segments lie upstream of two tandem diversity (D), joining (J) and constant (C) clusters and the genomic organization of the IgH locus presented as V n -D n -J n -C τ -D n -J n -C μ -C δ , similar to some other teleost fish. However, unlike some other teleost fish, ten V H , ten D and nine J genes were observed in this locus, which suggest teleost Igs might be conserved and diverse. Thus, it would be interesting to determine how Igs divide among themselves in immune response to different antigens. To address this hypothesis, we have developed three models by bath infection with parasitic, bacterial and fungal pathogens, respectively. We found that IgM, IgD and IgT were highly upregulated in the head kidney and spleen after infection with Ichthyophthirius multifiliis (Ich), suggesting that the three Igs might participate in the systemic immune responses to Ich. Moreover, the high expression of IgT in mucosal tissue, such as skin or gills, appeared after being infected with three different pathogens infection, respectively, in which the expression of IgT increased more rapidly in response to Ich infection. Interestingly, the expression of IgD showed a higher increase in spleen and head kidney being challenged with fungi, suggesting that IgD might play an important role in antifungal infection. Highlights • The IgH locus in the loach was illustrated for the first time. • Three infection models with bacteria, parasite and fungus were established, respectively. • The expressions of teleost Igs were detected in responses to fungal infection. [ABSTRACT FROM AUTHOR]

Abstract In mammalian, T-cell receptors (TCRs) play a key role in recognizing the presented antigen from external to protect organisms against environmental pathogens. To understand the potential roles of TCRγ and TCRδ in dojo loach (Misgurnus anguillicaudatus), Ma- TCRγ and Ma- TCRδ cDNAs were cloned and their gene expression profiles were investigated after bacterial, parasitic and fungal challenge. The open reading frame (ORF) of Ma- TCRγ and Ma- TCRδ cDNAs contained 948 and 867 bp, encoding 316 and 288 amino acid residues, respectively. Structurally, Ma- TCRγ and Ma- TCRδ were consisted of a signal peptide, a variable region, a constant region (IgC), a connecting peptide (CPS), a transmembrane region (TM) and a cytoplasmic domain (CYT), which were similar to those of other vertebrates. Multiple sequence alignment and phylogenetic analysis showed Ma- TCRγ and Ma- TCRδ were closely related to fish of Cyprinidae family. Ma- TCRγ and Ma- TCRδ were widely expressed in all tested organs/tissues, as the highest expressions of Ma- TCRγ and Ma- TCRδ were detected in kidney and gill, respectively. In addition, three infection models of dojo loach with bacteria (F. columnare G 4), parasite (Ichthyophthirius multifiliis) and fungus (Saprolegnia sp.) were constructed. The morphological changes of gills and skin after challenged with F. columnare G 4 and Ichthyophthirius multifiliis were investigated. Compared to F. columnare G 4 infection, mRNA expression of both TCRγ and TCRδ showed higher sensitivity in classical immune organs (kidney and spleen) and mucosal tissues (skin and gill) after challenge with Ichthyophthirius multifiliis and Saprolegnia sp. Our results first indicated that TCRγ and TCRδ of dojo loach might function differently in response to challenge with different pathogens. Highlights • Full-length cDNA sequences of TCRγ and δ genes were firstly cloned and identified from dojo loach (Misgurnus anguillicaudatus). • Three infection models of dojo loach with Flavobacterium cloumnare G 4 , Ichthyophthirius multifiliis and Saprolegnia sp. were constructed. • The Ma- TCRγ and Ma- TCRδ mRNA were highly expressed in skin and gill, and up-regulated in responses to challenge with bacteria, parasite and fungus. [ABSTRACT FROM AUTHOR]

