cDNA cloning and expression characterization of serum transferrin gene from oriental weatherfish Misgurnus anguillicaudatus

In this study, the cDNA coding serum transferrin (stf) of Misgurnus anguillicaudatus (mastf) was cloned. mastf cDNA is composed of 2326 bp with a 2007 bp open reading frame encoding 668 amino acids. The deduced Mastf protein consists of a signal peptide, two lobes (N and C‐lobes) and signature motifs of transferrin (Tf) family. The results of tissue distribution indicated that mastf mRNA was predominantly expressed in the liver. The results indicate that the mastf expression increased significantly in liver, blood, spleen and head kidney after the challenge with Aeromonas sobria, acting as a positive acute protein, suggesting that mastf is related to the immune response. The cloning and expression analysis of mastf further demonstrates the evolutionary conservation of Stf and immune function in vertebrates.     

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.     

Land-use change and irrigation systems in the agricultural landscape of terraced paddy fields in Awaji Island, central Japan

We analyzed changes in land use from 1963 to 2000 in the northern part of Awaji Island in central Japan, using topographical maps and aerial photographs. We compared these changes between two different irrigation systems: tazu, in which a community group jointly owns the rights to a water source, and private management. The study area consisted of 57.3% paddy field, 22.2% woodland, 7.2% urban land use, 6.3% grassland, 5.8% water body, and 1.2% dry field in 1963. In 2000, the area consisted of 44.3% paddy field, 21.0% woodland, 12.6% urban land use, 11.6% grassland, 5.3% water body, and 5.0% dry field. The proportion of agricultural use had decreased markedly over the 37-year period. In the tazu area, 38.1 and 37.7% of former paddy field had changed into urban land use and grassland, respectively, and 19.3% of former paddy field had become dry field by 2000. In contrast, 30.6% of former rice paddy had become dry field in the private management area. About half of the former rice paddy had changed into grassland (26.3%) and urban land use (24.0%) in the private area, and 18.6% had become woodland in 2000. In the tazu area, there were fewer abandoned fields, because farmers still manage their fields according to strict regulations. In the private management area, the set-aside or abandoned fields had changed into woodlands as a result of vegetation succession, because farmers had abandoned fields located far from their homes or on steep slopes. These results suggest that the irrigation system may influence the pattern of change in land use.     

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)

This 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.     

泥鳅性腺发生和分化的组织学研究

通过人工繁殖技术获得泥鳅幼体,采用石蜡显微切片技术对幼体性腺发生和分化的组织学特征进行了系统观察.结果表明泥鳅性腺发生于出膜后12 d,40日龄卵巢开始分化,至55日龄卵巢分化完全;精巢于出膜后55 d左右开始分化,100 d左右分化完全.卵巢分化早于精巢.从性腺分化开始,将要发育为卵巢的性腺还表现为体积快速增大,向体腔中间靠拢,横截面变宽;而将要发育为精巢的性腺则呈两端尖中间稍突的梭形,增生并不明显.这些特征可能与雌雄性腺的发育及生殖细胞的分化速度有关,可以作为泥鳅性腺早期分化的形态特征.     

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.     

Molecular cloning and expression analysis of myd88 from oriental weatherfish (Misgurnus anguillicaudatus) in response to bacterial challenge

Myeloid differentiation factor 88 (Myd88) plays an important role in both innate and adaptive immune response. In this study, the full-length complementary DNA (cDNA) of myd88 from Misgurnus anguillicaudatus was characterized. The myd88 cDNA is 1333 bp in length and contains an 855 bp open reading frame encoding a predicted protein of 284 amino acids. The predicted protein possesses typical Myd88 domain structural features including a death domain in the N-terminus, and box 1, 2, and 3 motifs of the Toll/IL-1 receptor domain in the C-terminus. Quantitative real-time PCR (qRT-PCR) revealed that myd88 messenger RNA (mRNA) was ubiquitously expressed in all examined tissues, especially highly in brain, kidney, blood, intestines and liver. qRT-PCR and western blotting were used to determine the mRNA and protein levels of Myd88 after Aeromonas veronii challenge, respectively. The Myd88 was remarkably upregulated in response to infection of A. veronii. These results suggested that Myd88 may play a vital role during the immune response of M. anguillicaudatus against bacterial infection.     

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.).     

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.     

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”.     

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     

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.     

Population genetic structure,diversity and stocking effect of the oriental weatherloach (<Emphasis Type="Italic">Misgurnus anguillicaudatus</Emphasis>) in an isolated island

Genetic endemism of island organisms and the threat to such organisms provided by artificially introduced genes are aspects of major interest in evolutionary and conservation studies of fishes. In this paper the genetic population structure of the oriental weatherloach, Misgurnus anguillicaudatus, in Sado Island of Japan was elucidated by phylogeographic analysis based on partial mitochondrial control region sequences. The specimens were sampled at 62 sites in Sado Island and 14 sites on the mainland close to the island. We found various haplotypes of different origins, most of which had already been reported from the mainland and other places of Japan. This suggests that the loach has been historically introduced to the island from various regions of Japan. Of the 62 sites on the island, cultured/nonnative individuals were confirmed to have been stocked at eight specific sites for feeding of re-introduced Japanese crested ibis (Nipponia nippon). By a Mantel test, geographical and genetic distances were not significantly correlated among the local populations in Sado Island. However a significant correlation was found when the eight stocked local populations were excluded from the analysis. This implied that the genetic distribution pattern of the loach on the island has been disturbed by the stocking. In addition, the nucleotide diversity values of stocked local populations were significantly higher than those of other local populations, also a likely outcome of the stocking. In conclusion, the loach on the island likely had their origins in multiple historical introductions and colonizations, where more recent stocking for the ibis has caused further genetic disturbance to their local populations.     

间苯二酚与邻苯二酚对泥鳅血清卵黄蛋白原的诱导作用

为探讨间苯二酚(m-dihydroxybenzene)与邻苯二酚(o-dihydroxybenzene)对雄性泥鳅Misgurnus anguillicau-datus血清卵黄蛋白原(Vitellogenin,Vtg)的诱导作用,将雄性泥鳅分别暴露于4种不同质量浓度间苯二酚(5.287mg/L、6.531mg/L、8.072mg/L、10.000mg/L)和邻苯二酚(16.708mg/L、17.742mg/L、18.836mg/L、20.00mg/L)中持续7d、14d、21d和28d,采用碱不稳定性蛋白结合磷法检测血清Vtg水平的变化。结果显示,与对照组相比,泥鳅在低质量浓度间苯二酚(5.287mg/L)和邻苯二酚(16.708mg/L)中暴露7d时,血清Vtg水平均有极显著升高,并且随着暴露浓度的增加,Vtg水平逐渐升高;在同一暴露浓度下,Vtg水平随暴露时间的延长逐渐升高;2种酚类化合物相比,暴露在间苯二酚中的泥鳅血清Vtg水平高于邻苯二酚。上述结果说明,2种化合物均可诱导雄性泥鳅肝脏合成Vtg,具有一定的环境雌激素效应,其诱导作用随着暴露剂量的增大和时间的延长逐渐增强。     

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.     

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.     

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.