Microsatellite-centromere mapping in the loach, Misgurnus anguillicaudatus

Primer sets for 15 polymorphic microsatellite loci were developed in the loach, Misgurnus anguillicaudatus (Cobitidae) by molecular cloning and sequencing techniques. Mendelian inheritance was confirmed for the 15 loci by examining the genotypic segregation produced with the primer sets in two full-sib families. The loci were mapped in relation to their centromere in four gynogenetic diploid lines, which were induced by inhibition of the second meiotic division after fertilization with genetically inert sperm. Microsatellite-centromere recombination rates ranged between 0.06 and 0.95 under the assumption of complete interference. Thus, these loci are distributed from the centromeres to the telomeres of their respective chromosomes. The success of mitotic gynogenesis, produced by suppression of the first cleavage, was verified by homozygosity at three diagnostic microsatellite loci that exhibited high gene-centromere meiotic recombination rates in the same family. The differences in heterozygosity levels observed with these markers were attributed to differences in the temporal application of heat shock following inert sperm activation.     

The preparation and characterization of an antimicrobial polypeptide from the loach,Misgurnus anguillicaudatus

Using Sephadex G-50 gel filtration, DEAE-52 cellulose ion-exchange chromatography, and an improved polyacrylamide gel electrophoresis together with electroelution, a novel polypeptide with antimicrobial activity in vitro was isolated and characterized from loach, Misgurnus anguillicaudatus. The polypeptide, named MAPP, contains about 94 residues containing l0 different amino acids, of which cysteine was the most abundant. No alkaline residue was found in MAPP. MAPP is a single-chain polypeptide with Mw of about 9800Da and pI of about 4.78; the N-terminus of MAPP was CFGWN. MAPP showed good inhibition of various bacteria including Bacillus subtilis, Escherichia coli, and Staphylococcus aureus. MAPP is thermally stable with more than 70% inhibitory bioactivity remaining after treatment at 60 degrees C for 30min. In addition, MAPP could inhibit the autoxidation of pyrogallol with a high efficiency. Similarity searches by comparing amino acid composition, MS-fingerprint, and the N-terminus of MAPP demonstrated that no protein exactly matched MAPP in databases around the world.     

Embryonic stages from cleavage to gastrula in the loach Misgurnus anguillicaudatus

Early developmental staging from the zygote stage to the gastrula is a basic step for studying embryonic development and biotechnology. We described the early embryonic development of the loach, Misgurnus anguillicaudatus, based on morphological features and gene expression. Synchronous cleavage was repeated for 9 cycles about every 27 min at 20 degrees C after the first cleavage. After the 10th synchronous cleavage, asynchronous cleavage was observed 5.5 h post-fertilization (hpf), indicating the mid-blastula transition. The yolk syncytial layer (YSL) was formed at this time. Expressions of goosecoid and no tail were detected by whole-mount in situ hybridization from 6 hpf. This time corresponded to the late-blastula period. Thereafter, epiboly started and a blastoderm covered over the yolk cell at 8 hpf. At 10 hpf, the germ ring and the embryonic shield were formed, indicating the stage of early gastrula. Afterward, the epiboly advanced at the rate of 10% of the yolk cell each hour. The blastoderm covered the yolk cell completely at 15 hpf. The embryonic development of the loach resembled that of the zebrafish in terms of morphological change and gene expression. Therefore, it is possible that knowledge of the developmental stages of the zebrafish might be applicable to the loach.     

Distribution and reproductive capacity of natural triploid individuals and occurrence of unreduced eggs as a cause of polyploidization in the loach,Misgurnus anguillicaudatus

Loaches (Misgurnus anguillicaudatus) were collected from 35 localities in Japan and assayed by flow cytometry to determine ploidy status. No tetraploids were found, with samples from 33 localities having no or few (1.2–3.2%) triploids. Samples collected from Ichinomiya Town, Aichi Prefecture, showed a relatively high rate of triploidy (7.7%). Samples collected from a fish farm in Hirokami Village, Niigata Prefecture, also showed high proportions of triploids (2.0–15.8%), these triploid males being sterile, but the females producing both large-sized triploid and small-sized haploid eggs. Such eggs developed bisexually rather than gynogenetically, giving rise to viable tetraploid and diploid offspring after normal fertilization. Of eight diploid females obtained from the same locality, one produced a high incidence of viable diploid gynogens (55%) after gynogenetic induction by fertilization with UV-irradiated spermatozoa. These observations indicated the presence of diploid fish which produced both diploid and haploid eggs. Thus, triploid and diploid individuals were also produced after fertilization with haploid spermatozoa. These results suggested that the occurrence of such unreduced eggs may be a cause of natural polyploidization in this species.     