Abstract In vertebrates, the T cell receptor (TCR) plays a crucial role in immune system. To date, the roles of fish TCRs in response to pathogen infection are still poorly understood. In the present study, we firstly cloned and identified the TCRα and TCRβ from dojo loach (Misgurnus anguillicaudatus) by RACE approaches. The full-length cDNAs of Ma- TCRα and Ma- TCRβ include an open reading frame (ORF) of 723 and 879 bp encoding a polypeptide of 241 and 293 amino acids, respectively. Structural analysis indicated that Ma- TCRα and Ma- TCRβ had a signal peptide, IgV domain, IgC domain, a connecting peptide (CPS), a transmembrane region (TM) and a cytoplasmic (CYT), which are similar to their counterparts described in other teleost. Phylogenetic analysis supported that Ma- TCR Cα and Ma- TCR Cβ were closely related to the Cα and Cβ region of Cyprinidae family, respectively. Transcriptional expression analysis indicated that Ma- TCRα and Ma- TCRβ mRNAs were ubiquitously expressed in a wide array of tissues and most abundantly found in skin, brain, kidney, gill and spleen. The expression patterns of Ma- TCRα and Ma- TCRβ after bacteria (F. columnare G 4), parasite (Ichthyophthirius multifiliis) and fungus (Saprolegnia) infection were detected by qRT-PCR. Additionally, the morphological changes of gill and skin following the three infection models were investigated. The results clearly indicated that Ma- TCRα and Ma- TCRβ was significant up-regulated not only in spleen and kidney, but also in skin and gill. In summary, our present findings suggested that Ma- TCRα and Ma- TCRβ might play significantly roles in the modulation of immune response and protect loach from different pathogens infection. Highlights • The full-length cDNA of TCRα and TCRβ was firstly cloned and identified from the loach (Misgurnus anguillicaudatus). • Three infection models of the loach with bacteria, parasite and fungus were constructed for the first time. • The Ma- TCR Cα and Ma- TCR Cβ mRNA were highly expressed in mucosal tissues including skin and gill. • Ma- TCR Cα plays a more important role than Ma- TCR Cβ in recognizing bacterial and parasitic infections. [ABSTRACT FROM AUTHOR]

Abstract The effects of different concentrations of copper sulfate on diploid and triploid fin cell lines (named DIMF and TRMF, respectively) in Misgurnus anguillicaudatus were studied. The LC 50 of copper sulfate estimated by an MTT assay was 268.39 in DIMF cells, and 311.54 μmol/L in TRMF cells, respectively. Activity of superoxide dismutase (SOD) in DIMF cells gradually increased as the concentration of copper sulfate increased (up to 200 μmol/L), and then gradually decreased. SOD activity in triploid loach fin cells, as well as glutathione peroxidase (GSH-Px) and glutathione-S-transferase (GST) activity in both diploid and triploid cells, decreased as the concentration of copper sulfate increased, which suggested that excessive copper exposure at the concentrations tested in this study was detrimental to anti-oxidative capability. In general, SOD, GST and GSH-Px activity was higher in triploid fin cells than in diploid cells. DNA breaks were observed by comet assays after 24 h exposure to 400 and 800 μM copper; DNA percent in the comet's tail was lower in TRMF than in DIMF. Ultrastructurally, there were no significant differences in the organelles of both cells, although a higher number of vesicles were observed in TRMF cells after copper exposure. Pathological changes induced by copper sulfate were similar in DIMF and TRMF cells, and were indicative of cell necrosis. Results above suggested that excessive copper sulfate exposure would lead to antioxidant enzymes activity reduction, along with antioxidant defenses disruption and superoxide radicals increasing, and then to DNA damage, ultrastructural changes and necrosis features in DIMF and TRMF M. anguillicaudatus fin cells. Triploid cell lines had higher resistance to copper than their diploid counterparts especially at higher concentrations of copper due to larger cells and higher intracellular content of detoxification enzymes to resist the toxicity of heavy metals. Graphical abstract Fin cell lines of diploid (DIMF) and triploid (TRMF) Misgurnus anguillicaudatus were used to detect the toxic effect of copper sulfate. Results showed that excessive copper sulfate exposure would lead to antioxidant enzymes activity reduction along with antioxidant defenses disruption and superoxide radicals increasing, and then to DNA damage, ultrastructural changes and necrosis features in DIMF and TRMF M. anguillicaudatus fin cells. Triploid cell lines had higher resistance to copper than their diploid counterparts especially at higher concentrations of copper due to larger cells and higher intracellular content of detoxification enzymes to resist the toxicity of heavy metals. Unlabelled Image Highlights • Copper sulfate reduced SOD, GST, and GSH-Px activity in loach cells. • Copper sulfate induced cellular damage, ultrastructural changes, and necrosis due to oxidative stress. • Triploid loach cell lines had higher resistance to copper than diploid cells. [ABSTRACT FROM AUTHOR]

