Misgurnus Anguillicaudatus Research Articles

Clonal reproduction assured by sister chromosome pairing in dojo loach, a teleost fish.

Wild-type dojo loach (Misgurnus anguillicaudatus) commonly reproduces bisexually as a gonochoristic diploid (2n = 50), but gynogenetically reproducing clonal diploid lines (2n = 50) exist in certain districts in Japan. Clones have been considered to develop from past hybridization event(s) between two genetically diverse groups, A and B, within the species. Fluorescence in situ hybridization analyses using the repetitive sequence "ManDra" as a probe clearly distinguished 25 chromosomes derived from group B out of a total of 50 diploid chromosomes of the clone, providing strong molecular cytogenetic evidence of its hybrid origin. In meiosis, diploid wild-type showed 25 bivalents, while diploid clones revealed 50 bivalents, indicating the presence of 100 chromosomes. In meiotic chromosome spreads in sex-reversed clonal males, ManDra signals were detected in 25 out of 50 bivalents, and only one out of two bivalents possessing major ribosomal RNA coding regions exhibited two positive ManDra signals. In clonal females, ManDra signals were detected in approximately 25 out of 50 bivalents. Thus, unreduced gametes should be generated by the pairing between sister chromosomes doubled from each ancestral chromosome from the different groups by premeiotic endomitosis. Sister chromosome pairing should assure production of unreduced isogenic clonal gametes due to the absence of the influence of recombination or crossing over.

Gonadal transcriptome analysis of hybrid triploid loaches (Misgurnus anguillicaudatus) and their diploid and tetraploid parents.

Hybrid triploid loaches (Misgurnus anguillicaudatus) were generated from natural tetraploid and diploid loaches. We studied the gonads of parents and offspring from direct and reciprocal crosses through histological and transcriptome analyses. The triploid offspring had inferior ability to form sperm and egg cells compared with diploid forms. After sequencing the transcriptomes, 655,109,158 clean reads were obtained, and 62,821 unigenes and 178,962 transcripts were assembled. Of these unigenes, 23,189 were annotated in the GO database, 18,525 in the KEGG database and 24,661 in the KOG database. 36 fertility-related genes were found to be differentially expressed between the direct cross (2n × 4n) progenies and their parents, while 53 fertility-related genes between the reciprocal cross (4n × 2n) progenies and their parents. Following protein-protein interaction network analyses, 54 differentially expressed genes, including PLCB4, cyp17a1 and Pla2g4d, were mined, yielding candidate genes involved in the poor fertility of hybrid triploid loaches. This is the first report of transcriptomes of gonads of hybrid triploid loaches and their parents, offering a substantial contribution to sequence resources for this species and providing a deep insight into the molecular mechanism controlling the fertility of hybrid triploid fish.

Performance of eDNA assays to detect and quantify an elusive benthic fish in upland streams

The sensitivity and specificity of eDNA-based monitoring, coupled with its potential utility to estimate population density or biomass, makes it a useful tool in invasive species management. In this study, we investigated the potential of the eDNA method to improve the detection of the elusive invasive fish, oriental weatherloach (Misgurnus anguillicaudatus), in a river system where a density gradient of the species occurs. We compared detection rates between eDNA and conventional monitoring methods and examined the relationship between eDNA and abundance in a flowing environment. The eDNA method had a higher site detection rate than conventional methods (63 vs. 38%). Weatherloach eDNA was detected at all sites where the fish has been previously caught and none of the sites where the species has not been caught for the past 7 years. There was an increasing density trend going downstream based on long-term conventional monitoring, but the eDNA concentration in water samples reflected this trend only in a continuous section of the river where impoundments were absent. We did not find a positive relationship between eDNA concentration and contemporary abundance estimates in our study area. A high eDNA concentration was recorded at a site (DVC) which was designated a low density site based on long-term catch data. This discrepancy was a likely result of physical habitat characteristics which influenced the efficiency of the conventional methods used. This study highlighted the challenges of inferring density from eDNA data in flowing water because habitat features may confound results, necessitating careful consideration for results to be useful to management.

Complete mitochondrial genome of natural diploid loaches Misgurnus anguillicaudatus from the Taihu Lake

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%.

Molecular characterization of elongase of very long-chain fatty acids 6 (elovl6) genes in Misgurnus anguillicaudatus and their potential roles in adaptation to cold temperature

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.

Molecular characteristics of mitochondrial DNA and phylogenetic analysis of the hybrid loach of Misgurnus anguillicaudatus (female) and Paramisgurnus dabryanus ssp. (male)

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.

Enantiospecific toxicity, distribution and bioaccumulation of chiral antidepressant venlafaxine and its metabolite in loach (Misgurnus anguillicaudatus) co-exposed to microplastic and the drugs.

