Dojo Loach Misgurnus Anguillicaudatus Research Articles

Nucleotide Sequence and Chromosome Mapping of 5S Ribosomal DNA from the Dojo Loach, Misgurnus anguillicaudatus

There are 2 genetically divergent groups in the dojo loach Misgurnus anguillicaudatus: A and B. Although most wild-type diploids reproduce sexually, clonal diploids (clonal loach) reproduce gynogenetically in certain areas. Clonal loaches produce unreduced isogenic eggs by premeiotic endomitosis, and such diploid eggs develop gynogenetically following activation by the sperm of sympatric wild-type diploids. These clonal loaches have presumably arisen from past hybridization events between 2 different ancestors. The genomic differences between these 2 groups have not been completely elucidated. Thus, new genetic and cytogenetic markers are required to distinguish between these 2 groups. Here, we compared the 5S rDNA region to develop markers for the identification of different dojo loach groups. The nontranscribed sequence (NTS) of the 5S rDNA was highly polymorphic and group-specific. NTSs were found in clades of 2 different groups in clonal loaches. In contrast, we did not find any group-specific sequences in the coding region of the 5S rRNA gene. Sequences were located near the centromere of the short arm of the largest submetacentric chromosomes in groups A and B and clonal loaches. Thus, the 5S rDNA of the dojo loach is conserved at the chromosomal location. Whereas, the sequences of the NTS regions evolved group-specifically in the dojo loach, with the sequences of both groups being conserved in clonal loaches.

Genome survey of Misgurnus anguillicaudatus to identify genomic information, simple sequence repeat (SSR) markers, and mitochondrial genome.

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 for M. anguillicaudatus is currently 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 1105.97Mb 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,the complete mitochondrial genome was sequenced. Based on the Maximum Likelihood and Bayesian inference analyses, M. anguillicaudatus yingde in this study was the "introgressed" mitochondrial type. Seventy microsatellite loci were randomly selected from detected SSR loci to test polymorphic, of which, 20 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 20 loci were highly informative (PIC > 0.700). Eight loci deviated from Hardy-Weinberg equilibrium after Bonferroni correction (P < 0.05). This is the first report of genome survey sequencing in M. anguillicaudatus, genome information, mitochondrial genome, andmicrosatellite markers will be valuable for further studies on population genetic analysis, natural resource conservation, and molecular marker-assisted selective breeding.

Development and characterization of 56 SNP markers in Misgurnus anguillicaudatus

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 < 0.05). The first set of SNP markers developed from M. anguillicaudatus will provide valuable information in further population genetic analysis and natural resource conservation.

Comparative analysis of mitochondrial genomes in genetically distinct groups of the dojo loach Misgurnus anguillicaudatus

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.

Fertility and gamete ploidy of allopentaploid offspring of tetraploid dojo loach Misgurnus anguillicaudatus females and large-scale loach Paramisgurnus dabryanus males

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.

Intra-ooplasmic injection of a multiple number of sperm to induce androgenesis and polyploidy in the dojo loach Misgurnus anguillicaudatus (Teleostei: Cobitidae)

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.

Molecular and morphological analyses revealed a cryptic species of dojo loach Misgurnus anguillicaudatus (Cypriniformes: Cobitidae) in Japan.

Although it has been reported that populations of the Japanese dojo loach Misgurnus anguillicaudatus (Cypriniformes: Cobitidae) belong to two distinct mitochondrial (mt)DNA (Type I and Type II), the taxonomic status of the species remains unresolved. To address this question, nuclear DNA and morphological analyses were performed on M. anguillicaudatus population in the Nakaikemi Wetland, where Type I and Type II lineages are sympatric. Results suggest the existence of a cryptic species (Type I) within the Japanese dojo loach.

