Morphological features of mucous secretory organ and mucous secretion of loach Misgurnus anguillicaudatus skin for friction drag reduction

We examined the functional morphology of loach Misgurnus anguillicaudatus skin by using synchrotron X-ray micro-computed tomography (SR-μCT) and high-contrast staining using osmium tetroxide or phosphotungstic acid (PTA), which enhances the image contrast of soft tissues. The captured high-spatial resolution images revealed that the surface ornamentations were stuck in the basement membrane of the loach scales. The ornamentations consisting of grooves (radii) and ridges (circuli) that can move freely and bend flexibly. The cross-sectional lateral microstructures of flat, concave and convex loach skins were observed from a live image of loach skin obtained through dark-field optical coherence tomography (OCT) imaging. The thickness of loach skin was changed with varying empty space between the mucous-cell layer and the scales by bending motion of loach. In addition, through direct measurement of drag reduction of loach skin, the mucous layer was found to have a strong influence on the reduction of skin friction. The present results enhance the understanding of the functional morphologies of mucous layer of loach to secrete mucus for skin friction reduction.     

Comparative study of superoxide dismutase and glutathione peroxidase activity in embryos and skeletal muscles of adult fish

The activity of the antioxidant enzymes superoxide dismutase and glutathione peroxidase in loach and sturgeon embryogenesis as well as in red and white skeletal muscles of loach was studied. The specific activity of cytoplasmic and mitochondrial forms of superoxide dismutase in developing sturgeon embryos was higher than in loach embryos, which may be due to oxygen conditions under which these species develop in nature. A similar dependence was also observed for the activity of glutathione peroxidase in embryos of these fish species. A comparative study of specific superoxide dismutase activity in loach and sturgeon embryos and in loach skeletal muscles showed that the activity of cytoplasmic superoxide dismutase is maximum in red and white muscles and minimum in loach embryos, whereas the activity of the mitochondrial form of this enzyme is maximum in red skeletal muscles.     

Spermatozoa of the loach Misgurnus fossilis as a test system for identification of new centromere proteins

We studied the possibility of using the spermatozoa of the loach Misgurnus fossilis L. for identification of centrosome proteins. It has been shown that the centrosome of the loach spermatozoa consists of a pair of centrioles of the standard structure and contains the marker protein gamma-tubulin, cytoplasmic microtubules branch out from it, and it does not contain any additional structures characteristic of the centrosomes of spermatozoa of many other fishes. A preparation enriched with intact centrosomes has been obtained from the loach spermatozoa. These centrosomes contained gamma-tubulin although they lost their ability to induce polymerization of microtubules. The preparation of loach centrosomes was successfully used to obtain a set of monoclonal antibodies against the mammalian centrosome. A new protein kinase LOSTEK was identified with the help of one of these monoclonal antibodies, SN2-3D2, which was localized in the centrosome and on then microtubules in both loach spermatozoa and cultured mammalian cells. Hence, the loach spermatozoa are a promising object for identification of new proteins of the mammalian centrosome.     

Reproduction and fecundity of the golden loach, Sabanejewia baltica Witkowski, 1994 from Bug River in Poland

In this paper we report for the first time the results of histology of the golden loach (S. baltica) gonads (25 females and 8 males) and absolute fecundity of females from the Bug River during the reproductive season. The golden loach has an asynchronous ovary and spawns in batches. The absolute fecundity of the golden loach ranged from 1507 to 7220 eggs (3050±1377). We hypothesize that the golden loach spawns twice a year.     

Examination of unidentifiable spined loach individuals found in the overlap zone of two tetraploid species within a single river in Japan

An endangered tetraploid spined loach species, Cobitis takenoi (Cypriniformes: Cobitidae; hereafter called Tango loach) is known to inhabit only a single river in Kyoto Prefecture, Japan. Since Tango loach was discovered recently, in 2010, and only described in 2016, its morphology, ecology, and genetics are not well studied. Another tetraploid spined loach species Cobitis sp. BIWAE type A (hereafter, called Ohshima loach) inhabits the same river. The two loaches are reported as morphologically distinguishable from each other. Although the habitats of the two species in the river are segregated (Ohshima loach and Tango loach inhabit the upper and lower reaches, respectively), they overlap to a small degree in the boundary area. Recently, some individuals with morphological characteristics that are intermediate between the two species were found in the overlap zone. It was suspected that hybrids between the two species were produced since breeding seasons of the two species overlapped. To investigate whether the two species produce hybrids, we performed mitochondrial and nuclear DNA analyses on the unidentifiable individuals. Eight individuals unidentifiable to the species level collected in the river between 2017 and 2018 were examined and compared with the Tango and Ohshima loach species. Using mitochondrial DNA (mtDNA) cytochrome b analysis, we found that six individuals had mtDNA types identical to Tango loach and two individuals had mtDNA types identical to Ohshima loach. Furthermore, sequencing analysis of nuclear recombination activating gene 1 (RAG-1) revealed that each species had species-specific alleles. The phylogenetic analysis indicated that alleles in Tango loach were divided into two clusters and those from Ohshima loach formed a single cluster. There were no discrepancies in the combination between mtDNA and nuclear DNA species types within each specimen. DNA fingerprinting analysis (AFLP) showed that the species-unidentifiable individuals exhibited distinctly segregated genetic groups corresponding with Tango and Ohshima loaches. In summary, no hybrids were detected from among any unidentifiable individual examined in this study. New conventional genetic method for discriminating the two sympatric loach species developed here can be effective tool for the conservation of the Tango loach since there was no strict diagnostic morphological character between them.     