Highlights • The full-length IL15 and IL15Rα cDNA sequences of dojo loach (Misgurnus anguillicaudatus) were identified. • Three infection models with Flavobacterium cloumnare G 4 , Ichthyophthirius multifiliis and Saprolegnia parasitica were constructed. • The expression levels of Ma -IL15 and Ma -IL15Rα in systematic and mucosal tissues were significantly elevated within three infection models. • The expression levels of Ma -IL15 and Ma -IL15Rα showed consistent up-regulation after fungal infection. Abstract Interleukin 15 (IL15) is a pleiotropic cytokine that participates in innate and adaptive immunity along with its receptor α-chain (IL15Rα). In order to investigate the potential roles of IL15 and IL15Rα in dojo loach (Misgurnus anguillicaudatus), we firstly cloned the cDNA sequence of Ma- IL15 and Ma- IL15Rα, which contain 1096bp and 1236bp and code proteins of 193 amino acids and 210 amino acids, respectively. A short signal peptide and Pfam IL15 domain were found in Ma -IL15, while a highly conserved sushi domain existed in Ma -IL15Rα. Ontogeny analysis indicated that significantly increased expression of Ma -IL15 and Ma - IL15Rα mRNA were detected in larvae from 1d to 7d post hatching, while relative high expression levels were detected in both systematic and mucosal immune-related tissues of adult dojo loach. Then three dojo loach infection models with F. columnare G 4 , I. multifiliis and Saprolegnia parasitica were constructed, which resulted in increased skin goblet cells and serious lesions in gill. Ma -IL15 and Ma -IL15Rα showed different expression patterns in different tissues during three infection models. Ma -IL15Rα mRNA was found to be more significantly elevated than Ma -IL15 after infection with F. columnare G 4 in all examined tissues including kidney, spleen, gill and skin. I. multifiliis infection induced higher expression of Ma -IL15 in mucosal tissues including skin and gill, while it mainly increased Ma -IL15Rα expression in kidney. Moreover, our study firstly evaluated the influence of fungal infection on IL15 and IL15Rα expression in teleost, and it is interesting to find that both Ma -IL15 and Ma -IL15Rα expression showed consistent up-regulation after Saprolegnia parasitica infection compared to two other infection models. Therefore, our results suggest that Ma -IL15 and Ma -IL15Rα possess important defensive roles in systematic and mucosal tissues of dojo loach during bacterial, fungal and parasitic infection. [ABSTRACT FROM AUTHOR]

Misgurnus anguillicaudatus is a highly valued, aquaculture-relevant food fish in East Asian countries. Because of overfishing and environmental pollution, the number of wild Nansi loach has been sharply decreased in these years. In this study, the complete mitochondrial genome of Nansi loach was obtained by PCR. The genome is 16,646 bp in length, including 2 ribosomal RNA genes. 13 proteins-coding genes, 22 transfer RNA genes, and a non-coding control region, the gene composition and order of which was similar to most reported from other vertebrates. Sequence analysis showed that the overall base composition is 29.8% for A, 28.2% for T, 25.6% for C, and 16.4% for G. The phylogenetic tree showed that Misgurnus family got together for one branch, which includes Nansi M. anguillicaudatus, and the other loaches had their own branches. Also, the mitochondrial genome sequence of M. anguillicaudatus was aligned by BLAST, compared with Cobitinae the sequence similarity could reach >95%, and the similarity to Misgurnus was >99%.

The hybrid loach of M. anguillicaudatus (female) and M. bipartitus (male) have 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 16,647 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 which was similar to most reported from other vertebrates. Sequence analysis showed that the overall base composition is 29.75% for A, 28.28% for T, 25.61% for C, and 16.36% 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 sequence of the hybrid loach was aligned by BLAST, compared with Cobitinae the sequence similarity could reach >95%, and the similarity to Misgurnus was >99%. The hybrid loach follows the matrilineal inheritance.

Sox4, a member of the SoxC subfamily which of the Sox family, plays important roles in the development of the vertebrate gonad and nervous system. We have cloned a Sox4 homologue from the brain of Misgurnus anguillicaudatus using homologous cloning and rapid amplification of cDNA ends. We named the cloned gene as MaSox4. The full-length cDNA was 2122 bp, containing a 718 bp 5′-untranslated region and a 267 bp 3′-untranslated region. The open-reading frame of the cloned gene encoded 378 amino acids and contained a characteristic HMG-box DNA-binding domain with the specific motif (RPMNAFMVW). Phylogenetic analysis indicated that MaSox4 is highly homologous to Sox4 in different species. Protein sequence analysis showed that MaSox4 is a nonsecretory hydrophilic protein. Quantitative real-time reverse transcription polymerase chain reaction and in situ hybridization assay revealed that MaSox4 was ubiquitously expressed during embryogenesis and is present in various adult tissues, especially in the central nervous system. Our study suggests that MaSox4 is highly conserved among vertebrates’ evolution and might be involved in developmental processes such as embryogenesis, neurogenesis and gonad development. [ABSTRACT FROM AUTHOR]