Protein polymorphism and geographic variation in the loach Misgurnus anguillicaudatus

Electrophoretic variations of enzymatic and non-enzymatic proteins were investigated in 8 populations of the loach Misgurnus anguillicaudatus. The cathodic hemoglobin and the muscle protein in the region II were shown to be polymorphic, in addition to the five polymorphic proteins previously reported (the anodic hemoglobin, muscle protein in the region III, 6-phosphogluconate dehydrogenase, phosphoglucomutase and aspartate aminotransferase; Kimura, 1976 & 1977). The muscle proteins in the regions I, IV and V were monomorphic. The values of genetic distances between loach populations, calculated over 10 loci, indicated that M anguillicaudatus should be classified into two local races.     

Protein polymorphism and geographic variation in the loach Misgurnus anguillicaudatus

Electrophoretic variations of enzymatic and non-enzymatic proteins were investigated in 8 populations of the loach Misgurnus anguillicaudatus . The cathodic hemoglobin and the muscle protein in the region II were shown to be polymorphic, in addition to the five polymorphic proteins previously reported (the anodic hemoglobin, muscle protein in the region III, 6-phosphogluconate dehydrogenase, phosphoglucomutase and aspartate aminotransferase; Kimura, 1976 & 1977). The muscle proteins in the regions I, IV and V were monomorphic. The values of genetic distances between loach populations, calculated over 10 loci, indicated that M. anguillicaudatus should be classified into two local races.     

Electrophoretic patterns of aspartate aminotransferase in the loach Misgurnus anguillicaudatus

The loach Misgurnus anguillicaudatus is a fresh-water teleost and is known to be widely distributed through Japan, Formosa, Korea and into eastern Asia. The author has reported electrophoretic variation in five different proteins in the loach (Kimura, 1976a, b, c). This note describes a sixth example, electrophoretic variation of aspartate aminotransferase (AAT; EC 2.6.1.1.).     

Feeding behaviour of the Japanese loach,Misgurnus anguillicaudatus (Cobitididae)

The feeding behaviour of a Japanese loach,Misgurnus anguillicaudatus was experimentally analyzed. From observations the behaviour patterns were described. The adult fish responded to a piece of thread soaked with filteredTubifex homogenate (Tubifex solution) and showed feeding behaviour. But they ignored an unsoaked piece of thread. LivingTubifex kept in a glass cell failed to elicit feeding behaviour in the fish. A gauze-covered vessel withTubifex solution buried in the sand substrate released feeding behaviour in fish in its vicinity. It follows that the feeding behaviour of this fish is induced by some chemical stimuli. WhenTubifex solution was introduced at the central area of the plastic vessel with a fish, the fish suddenly started the Feeding-crawl and showed Gulp which was composed of Dig and Twist. Using sand particles, it was shown that intake of materials into the digestive tract occurs only at the moment of Twist and not any other time. The standard feeding patterns ofMisgurnus anguillicaudatus are thus quick inhalation of organic matter together with substrate, occurring at the moment of Twist, which is released at the place where the fish received the appropriate chemical stimuli.Misgurnus anguillicaudatus is regarded as a typical “detritus feeder”.     

Hemopoietic sites and development of eosinophil granulocytes in the loach,Misgurnus anguillicaudatus

Summary Unique eosinophils, each of which contained only one eosinophilic granule, have been found in the peripheral blood of the loach (itMisgurnus anguillicaudatus). Several loach organs have been studied by light and electron microscopy to determine the hemopoietic site of this cell type. Eosinophils are produced mainly in the spleen and to a small extent in the kidney, but not in other organs.Presumed myeloblasts are identified as large lymphoid cells containing a number of small-dense granules (diameter, 0.12–0.16 m) in the cytoplasm. These granules have been observed throughout eosinophilopoiesis but they are most abundant in the promyelocyte stage. The largest cells have been identified as myelocytes which contain a number of large granules (diameter, 0.7–1.4 m) with electron-dense crystalline cores. These large granules are present from the myelocyte to metamyelocyte stage. Metamyelocytes differ from myelocytes in having more large granules. Mature eosinophils are morphologically similar to metamyelocytes but are characterized by the presence of only one very large electron-dense granule (diameter, 2.5–2.8 m) with a crystalline core.The nature of these granules has been studied by enzyme digestion using pepsin and trypsin. The results indicate that the crystalline cores are almost pure protein.     