Distribution of the oriental weatherloach, Misgurnus anguillicaudatus, in paddy fields and its implications for conservation in Sado Island, Japan

The distribution of the oriental weatherloach, Misgurnus anguillicaudatus, together with related environmental factors were surveyed at 185 paddy field locations on Sado Island in Japan. This was associated with the reintroduction of the Japanese crested ibis, Nipponia nippon, which prefers to feed on the loach. Loach were found to be present at 90 locations (49%). Analysis with GIS, GLM and AIC revealed that positive factors for the loach distribution included the presence of an earth ditch, the connections at outlets and the proportion of paddy field area within an 800 m radius. Conversely, pumping-up water irrigation and flow irrigation affected loach distribution negatively. In an interview survey that was conducted concurrently, older farmers in the area recalled that the loach had once been distributed over almost the entire island (89/96, 93%) about half a century ago. The farmers also suggested that the impact of agricultural chemicals was one of the main reasons for loach reduction or local extinction. A map of the current potential loach distribution was made using the best fit model from GIS variables. The loach was expected to be distributed in the Kuninaka region with high probability (60–100%) and in patches in the Osado and Kosado regions with low–medium probability (10–50%). As a feasible scenario for the conservation of the loach, another predictive map of the loach distributions was made using a model fit based on the GIS variables and outlet connection, in which the all of the outlets were presumed to be connected to the ditches without gaps. In this case, the loach were expected to be distributed almost throughout the paddy fields with medium–high probability (40–100%). In another scenario that presumed the complete land consolidation of all the paddy fields, the probability of loach occurrence was low (0–30%) throughout the island with the exception of the island’s center in the Kuninaka region.     

Evidence of a secondary interspecific mitochondrial DNA introgression in the pond loach Misgurnus dabryanus (Teleostei: Cobitidae) population introduced in Japan

The pond loach Misgurnus dabryanus is a freshwater fish with a distribution range spanning the eastern part of the Asian continent, the Korean Peninsula, and Taiwan. The pond loach was transplanted to the Japanese archipelago through the co-inclusion with dojo loach (Misgurnus anguillicaudatus species complex) populations, which were imported live from China for food materials, and it is currently distributed widely across Japan. A previous mitochondrial DNA (mtDNA) analysis revealed that a pond loach population in Ehime Prefecture (Shikoku Island, Japan) included two highly diverged mtDNA groups (Groups I and II). To examine the origin of these two distinct forms of mtDNA within the Japanese pond loach population, we performed phylogenetic analyses using sequences based on the mtDNA of cytochrome oxidase b (cyt b) and the nuclear DNA recombination activating gene 1 (RAG-1). We also conducted a random amplified polymorphic DNA (RAPD) analysis to examine the establishment of reproductive isolation between sympatric pond loaches with two different mtDNA groups. Our mtDNA phylogenetic results indicated that the two diverged pond loach mtDNA sequences showed polyphyletic relationships among Misgurnus species and its related genus Cobitis. In contrast, there were no clear divergence in nuclear DNA among the pond loaches irrespective of their mtDNA groups, and they all formed monomorphic clades in the phylogenetic relationships among the species. The discrepancy between the mtDNA and nuclear DNA genes support that the existence of two diverged forms of DNA within the pond loach population could be attributed to past mtDNA introgressions from other species rather than convergent evolution. Previous mtDNA phylogenetic studies among Cobitidae revealed that the dojo loach also consisted of two genetically diverged polyphyletic clades: an original Misgurnus mtDNA and an introgressed mtDNA from Cobitis species. In our mtDNA result, the Group II haplotype of the pond loach was included in the mtDNA from the introgressed dojo loach. This suggested that the Group II haplotype was derived from introgressed dojo loach mtDNA. The close relationships between the introgressed dojo loach and the pond loach mtDNA indicated that this secondary introgression had recently occurred via hybridization in a recent artificial aquaculture or transportation process. Common RAG-1 alleles and RAPD bands were shared between the sympatric pond loaches with original and introgressed mtDNAs. This indicates that the introgressed mtDNA haplotype is included as one of the polymorphic genotypes within the pond loach populations, and does not represent existence of different cryptic species.     