Elongase of very long-chain fatty acids 6 (ELOVL6) is a rate-limiting enzyme catalyzing elongation of saturated and monounsaturated long-chain fatty acid. Although functional characteristics of Elovl6 have been demonstrated in mammal, the role of elovl6 in fish remains unclear. In this study, we firstly cloned three isoforms of elovl6 ( elovl6a , elovl6b and elovl6 - like ) from loach ( Misgurnus anguillicaudatus ). Molecular characterizations of the three elovl6 isoforms in loach and their expressions of early life stages and different tissues were then determined. We also functionally characterized the three elovl6 isoforms using heterologous expression in baker's yeast. Results obtained here showed the three elovl6 proteins in loach can elongate C16:0 and C16:1 to C18:0 and C18:1, respectively. At last, to confirm the role of three loach elovl6 isoforms for elongation of fatty acids in adaption to cold stress, differences in skin histological structures, body fatty acid compositions, expressions of four hepatic lipogenesis or lipolysis related genes, and expressions of the three elovl6 isoforms and their related gene uncoupling protein 1 ( ucp1 ) in different tissues were investigated in the loach reared in two different water temperatures (28 °C and 4 °C) for ten days. Cold stress increased ratios of C18/C16 and C20:5n−3/C18:3n−3 in loach body, and induced expressions of hepatic acyl-CoA delta-9 desaturase 1 ( scd1 ), sterol-regulator element-binding protein 1 ( srebp1 ), carnitine palmitoyltransferase 1 ( cpt1 ) and fatty acid synthase ( fas ). Meanwhile, significant differences were found in expressions of the three elovl6 isoforms in different tissues between 28 °C and 4 °C groups. Overall, this study suggests that the three elovl6 isoforms in loach have ability to elongate C16 to C18, and elovl6 proteins in loach may play a role in adaptation to cold stress. [ABSTRACT FROM AUTHOR]

Summary: Natural tetraploid loach (Misgurnus anguillicaudatus) living in the Changjiang River basin and adjacent area in China has been considered a true genetic tetraploidy (4n = 100) with four sets of homologous chromosomes. Here, we reported its further supporting evidence provided by the cold‐shock androgenesis. Viable androgenetic progeny appeared when eggs were cold‐shocked at 3°C for 60 min, starting 5 min after fertilization with sperm of natural tetraploid males, although the survival rate was significantly lower than control group. The majority (87%) of androgenetic embryos were diploid (2n = 50) and all‐paternal inheritance was verified in larval stage by microsatellite genotypes. Microscopic observations confirmed the elimination of both egg nucleus and second polar body from a fertilized egg, followed by the cleavage exclusively with sperm nucleus. Thus, the appearance of normal diploid androgenetic progeny from sperm of natural tetraploid loach revealed the presence of four chromosome sets in tetraploid males. [ABSTRACT FROM AUTHOR]

Hemato-biochemical and histopathological alterations in mud loach (Misgurnus mizolepis) infected with Aeromonas sobria were characterized in the present study. Fish infected with 1 × 106 colony forming units/mL of A. sobria (25 fish × 3 replicates × 3 tanks) and uninfected (control) fish (25 fish × 3 replicates × 3 tanks) were kept in laboratory tanks with adequate water parameters, and euthanized at 1, 3, and 5 days post-infection (PI). Infected fish became lethargic and developed rapid opercular movements, pale gills, and cutaneous petechial hemorrhages during the 5-day experimental period. Hemato-biochemical parameters, namely hematocrit, hemoglobin, total protein, total calcium, alkaline phosphatase (ALP) activity, and glucose level were significantly reduced, whereas urea (UA) level, and aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) activities were markedly increased in infected fish compared to control fish. Total leukocyte count was higher of infected than in control fish by day 3 PI, but dramatically decreased on day 5 PI. The leukogram showed the predominance of lymphocytes and neutrophils during the experimental period. Histologically, kidney and spleen showed extensive hemorrhage with cellular necrosis. Liver cells showed severely necrotic foci with karyopyknosis, karyorrhexis, karyolysis, and hyperchromatosis of the nuclear membrane. Thus, A. sobria infection causes hemato-biochemical changes such as anemia, hypoglycemia, hypoproteinemia, hypocalcemia, increased AST, ALT, and LDH activities, increased UA concentrations, and reduced ALP activity, and lead to compromised disease resistance, tissue damage, and low survival rates. [ABSTRACT FROM AUTHOR]

The co-existence of several ploidy types in natural populations makes the cyprinid loach Misgurnus anguillicaudatus an exciting model system to study the genetic and phenotypic consequences of ploidy variations. A first step in such effort is to identify the specific ploidy of an individual. Currently popular methods of karyotyping via cytological preparation or flow cytometry require a large amount of tissue (such as blood) samples, which can be damaging or fatal to the fishes. Here, we developed novel microsatellite markers (SSR markers) from M. anguillicaudatus and show that they can effectively discriminate ploidy using samples collected in a minimally invasive way. Specifically, we generated whole genome transcriptomes from multiple M. anguillicaudatus using the Illumina paired-end sequencing. Approximately 150 million raw reads were assembled into 76,544 non-redundant unigenes. A total of 8,194 potential SSR markers were identified. We selected 98 pairs with more than five tandem repeats for further assays. Out of 45 putative EST-SSR markers that successfully amplified and harbored polymorphism in diploids, 11 markers displayed high variability in tetraploids. We further demonstrate that a set of five EST-SSR markers selected from these are sufficient to distinguish ploidy levels, by first validating them on 69 reference specimens with known ploidy levels and then subsequently using fresh-collected 96 ploidy-unknown specimens. The results from EST-SSR markers are highly concordant with those from independent flow cytometry analysis. The novel EST-SSR markers developed here should facilitate genetic studies of polyploidy in the emerging model system M. anguillicaudatus. [ABSTRACT FROM AUTHOR]