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

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

Molecular characteristics of mitochondrial DNA and phylogenetic analysis of the loach (Misgurnus anguillicaudatus) from the Poyang Lake

The goal of our study was to investigate the molecular characteristics of mitochondrial DNA (mtDNA) and phylogenetic construction of the weather loach (Misgurnus anguillicaudatus) in Poyang Lake. The complete mitochondrial genome was 16,634?bp, and the gene order was identical to that of teleost fishes. Compared with the previous reported weather loach in China, there were numerous nucleotide substitutions and length polymorphisms on the structural genes of mitochondrial DNA in the loach from the Poyang Lake. The Phylogenetic tree indicated that the loach had its own molecular characteristics and was somewhat different from those in other regions of China. Fourteen unique haplotypes of the cytochrome b (cyt b) gene were obtained from 300 weather loaches. The Phylogenetic tree based on the cyt b gene showed that the loaches were substructured into two different populations in The Poyang Lake. Results indicated that the loaches in Poyang Lake not only showed the same phylogeny as the loaches in other areas of China, but also generated its own unique phylogenetic relationships.     

Geographical variation of lactate dehydrogenase and phosphoglucomutase in the loach Misgurnus anguillicaudatus

One of the two loci controlling muscle lactate dehydrogenase (LDH) was shown to be polymorphic in the loach Misgurnus anguillicaudatus. The result of the survey on four populations suggests that the differences in the LDH gene frequencies are correlated with the geographical distribution of the loach. A fourth phosphoglucomutase allele ( PGM ^D) was found in one of the loach populations examined.     

Geographic variation of muscle adenylate kinase in the loach Misgurnus anguillicaudatus

The electrophoretic pattern of the loach muscle adenylate kinase was composed of one or two major bands. Each major band was preceded by two minor bands. Three codominant alleles were postulated to segregate in loach. Each allele coded for one major band with different mobility.
Adenylate kinase (AK. E.C. 2.7.4.3.) catalyses the reaction 2ADP ATP + AMP. and is known as a heat stable protein constituent of skeletal muscle. Electrophoretic variation of AK has been reported in the pika Ochotona r. rufescens (Vergnes et al. 1974). the teleostean fish Zoarces viviparus (Frydenberg & Si-monsen, 1973), the mussel Mvtilus edulis (Ahmad et al. 1977). and the tunicate Ciona intestinalis (Schmidtke & Engel. 1980). In this note, individual variation of AK in muscle extracts of the fresh-water fish Misgurnus anguillicaudatus is described.
Loach were collected in ponds or purchased from fish shops (Table 1 & Fig. 1). Three populations (OS. AS and KN) were purchased from fish shops in Osaka. Akashi and Kanazawa cities, respectively. Their exact sampling locations were not known. For reference, the locations of these cities are indicated by an open circle in Fig. i. Fish were collected and stored frozen at – 20 ^oC at the sampling time given in Table 1. Muscle extracts were prepared and examined in the period February-April 1981.The method for preparing muscle extract and-the starch gel electrophoretic procedures were the same as those reported previously (Kimura, 1976). The amine-citrate buffer system as described by Clayton & Tretiak (1972) was used. AK was stained by the method of Allendorf et al. (1977). After electrophoresis, an inhibition test was also performed by immersing the gel in 10^-3 M 5.5'-dithiobis-(2-nitrobenzoate) solution for 30 minutes at room temperature.
Under the electrophoretic condition used in the present study, all of the AK     

Haematological characterization of loach Misgurnus anguillicaudatus: comparison among diploid, triploid and tetraploid specimens

The purpose of this study was to determine whether diploid, triploid and tetraploid loach (Misgurnus anguillicaudatus) differed in terms of their main haematological and physiological characteristics. Diploid and tetraploid fish were produced by crossing of natural diploids (2n x 2n) and natural tetraploids (4n x 4n), respectively. Triploid fish were produced by hybridization between diploid males and tetraploid females. The blood cells were significantly larger in polyploids, and the volumetric ratios of erythrocytes and leucocytes (thrombocyte and neutrophil) in tetraploids, triploids and diploids were consistent with the ploidy level ratio of 4:3:2. No significant differences were observed in haematocrit among polyploids. The erythrocyte count decreased with increased ploidy level, while total haemoglobin, mean cell volume, mean cellular haemoglobin content, and mean cell haemoglobin concentration all increased with increase in ploidy level. Erythrocyte osmotic brittleness declined in polyploids so that polyploid erythrocytes were more resistant to osmotic stress than diploid ones. Overall, loach with higher ploidy levels showed evidence of some advantages in haematological characteristics.     