Habitat factors affecting the distribution and abundance of the spinous loach <Emphasis Type="Italic">Cobitis shikokuensis</Emphasis> in southwestern Japan

We examined habitat factors related to the distribution and abundance of the spinous loach Cobitis shikokuensis, an endangered benthic fish, in the Shigenobu River system, southwestern Japan. In the study river, the spinous loach was distributed widely along the main stem, from headwater to near the mouth, whereas it was rarely found in tributary streams. Classification tree analysis showed that the presence/absence of spinous loach was explained by a combination of percent pebble and length of river fragment between artificial barriers. Spinous loach incidence was high in sites with abundant pebble (>27.7%), but low in sites with short river fragment (≤0.97 km) even if pebbles were abundant. A regression tree model for loach density retained only percent pebble as a single best predictor, with sites with higher percent pebble (>40.4%) having higher density. These results suggest that substrate condition is an important factor determining the distribution and abundance of spinous loach and also that habitat fragmentation by artificial barriers has great potential to threaten the spinous loach population in this river. Considering the highly fragmented situation of the study river and prevention of upstream migration by barriers, we conclude that maintenance of suitable habitats in upper reaches has high priority for conservation of the spinous loach.     

枯草芽孢杆菌对泥鳅养殖池塘水水质的改善研究

通过在泥鳅(Misgurnus anguillicaudatus Cantor)养殖水体中施加一定浓度梯度枯草芽孢杆菌(Bacillus subtili Cohns),测定了水化学指标并观察泥鳅生长情况。试验结果表明:枯草芽孢杆菌能够稳定养殖水体的pH值,对COD_Mn、TN和NO₃^--N降解极为显著,其降解速率分别为1.2、0.24和2.7g·L^-1·d^-1,TP在一定范围内波动;当菌液浓度大于149mL·L^-1时,随着浓度的增加,停食后的泥鳅体重变化减小,因此,枯草芽孢杆菌对泥鳅养殖池塘水水质改善效果明显。     

Spermatozoa of the Loach Misgurnus fossilis in the Identification of New Centrosome Proteins

We studied the possibility of using the spermatozoa of the loach Misgurnus fossilis L. in the identification of centrosome proteins. It has been shown that the centrosome of the loach spermatozoa consists of a pair of centrioles of the standard structure and contains the marker protein -tubulin, cytoplasmic microtubules branch out from it, and it does not contain any additional structures characteristic of the centrosomes of spermatozoa of many other fishes. A preparation enriched with intact centrosomes was obtained from the loach spermatozoa. These centrosomes contained -tubulin, although they had lost their ability to induce the polymerization of microtubules. The preparation of loach centrosomes was successfully used to obtain a set of monoclonal antibodies against the mammalian centrosome. A new protein kinase LOSTEK was identified with the help of one of these monoclonal antibodies, SN2-3D2, which was localized in the centrosome and then on microtubules in both loach spermatozoa and cultured mammalian cells. Hence, the loach spermatozoa are a promising means to identify new proteins in the mammalian centrosome.     

Spatial and ontogenetic variability in the microhabitat use of stream-dwelling spined loach (Cobitis taenia) and stone loach (Barbatula barbatula)

Ontogenetic and spatial variability in microhabitat use of spined loach Cobitis taenia (Linnaeus), considered as one species for the purposes of this study, and stone loach Barbatula barbatula (Linnaeus) were examined in the River Great Ouse basin, England, using multivariate and habitat suitability methods, including a technique for handling spatial variation in collections of preference curves. Distinct ordinations of spined age classes and stone loach developmental stages, respectively, in canonical correspondence analysis of species × variables × samples relationships suggest that the two species occupy completely different microhabitats; however, young‐of‐the‐year spined loach occurred more often than expected with all developmental stages of stone loach except young larvae. Water velocity and filamentous algae were the most influential microhabitat variables, the latter decreasing in importance with increasing age of both fish species. Preferred water velocities generally decreased with age in spined loach and increased in stone loach, with substratum size generally increasing with fish age in both species. Spatial variation in microhabitat preferences was great in both species but less so in the spined loach, suggesting that limited plasticity in habitat use could account, at least in part, for the latter species’ limited distribution and abundance in the catchment. Preference curves for a species, if generated and verified for all life intervals and all seasons, could be used as a management tool for a given stream or sector of river basin. But preference curves should be generated for each location to ensure that river management decisions with regard habitat and species conservation consider local‐level species requirements. Thus, a multi‐(eco)species and multi‐scale approach is required in habitat suitability assessments.     