Abstract Neutrality plots revealed that selection probably dominates codon bias, whereas mutation plays only a minor role, in shaping the codon bias in three loaches, Misgurnus anguillicaudatus , M. mohoity , and M. bipartitus. These three species also clearly showed similar tendencies in the preferential usage of codons. Nineteen, nine, and 14 preferred codon pairs and 179, 182, and 173 avoided codon pairs were also detected in M. anguillicaudatus , M. bipartitus , and M. mohoity , respectively, and the most frequently avoided type of cP3-cA1 dinucleotide in these species was nnUAnn. The expression-linked patterns of codon usage revealed that higher expression was associated with higher GC 3 , lower ENC, and a smaller proportion of amino acids with high size/complexity (S/C) scores in these three species. These results elucidate selectively driven codon bias in Misgurnus species, and reveal the potential importance of expression-mediated selection in shaping the genome evolution of fish. Highlights • Selection dominates codon bias, whereas mutation plays only a minor role, in shaping the codon bias in the loaches. • Three loach species clearly show similar tendencies in the preferential usage of codons. • Higher expression was associated with higher GC3, lower ENC (effective number of codons) in the loaches. • Highly expressed gene was linked to a higher frequency of amino acids with low metabolic costs in the loaches. [ABSTRACT FROM AUTHOR]

The polymeric immunoglobulin receptor (pIgR) is an essential component of the mucosal immune system in jawed vertebrates including teleost fish, which mediate transepithelial transport of secretory immunoglobulins (sIgs) to protect organisms against environmental pathogens. In this study, we firstly cloned and identified the pIgR from dojo loach ( Misgurnus anguillicaudatus ). The full-length cDNA of Ma -pIgR was of 1145 bp, containing an open reading frame (ORF) of 1101 bp encoded a predicted protein of 336 amino acids. The structure of Ma -pIgR is comprised of a signal peptide, a transmembrane region, an intracellular region and an extracellular region with two Ig-like domains (ILDs), which are similar to their counterparts described in other teleosts. Multiple sequence alignment and phylogenetic analysis showed the dojo loach is closely related to the fish family Cyprinidae. The transcriptional level of Ma -pIgR was detected by quantitative real-time PCR (qRT-PCR) in different tissues and high expression was found in liver, skin, kidney, eye, fin and gills. Two infection models of the loach with bacteria ( Aeromonas hydrophila ) and parasite ( Ichthyophthirius multifiliis ) were constructed for the first time. Histological studies showed the goblet cells in skin significantly increased and the ratio of gill length to width also significantly changed after challenged with A.hydrophila . Both challenge experiments resulted in the significant up-regulated expression of Ma -pIgR not only in kidney and spleen, but also in skin and gills. Our results suggest that pIgR may play an important role in skin and gill mucosal immunity to protect the loach against bacteria and parasite. [ABSTRACT FROM AUTHOR]

In this study, full-length cDNA sequences of elovl4a and elovl4b from loach Misgurnus anguillicaudatus were cloned. The full-length cDNAs of loach elovl4a and elovl4b were 2423 and 2054bp, encoding 315 and 300 amino acids, respectively. The deduced amino acid sequences of elovl4a and elovl4b in loach both shared the highest identity with those of Danio rerio , whereas lower identity score between loach elovl4a and elovl4b was present. Temporal expression and tissue expression of loach elovl4a and elovl4b were studied by reverse transcriptase PCR. Results of the tissue expression analyses suggested different functions of loach elovl4a and elovl4b. Functional characterizations of loach elovl4a and elovl4b on synthesis of fatty acids, especially elongating C18 polyunsaturated fatty acids (PUFAs) to longer-chain fatty acids, were studied by heterologous expression in Saccharomyces cerevisiae. Loach elovl4a and elvol4b enzymes were able to elongate all fatty acids tested including 18:2n-6, 18:3n-3, 18:3n-6, 20:4n-6 and 20:5n-3. At last, expression levels of the two elovl4 genes of loach fin cells incubated with 18:2n-6 and 18:3n-3 of different concentrations were measured. Expressions of elovl4a and elovl4b of loach fin cells were significantly up-regulated by 18:2n-6 and 18:3n-3. The results obtained here indicated that loach elovl4 could effectively elongate C18 PUFAs. This was a systematic report of elovl4's elongating functions towards C18 and provided an alternative pathway for C20 biosynthesis in fish species. [ABSTRACT FROM AUTHOR]