Genetically diversified populations in the loach Misgurnus anguillicaudatus inferred from newly developed microsatellite markers

The loach Misgurnus anguillicaudatus is a freshwater fish which include natural polyploids and clones besides most bisexually reproducing diploids. Recent genetic studies using allozymes differentiated the populations in northern region of Hokkaido from other populations in Japan. Here, we developed 23 new sets of polymerase chain reaction primers to detect polymorphic microsatellites loci and tested them in 20 individuals from Memanbetsu town (northern Hokkaido) and Kita village (southern Hokkaido). These markers can be potentially diagnostic for identification of two populations.     

Comparison of vertical and horizontal swimming behaviour of the weather loach Misgurnus anguillicaudatus

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.     

Geographical variation of lactate dehydrogenase and phosphoglucomutase in the loach Misgurnus anguillicaudatus.

One of the two loci controlling muscle lactate dehydrogenase (LDH) was shown to be polymorphic in the loach Misgurnus anguillicaudatus. The result of the survey on four populations suggests that the differences in the LDH gene frequencies are correlated with the geographical distribution of the loach. A fourth phosphoglucomutase allele (PGMD) was found in one of the loach populations examined.     

Ploidy manipulation using diploid sperm in the loach, Misgurnus anguillicaudatus: a review

This paper assesses the present state of the art of ploidy manipulation in the loach, Misgurnus anguillicaudatus (Teleoste: Cobitidae). Diploid sperm can be obtained from natural tetraploid individuals with four sets of homologous chromosomes. Using diploid sperm, various polyploids and androgenetic diploids have been produced. Cryptic clonal lineages are also recognized in wild populations of the loach. They produce unreduced diploid eggs genetically identical to somatic cells of the mother fish and most diploid eggs develop gynogenetically as a member of the clone. However, some eggs develop to triploid and/or diploid-triploid mosaic individuals by incorporation of sperm nucleus. Diploid-triploid mosaic males exclusively generate fertile diploid sperm with clonal genotypes. Such diploid sperm can also be obtained from artificially sex-reversed clonal individuals. Recent population studies suggested that Japanese M. anguillicaudatus might not be a single species, but a complex involving cryptic species, because wild populations exhibited genetic differentiation at interspecific level. This implies possible relationship between atypical reproduction and natural hybridization in the loach.     

Developmental stages and germ cell lineage of the loach (Misgurnus anguillicaudatus)

The staging of embryonic and larval development, and the germ cell lineage of the loach, Misgurnus anguillicaudatus, are described. Fertilized eggs were obtained by artificial insemination. For the convenience of detailed observation and photography of the external appearance, we use dechorionated embryos. Through a series of operations, these embryos were cultured at 20 degrees C in an incubator. Embryonic and larval development of the loach was divided into five periods: cleavage, blastula, gastrula, segmentation, and hatching. Stages were assigned within each of these periods. Developmental stages were determined and named by morphological features and somite number. The staging series were photographed and tabulated. The germ cell lineage was then elucidated by whole mount in situ hybridization of mRNA expression of the germ-cell-specific marker vasa and histological analysis. Primordial germ cells (PGCs) of the loach derived from the cleavage furrows of 8-cell stage embryos began proliferation in the late blastula period and migrated to the gonadal anlagen through a migration pathway similar to that of the zebrafish. However, it is characteristic of the loach that PGCs migrate a long distance and stay in the posterior part of the yolk-extension region.     

Genetic diversity and parentage assignment in Dojo loach,Misgurnus anguillicaudatus based on microsatellite markers

In this study, genetic diversity and structure of three Misgurnus anguillicaudatus populations from three different geographical locations in China (Hunan, Hubei and Henan province) were investigated using microsatellite markers. High level of genetic diversity of all three populations was revealed by expected heterozygosity (H_e), observed heterozygosity (H_o) and allele number. Significant genetic differentiations were found between all pairs of populations. The efficiency of eight microsatellite markers in parentage assignment of 540 progeny from twenty full-sib families was evaluated. Simulation based on allele frequency data demonstrated that probabilities of exclusion per locus range from 0.313 to 0.825 when no parent information is available and 0.504 to 0.904 when one parent is known. The assignment success rate based on the real data using eight markers was 96.85%. This study indicates that these M. anguillicaudatus resources are valuable genetic and breeding material for aquaculture and the microsatellite markers will be useful for investigation of genetic background and molecular marker-assisted selective breeding in this species.