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

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

Effects of dietary phospholipid levels on growth performance, fatty acid composition and antioxidant responses of Dojo loach Misgurnus anguillicaudatus larvae

A 30-day feeding trial was conducted to determine the effects of different levels of dietary phospholipids (PLs) on growth performance, fatty acid composition and antioxidant enzyme activities of loach Misgurnus anguillicaudatus larvae. After 15 days post-hatching, larvae were fed five isoproteic and isolipidic formulated diets with different levels of PLs: 0% (PLs0.8), 2% (PLs1.6), 4% (PLs1.9), 6% (PLs2.3) and 8% (PLs3.0). Results showed that dietary PLs supplementation significantly improved survival and growth of larvae. Survival rate (SR) and body weight gain (BWG) in larvae fed the PLs2.3 diet were significantly higher than those fed PLs0.8, PLs1.6 and PLs1.9 diets, and no differences on SR and BWG were observed between PLs2.3 and PLs3.0 groups, indicating that a supplement of more than 2.3 g 100 g− 1 of PLs in diet was required for loach larvae. Dietary PLs supplementation significantly increased fat content and the ratio of neutral lipid (NL)/polar lipid (PL) in the whole body. Concentrations of 20:5n-3 and total n-3 fatty acids in the whole body significantly increased in NL and significantly decreased in PL with incremental dietary PLs levels. Dietary PLs supplement significantly increased superoxide dismutase activity and reduced activities of catalase and glutathione peroxidase in the whole body. These results indicated that supplementation of more than 2.3 g 100 g− 1 of PLs in diet could improve growth and survival of loach larvae.

The complete mitochondrial genomes of natural diploid and tetraploid loaches Misgurnus anguillicaudatus (Cypriniformes: Cobitidae)

Dojo loach Misgurnus anguillicaudatus, a small-sized freshwater fish species, is not only one of the most important cultured fish in East Asia, but also a promising model animal to study evolutionary biology for polyploidy. We sequenced the complete mitochondrial genomes from the natural diploid and tetraploid individuals of this species. The two different cytotypes loaches share the same length (16,646 bp) and structural organization of mitochondrial genome. The two genomes have 98.3% nucleotide sequence similarity, and the biggest nucleotide sequence divergence between homologous genes is observed in CYTB gene.

Comparative studies on lipid profiles and amino acid composition of wild and cultured Dojo loach Misgurnus anguillicaudatus obtained from southern Japan

The aim of this study was to determine the differences in the nutritional properties of wild and cultured loach Misgurnus anguillicaudatus obtained from southern Japan. Major parameters, such as whole-body proximate composition, lipid classes, and fatty acid and amino acid composition, were investigated. The whole-body total lipid content of cultured loach was twofold higher than that of wild loach and the proportion of neutral lipids was significantly higher in cultured loach than in wild loach. However, the polar lipid content was two- to threefold lower in cultured loach than in wild loach. Compared to the wild loach, higher contents of 20:5n-3 and 22:6n-3 were detected in cultured loach, but much lower contents of 20:4n-6. In terms of whole-body amino acid composition, wild and cultured loach showed similar profiles. In conclusion, the consumption of cultured loach will contribute to dietary n-3 highly unsaturated fatty acids intake, with a benefit to human health.

Annual changes in testicular development and plasma sex steroids in the captive male dojo loach Misgurnus anguillicaudatus

Testicular development in the captive male dojo loach Misgurnus anguillicaudatus was examined monthly in relation to the levels of plasma sex steroids [testosterone (T), 11-ketotestostrone (11-KT), and 17,20β-dihydroxy-4-pregnen-3-one (DHP)]. On the basis of testicular histology, the annual gonadal cycle was found to be divisible into 3 periods: the recovery and proliferation period, which mainly consists of early spermatogenic testis from August to November (reproductive phase I); the preparation period for the next spawning period, which mainly consists of late spermatogenic testis from December to April (reproductive phase II); and the mature period, characterized by a high proportion of mature testis from May to July (reproductive phase III). Individual variability in testicular development was high, and continuous spermatogenesis was observed throughout the year. High levels of plasma T, 11-KT, and DHP were observed during reproductive phase III. 11-KT began to increase in February, while T was present at low levels in reproductive phase II. These results suggest that the physiologically active season of testis development for breeding in the dojo loach is from May to July, although spermatogenesis occurs throughout the year.