The complement component 3 (C3) is a central component of complement system. All three pathways converge at formation of C3 convertases and share the terminal pathways of membrane attack complex (MAC) formation. In this study, three isoforms of C3 were discovered in Misgurnus anguillicaudatus , named “C3-1”, “C3-2” and “C3-3”, respectively. The full-length of C3-1 cDNA sequence was firstly identified and analyzed from dojo loach ( Misgurnus anguillicaudatus ). The Ma -C3-1 cDNA sequence comprised of 4509 bp encoding 1454 amino acids with a putative signal peptide of 20 amino acid residues. The deduced amino acid sequence showed that Ma -C3-1 has conserved residues and domain, which are known to be crucial for C3 function. Interestingly, an amino acid substitution of the highly conserved GCGEQ was discovered in Ma -C3-1. Phylogenetic analysis showed that Ma -C3-1 was closely related to Cyprinidae. The mRNA expression levels of three isoforms of C3 were detected in kidney, eye, spleen, gonad, heart, fin ray, gut, muscle, brain, gill, skin, blood and liver. The expression of Ma -C3-1 and Ma -C3-3 were mainly detected in liver, followed by spleen, gonad. However, the high expression of Ma -C3-2 was found in kidney, followed by blood and gonad. The morphological changes of gill and skin, and the expression pattern of these three isoforms C3 molecular following the infection with Aeromonas hydrophila were investigated. The mRNA expression levels of three C3 isoforms were up-regulated in the gill, skin, liver and spleen after infection with A.hydrophila . Similarly, challenge experiments resulted in significant up-regulated expression of other complement-relevant genes in gill, liver and skin, such as C4, C5, C8b, especially at 24 h and 36 h. These results suggest that complement system might play an important role not only in liver, but also in the mucosal tissues as gill and skin of teleost fish. [ABSTRACT FROM AUTHOR]

Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is a well-known method to quantify gene expression by comparing with the reference genes. Generally, housekeeping genes were set as references, as for their stable expression in varying conditions. Here, we try to evaluate few of such genes to identify suitable housekeeping genes as references for qRT-PCR analysis of gene expression in Misgurnus anguillicaudatus. This study evaluated the expression of four commonly used housekeeping genes, i.e. b-actin (ACTB), elongation factor 1 alpha (EF-1a), glyceraldehyde-3-phosphate (GAPDH) and 18S ribosomal RNA (18S rRNA), in gender difference, effects of tissue type, different developmental stages, chemical treatment of embryos/larvae with commonly used vehicles for administration and agents that represent known environmental toxicant. Rank ordering of expression stability was done using geNorm, NormFinder and BestKeeper algorithms. Results suggested that in the qRTPCR test, in all the experimental conditions, EF-1a could be selected as reference gene when analysing a target gene. For the study of different development stages, ACTB could be a candidate as reference gene. For the studies associated with different gender and tissue types, EF-1a would be better targeted as reference gene. Meanwhile, in toxicant treatment, expression of EF-1a seems to be more stable than others and could be considered as reference gene. This study could provide useful guidelines that can be expected to aid M. anguillicaudatus researchers in their initial choice of housekeeping genes for future studies and enable more accurate normalization of gene expression data. [ABSTRACT FROM AUTHOR]

Misgurnus anguillicaudatus is a highly valued, aquaculture-relevant food fish in East Asian countries. Because of overfishing and environmental pollution, the number of wild Taihu loach has been sharply decreased in these years. In this study, the complete mitochondrial genome of Taihu loach was obtained by PCR. The genome is 16,646 bp in length, including 2 ribosomal RNA genes. Thirteen proteins-coding genes, 22 transfer RNA genes, and a non-coding control region, the gene composition and order of which was similar to most reported from other vertebrates. Sequence analysis showed that the overall base composition is 29.8% for A, 28.2% for T, 25.7% for C, and 16.3% for G. The phylogenetic tree showed that Misgurnus family got together for one branch, which includes Taihu M. anguillicaudatus, and the other loaches had their own branches. Also the mitochondrial genome sequence of M. anguillicaudatus were aligned by BLAST, compared with Cobitinae the sequence similarity could reach >94%, and the similarity to Misgurnus was >99%.

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.