In present study, we investigated the enantioselective behaviors of the chiral antidepressant venlafaxine and its metabolite O-desmethylvenlafaxine in loach Misgurnus anguillicaudatus (M. anguillicaudatus), as well as effects of microplastic on toxicity, distribution and metabolism through a 40-day co-exposure. The contents of SOD and MDA in loach liver elevated when the loach was exposed to venlafaxine and O-desmethylvenlafaxine. Moreover, co-exposure with microplastic might lead to more adverse effect against loach. The distribution of venlafaxine and O-desmethylvenlafaxine were both detected in loach tissues and liver subcellular. The concentrations of venlafaxine and O-desmethylvenlafaxine were lower in water in microplastic-present treatment. Whilst, more contaminants were accumulated in liver through the "vehicle" (microplastic). Enantioselective behavior of venlafaxine and O-desmethylvenlafaxine occurred with R-enantiomers being preferentially degraded. With microplastic present, the bioaccumulation factor (BAF) of venlafaxine and O-desmethylvenlafaxine in loach tissue amplified more than 10 times. In liver subcellular structure, microplastic may help to transport more compounds into subtle areas and postpone the contaminants metabolism in organisms. The present study for the first time gained an insight into the potential ecological effects and environmental behaviors of combined pollutions of chiral pharmaceuticals and microplastic, which could supply important information for environment risk assessment of concurrent organic pollutants and microplastic.

Development of novel EST-SSR markers for ploidy identification based on de novo transcriptome assembly for Misgurnus anguillicaudatus.

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.

Monthly Change of the Length-weight Relationship of the Loach (<i>Misgurnus anguillicaudatus</i>) Population in Paddy Fields by Farming Practices

Monthly Change of the Length-weight Relationship of the Loach (<i>Misgurnus anguillicaudatus</i>) Population in Paddy Fields by Farming Practices

Genomic evidence for the population genetic differentiation of Misgurnus anguillicaudatus in the Yangtze River basin of China

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.

Possibility of habitat segregation between two sympatric species of Japanese dojo loach in the Naka-ikemi Wetland, Fukui Prefecture, Japan

Possibility of habitat segregation between two sympatric species of Japanese dojo loach in the Naka-ikemi Wetland, Fukui Prefecture, Japan

Viable diploid progeny induced from sperm of Chinese tetraploid pond loach by cold-shock androgenesis

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.

A preliminary investigation into the use of the invasive golden apple snail, Pomacea canaliculata (Lamarck, 1822), as a water purifier and food source in the breeding ponds of the oriental weatherloach Misgurnus anguillicaudatus (Cantor, 1842)

ABSTRACTThis study investigated the feasibility of using an invasive snail, Pomacea canaliculata, as a food source and water purifier for the commercial breeding of the loach Misgurnus anguillicaudatus. The predatory potential of M. anguillicaudatus (3.5–5.5 g) against hatchling snails was evaluated in aquaria and simulated paddy fields. Some hatchling snails left the water to avoid being preyed upon by the loaches, and approximately 10 hatchlings died per day in the presence of five loaches in aquaria, whereas a weaker snail control effect was observed in the simulated paddy fields. The growth of rice seedlings (Oryza sativa) was not reduced by the presence of hatchling snails alone, but the shoot biomass of seedlings coexisting with snails was promoted after introducing the loaches. Additionally, the presence of P. canaliculata adults improved the aquatic environment in the short term for loach breeding by decreasing the turbidity of the water. Importantly, M. anguillicaudatus (12–18 g) mortality decreased and its weight increased in the presence of adult snails.

Molecular characterization of peroxisome proliferator activated receptor gamma (PPARγ) in loach Misgurnus anguillicaudatus and its potential roles in fatty acid metabolism in vitro

Abstract Peroxisome proliferator activated receptor gamma (PPARγ) was firstly investigated in loach Misgurnus anguillicaudatus by RACE (rapid amplification of cDNA ends) and qPCR (real-time quantitative PCR). An in vitro cell culture experiment was then performed and showed that loach PPARγ positively affected expressions of four fatty acid metabolism related genes, namely fatty acid desaturase 2, lipoprotein lipase, elongase of very long chain fatty acids 5 and sterol regulatory element-binding protein-1c. At last, effects of C18:2n-6 (linoleic acid, LA) and C18:3n-3 (linolenic acid, LNA) on loach PPARγ expression were investigated. Loach fin cells cultured with 50 μM and 100 μM of LA or 100 μM LNA exhibited significantly lower PPARγ transcriptions than those cultured with 0 μM LA and LNA, showing that high-dose LA/LNA suppressed PPARγ expression in loach fin cells. Results of the two in vitro experiments suggested that PPARγ played an important role in fatty acid metabolism of the loach.

Trophic transfer of citrate, PVP coated silver nanomaterials, and silver ions in a paddy microcosm.