Annual Changes in Ovarian Development and Sex Steroid Levels in Cultured Female Dojo Loach Misgurnus anguillicaudatus

Annual Changes in Ovarian Development and Sex Steroid Levels in Cultured Female Dojo Loach Misgurnus anguillicaudatus

Effects of modern drainage systems on the spatial distribution of loach in rice ecosystems

Abstract Although rice fields support many aquatic species, recent agricultural intensification in Asia has led to loss of biodiversity, such as freshwater fish, in exchange for increased agricultural productivity. In Japan, modern drainage systems have been introduced in recent decades. This study investigated the effects of several aspects of modern drainage systems on the spatial distribution of dojo loach Misgurnus anguillicaudatus, a dominant fish species in Japanese rice fields. Multimodel inference approaches and hierarchical partitioning were used to estimate the direction and degree of the effect of each system on the spatial distribution of dojo loach. Three variables related to modern drainage systems (gaps between fields and ditches, the type of drainage ditch, and the presence of underground drainage pipes), flooding dates, and spatial autocovariates showed significant influence on the abundance of loach. In particular, gaps had the largest impact among all the variables on loach in the later part of the survey (June). Based on the results, the effectiveness of several conservation methods implemented for paddy‐dwelling fish in Japan was discussed. The knowledge will be a guide to the appropriate conservation of freshwater fish in Asia and other countries. Copyright © 2011 John Wiley &amp; Sons, Ltd.

Comparative studies on survival and growth performance among diploid, triploid and tetraploid dojo loach Misgurnus anguillicaudatus

To determine the cytotype with better traits for the aquaculture practices of the dojo loach Misgurnus anguillicaudatus from the viewpoint of fish farming improvement, factorial crosses (2n♀ × 2n♂, 2n♀ × 4n♂, 4n♀ × 2n♂, 4n♀ × 4n♂) were conducted between natural diploids (D) and tetraploids (T), producing DD, DT, TD, and TT groups (female listed first). The potential benefits of the different cytotypes in culture were evaluated by comparing growth performance and survival rate for a 15-month rearing trial under the same production conditions. The average fertilization rate in DT and TT was significantly lower than in the DD and TD groups, possibly indicating the poor fertilizing capacity of the tetraploid sires. Survival rate in DT and TD was slightly lower than in DD but significantly higher than in the TT groups. Tetraploid females produced obviously larger eggs than diploids and, subsequently, significantly longer initial body length of TT and TD than DD and DT fry. However, from the second month of the growth trial, TT suffered higher mortality than other cytotypes, which significantly influenced morphometric growth parameters. The TD group exhibited superior growth performance throughout the experiment. The mean body length of DT was comparable with that of DD fish during the first 7 months but began to outgrow DD from the 9th month. This study suggests that the relatively better growth of tetraploid and higher survival rate of diploid can be integrated via inter-ploidy hybridization to get TD triploids with better culture traits.

Comparison of haematology and serum biochemistry of cultured and wild Dojo loach Misgurnus anguillicaudatus

Haematological and serum biochemistry parameters were studied and compared between cultured and wild ecotypes of Dojo loach Misgurnus anguillicaudatus during the prime fishing season, i.e. May-August. Data were analysed for the impact of feeding regime and other ecological conditions on the physiology of fish. The results revealed that haemoglobin, cholesterol, total protein, creatinine and uric acid levels in the two ecotypes were significantly different (n = 56, df = 54, P < 0.05). In addition, red blood cell, glucose, triglyceride and urea nitrogen levels were significantly higher in cultured individuals (n = 56, df = 54, P < 0.01) than in their wild counterparts. In contrast, the white blood cell level in cultured fish was significantly (n = 56, df = 54, P < 0.01) lower than that in the wild ones. These differences can be attributed to the physiological acclimatization of the fish to their living conditions and feeding regime, which influences the energy metabolism and, consequently, the health of the fish.