Dojo loach ( Misgurnus anguillicaudatus) is one of the most important cultured freshwater fish in several East Asian countries. However, a little information is available in its nutritional requirements. Thus, this study was conducted to evaluate the effects of feeding varying levels of dietary protein and lipid on growth, fatty acid composition and antioxidant-related gene expressions in juvenile loach. Six practical diets at three levels of protein (30%, 40% and 50%) and two levels of lipid (6% and 12%) were fed to loach juveniles (initial weight 0.40 g) in triplicated groups (20 fish per replicated) for a period of 8 weeks. Results showed that regardless of lipid level, body weight gain of fish was significantly increased with incremental dietary protein level. Meanwhile, feed conversion ratio was significantly decreased by dietary protein levels, and the lowest value was observed in fish fed dietary protein levels of 50%, regardless of dietary lipid level. Moreover, the percentage of 22:6n-3 in viscera was significantly increased by different protein levels. The expression level of catalase was significantly increased with incremental dietary protein level with both lipid levels. Meanwhile, the expression level of hepatic nuclear factor erythroid 2-related factor 2 ( Nrf2) was downregulated with incremental dietary protein level with 6% of lipid level, but the expression was upregulated with incremental dietary protein level with 12% of lipid level. In conclusion, these data suggested that 6% lipid and 50% protein in diet was optimal for loach during early development stage. [ABSTRACT FROM AUTHOR]

Repetitive DNA sequences, ManDra and ManBgl, were isolated from the DraI and BglII digests of the genomic DNA of Misgurnus anguillicaudatus, respectively. A primer set of ManDra distinguished two genetically different groups (A and B) of M. anguillicaudatus by specific electrophoretograms. A primer set of ManBgl amplified the DNA of M. anguillicaudatus and M. mizolepis. The individuals of M. anguillicaudatus were divided into two groups depending on the fragment sizes, in which the groups A and B (B-1 and B-2) showed 400 and 460 bp, respectively. M. mizolepis was distinguished by a different pattern (400-, 460-, and 510-bp fragments). PCR-RFLP analyses of recombination activating gene 1 gave a clear difference between A or B-2 (443-bp fragment) and B-1 groups (296- and 147-bp fragments). Clonal lineages and hybrids between B-1 and B-2 groups could be identified by appearance of three fragments (443, 296, and 147 bp). The combined analyses using the above three nuclear markers discriminated among nuclear genomes of genetic groups (A, B-1 and B-2) of M. anguillicaudatus and M. mizolepis. In several localities, natural hybridizations between the group B-1 and B-2 loaches and introgressions of clonal mitochondrial genomes into the group B-1 loaches were detected. [ABSTRACT FROM AUTHOR]

A 60-day feeding trial was conducted to determine the effects of different dietary vitamin C levels on growth performance, immune response and antioxidant capacity of loach juveniles. Six isonitrogenous (58.6% of crude protein), isoenergetic (17.5 kJ g−1) practical diets supplemented with 0 ( VC0), 100 ( VC100), 200 ( VC200), 500 ( VC500), 1000 ( VC1000) and 5000 mg kg−1 ( VC5000) of VC (35% ascorbic acid equivalent) were fed to fish (mean initial weight 0.11 ± 0.02 g) in triplicate. Results showed that fish fed VC0 diet had significantly lower body weight gain ( BWG) and survival rate ( SR). However, BWG and SR improved significantly in fish fed VC100 and VC200 diet respectively. Whole body ascorbic acid concentration increased with incremental dietary VC levels from 0 to 100 mg kg−1. The activity of mucus alkaline phophatase was significantly increased by the dietary VC level. Incremental levels of VC significantly reduced activities of glutathione peroxidase ( GPx) and catalase. Moreover, fish fed diets containing more than 100 mg kg−1 VC significantly down-regulated the superoxide dismutase and GPx mRNA expression in liver. Meanwhile, the expressions of liver heat shock protein ( HSP70) and nuclear factor-erythroid 2-related-2 ( Nrf2) were affected by fish fed diets containing VC from 100 to 5000 mg kg−1. In conclusion, VC requirement of loach juveniles for optimum growth and functionally preventing lipid peroxidation was more than 200 mg kg−1 of diet. Moreover, high dose of VC supplementation did not show any detrimental effects on loach growth performance. [ABSTRACT FROM AUTHOR]