We used replicated paddy microcosm systems to estimate the tropic transfer of citrate-coated silver nanoparticles (AgNP citrate), polyvinylpyrrolidone (PVP)-coated AgNP (AgNP PVP), and silver ions (AgNO3) for 14 days under two exposure regimes (a single high-dose exposure; 60 μg L-1 and a sequential low-dose exposure at 1 h, 4 days and 9 days; 20 μg L-1 × 3 = 60 μg L-1). Most Ag ions from AgNO3 had dispersed in the water and precipitated partly on the sediment, whereas the two Ag NPs rapidly coagulated and precipitated on the sediment. The bioconcentration factors (BCFs) of Ag from AgNPs and AgNO3 in Chinese muddy loaches and biofilms were higher than those of river snails in both exposure conditions. These BCFs were more prominent for 14 days exposure (7.30 for Chinese muddy loach; 4.48 for biofilm) in the low-dose group than in the single high-dose group. Their retention of AgNPs and Ag ions differed between the two exposure conditions, and uptake and elimination kinetics of Ag significantly differed between AgNP citrate and AgNP PVP in the sequential low-dose exposure. Stable isotopes analyses indicated that the trophic levels between Chinese muddy loaches and biofilms and between river snails and biofilms were 2.37 and 2.27, respectively. The biomagnification factors (BMFs) of AgNPs and AgNO3 between Chinese muddy loaches and biofilms were significantly higher than those between river snails and biofilms under both exposure settings. The BMFs of AgNP citrate and AgNO3 between Chinese muddy loaches and biofilms were greater than those of AgNP PVP for 14 days in the single high-dose group, whereas the BMFs of AgNP PVP were greater than those of AgNP citrate and AgNO3 in the sequential low-dose group. These microcosm data suggest that AgNPs have the potential to impact on ecological receptors and food chains.

First Record of the Large-Scale Loach Paramisgurnus dabryanus (Cobitidae) in the United States

Abstract Exotic species have been implicated as a major threat to native freshwater fish communities in the Unites States. The San Francisco Estuary watershed has been recognized as one of the most invaded systems where exotics often dominate the fish community. On October 6, 2014, members of the U.S. Fish and Wildlife Service detected a previously unknown exotic fish in a disconnected pool immediately upstream from the Chowchilla Bifurcation Structure in the San Joaquin River, a major tributary of the San Francisco Estuary. A member of the U.S. Fish and Wildlife Service initially identified the fish as an Oriental Weatherfish Misgurnus anguillicaudatus using external morphological characteristics. We conducted additional fish sampling near the Chowchilla Bifurcation Structure in November 2014 and collected a total of six additional specimens in disconnected pool habitats. Unexpectedly, genetic and meristic techniques revealed that these specimens were Large-Scale Loach Paramisgurnus dabryanus. To our knowledge this is the first confirmed occurrence of Large-Scale Loach in the United States and the suspected pathway of introduction is release from aquaria. Very little is known about the population in the San Joaquin River. We recommend further evaluation of the ecology, distribution, and abundance of Large-Scale Loach to better understand their potential impact on the fish communities of the San Joaquin River and the likelihood of establishment throughout the United States.

The effects of two free-floating plants (Eichhornia crassipes and Pistia stratiotes) on the burrow morphology and water quality characteristics of pond loach (Misgurnus anguillicaudatus) habitat

Loach exhibit conspicuous drilling behaviors in the mud of shallow waters, yet their burrow morphology and the factors affecting this behavior have received little attention. We characterized the burrow morphology and water quality of the pond loach Misgurnus anguillicaudatus in three scenarios: in tanks without plants, tanks with the free-floating plant water hyacinth Eichhornia crassipes, and tanks with water lettuce Pistia stratiotes. Water hyacinth effectively removed water TN, COD, NO3-N and NH4-N, and water lettuce removed water TP and NH4-N. Water hyacinth and water lettuce markedly reduced water turbidity and DO, increased TOC and EC. Water hyacinth purified water more effectively than water lettuce, providing a suitable habitat for loach feeding, living and burrowing. The burrow structures were V-shaped, Y-shaped, inverted L-shaped, or complicated dendritic networks composed of multiple V shapes. The hyacinth treatment was characterized by the greatest burrow volume, length, depth, and structural complexity, but the opening size was reduced by dense root mat coverage. Burrows in the water lettuce treatment were characterized by intermediate volume, length, branches and sinuosity, but they had the largest opening and pit size. The control treatment had a flat bottom with the smallest, shortest burrows. This study indicates that free-floating plants improve habitat suitability and change burrow morphology and may be used to improve loach breeding methods.

Influence of Farming Practices on Weight-Length Relationship of the Loach (Misgurnus anguillicaudatus) Population in Rice Paddy Fields

Influence of Farming Practices on Weight-Length Relationship of the Loach (Misgurnus anguillicaudatus) Population in Rice Paddy Fields

Elovl4 can effectively elongate C18 polyunsaturated fatty acids in loach Misgurnus anguillicaudatus

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.

Polymeric immunoglobulin receptor in dojo loach (Misgurnus anguillicaudatus): Molecular characterization and expression analysis in response to bacterial and parasitic challenge

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.