We examined the toxic effects of glyphosate on diploid (DIMF) and triploid (TRMF) fin cell lines from the Oriental Weather Loach Misgurnus anguillicaudatus . The LC 50 values of glyphosate estimated by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay were 315.34 and 371.77 mg/L for DIMF and TRMF, respectively. Superoxide dismutase (SOD) and glutathione-S-transferase (GST) activities in DIMF and TRMF cells gradually increased and then decreased with increasing glyphosate concentrations, reaching a maximum at 240 mg/L glyphosate. In contrast, acetylcholinesterase (AChE) activities in DIMF and TRMF decreased with increasing concentrations of glyphosate in a concentration-dependent manner. SOD and AChE activities were generally significantly higher in TRMF compared with DIMF cells (P < 0.05). The rates of micronucleus and abnormal nuclei were significantly higher in DIMF and TRMF groups treated with 80–560 mg/L glyphosate compared with the control groups (P < 0.01). The highest micronuclei rates in both DIMF and TRMF cells (both 4.30‰) occurred at 400 mg/L glyphosate. There were no differences in the rates of micronuclei and abnormal nuclei between DIMF and TRMF cells at any glyphosate concentration. Cell damage, including chromatin condensation, nucleus distortion, and broken and reduced endoplasmic reticulum, mitochondria, and ribosomes, were found in both cells treated with the LC 50 concentration of glyphosate. Moreover, vacuolization and apoptotic bodies occurred in glyphosate-exposed DIMF and TRMF cells, indicating apoptosis. These results indicate that glyphosate in the range of tested concentrations represent a potential risk to loach through inhibiting proliferation of diploid and triploid cell lines and induces micronuclei and apoptosis. [ABSTRACT FROM AUTHOR]

Heat shock protein 70 (HSP70) and 90 (HSP90) are the most broadly studied proteins in HSP families. They play key roles in cells as molecular chaperones, in response to stress conditions such as thermal stress. In this study, full-length cDNA sequences of HSP70 , HSP90α and HSP90β from loach Misgurnus anguillicaudatus were cloned. The full-length cDNA of HSP70 in loach was 2332bp encoding 644 amino acids, while HSP90α and HSP90β were 2586bp and 2678bp in length, encoding 729 and 727 amino acids, respectively. The deduced amino acid sequences of HSP70 in loach shared the highest identity with those of Megalobrama amblycephala and Cyprinus carpio . The deduced amino acid sequences of HSP90α and HSP90β in loach both shared the highest identity with those of M. amblycephala . Their mRNA tissue expression results showed that the maximum expressions of HSP70 , HSP90α and HSP90β were respectively present in the intestine, brain and kidney of loach. Quantitative real-time PCR was employed to analyze the temporal expressions of HSP70 , HSP90α and HSP90β in livers of loaches fed with different levels of vitamin C under thermal stress. Expression levels of the three HSP genes in loach fed the diet without vitamin C supplemented at 0 h of thermal stress were significantly lower than those at 2 h, 6 h, 12 h and 24 h of thermal stress. It indicated that expressions of the three HSP genes were sensitive to thermal stress in loach. The three HSP genes in loaches fed with 1000 mg/kg vitamin C expressed significantly lower than other vitamin C groups at many time points of thermal stress, suggesting 1000 mg/kg dietary vitamin C might decrease the body damages caused by the thermal stress. This study will be of value for further studies into thermal stress tolerance in loach. [ABSTRACT FROM AUTHOR]

Experiments on the swimming kinetics and behaviour of weather loach Misgurnus anguillicaudatus showed that horizontal swim speed was significantly greater than swim speeds when ascending to or descending from the water surface to gulp air. Vertical swimming speeds during ascending or descending were similar. Misgurnus anguillicaudatus swam unsteadily during vertical movements compared with horizontal movements. [ABSTRACT FROM AUTHOR]

Diploids and tetraploids of the cyprinid loach Misgurnus anguillicaudatus coexist in many natural habitats in Asia. However, little is known about the biological and ecological differences between these two ploidy forms. We examined age, body size and growth rates of fish in a mixed ploidy population of M. anguillicaudatus in the Yangtze River basin in China. The sex ratios of both diploid and tetraploid M. anguillicaudatus were highly skewed toward females, who tended to be larger than males. The age distributions of the collected specimens clearly indicated that tetraploids lived longer than diploids. For example, we found a substantial number of tetraploids that were 5+ years old, while the oldest diploids were 4+ years old. Tetraploids were also longer and heavier than diploids for both sexes. Using the empirical body size and age data, we inferred the growth patterns of these fish with the von Bertalanffy growth function. The estimated asymptotic body lengths ( L ) indicated that tetraploids could indeed achieve larger body sizes than diploids. This difference was partially due to the increased growth rate, as demonstrated by lower growth coefficient ( K) and higher growth performance index ( φ'). In sum, we show that tetraploid M. anguillicaudatus exhibited significantly increased longevity and superior growth performance compared to diploids. These differences may contribute to the ecological competitiveness of tetraploid M. anguillicaudatus, thus enabling them to coexist with diploids in certain ecological settings. [ABSTRACT FROM AUTHOR]