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.     

Detection of hybridization between two loach species (<Emphasis Type="Italic">Paramisgurnus dabryanus</Emphasis> and <Emphasis Type="Italic">Misgurnus anguillicaudatus</Emphasis>) in wild populations

Artificial interspecific hybrids between large scale loach P. dabryanus and tetraploid pond loach M. anguillicaudatus (Cobitidae, Cypriniformes) are viable. To detect the occurrence of possible natural hybridization, genetic analyses by using microsatellite markers were performed for natural populations of large scale loach and pond loach, the reciprocal laboratory hybrids, and “supposed hybrids” with ambiguous morphology. The fertility of the artificial hybrids was also tested. At one diagnostic microsatellite (Mac50), one out of 20 “supposed hybrids” was identified to be F₁ hybrid between the two loach species because it had the same genotype as that of the laboratory hybrids. The triploid hybrids between the two species were confirmed to be female-sterile. The results show that rare hybridization has occurred between diploid large scale loach and tetraploid pond loach in nature although it may have little effect in genetic introgression. This study is helpful for fish conservation and encourages further investigation on natural hybridization and introgression of loaches.     

The Loach as an object of experimental embryological studies at the Department of Embryology

This paper provides a history of research at the Department of Embryology on the teleostean loach fishMisgurnus fossilis L. as a model. The use of the loach in education is briefly described.     

Environmental DNA analysis provides an overview of distribution patterns of two dojo loach species within the Naka-ikemi Wetland,Fukui Prefecture,Japan

Ichthyological Research - In the Naka-ikemi Wetland, a cryptic species of the dojo loach, Misgurnus sp. Type I (called Type I loach herein), inhabits along with the common nominal species,...     

Size‐related and diel variations in microhabitat use of three endangered small fishes in a Mediterranean coastal stream

This study analysed the microhabitat use of three endangered fish species, Andalusian toothcarp Aphanius baeticus , Iberian loach Cobitis paludica and sand smelt Atherina boyeri , in a coastal stream stretch. Plastic minnow traps were set both during daytime and at night on the bottom and at the surface. Depth and presence of effective refuge were recorded for each trap. To assess size‐related changes in microhabitat use individuals of each species were classified in three size classes. The three species preferentially used bottom positions in the water column, though this behaviour was more evident in the case of Iberian loach. While large Iberian loach remained active at night Andalusian toothcarp and sand smelt were strongly diurnal, especially larger individuals. The three species showed a clear ontogenetic change in microhabitat preferences towards deeper waters. Small Andalusian toothcarp and medium‐sized Iberian loach used deeper microhabitat in the presence of refuge. Large Andalusian toothcarp consistently preferred exposed microhabitat at any time. Andalusian toothcarp using refuge were smaller at any time, while Iberian loach followed this pattern only at night. The differential vulnerability of these species to different predators (aerial and aquatic; diurnal and nocturnal) could explain the observed patterns in microhabitat use. Fish tended to co‐occur in microhabitats either due to habitat characteristics independently of species or due to species independently of habitat. Andalusian toothcarp segregated spatio‐temporally from sand smelt and Iberian loach, but these species occurred independently of each other. According to these co‐occurrence patterns, Andalusian toothcarp would be more sensitive than Iberian loach or sand smelt to interspecific interactions.     

Production of a mutant of large-scale loach Paramisgurnus dabryanus with skin pigmentation loss by genome editing with CRISPR/Cas9 system

CRISPR/Cas9 system has been developed as a highly efficient genome editing technology to specifically induce mutations in a few aquaculture species. In this study, we described induction of targeted gene (namely tyrosinase, tyr) mutations in large-scale loach Paramisgurnus dabryanus, an important aquaculture fish species and a potential model organism for studies of intestinal air-breathing function, using the CRISPR/Cas9 system. Tyr gene in large-scale loach was firstly cloned and then its expressions were investigated. Two guide RNAs (gRNAs) were designed and separately transformed with Cas9 in the loach. 89.4% and 96.1% of injected loach juveniles respectively displayed a graded loss of pigmentation for the two gRNAs, in other words, for target 1 and target 2. We classified the injected loach juveniles into five groups according to their skin color phenotypes, including four albino groups and one wild-type-like group. And one of them was clear albino group, which was of high ornamental and commercial value. More than 50 clones for each albino transformant with a visible phenotype in each target were randomly selected and sequenced. Results obtained here showed that along with the increase of pigmentation, wild-type alleles appeared in the injected loach juveniles more often and insertion/deletion alleles less frequently. This study demonstrated that CRISPR/Cas9 system could be practically performed to modify large-scale loach tyr to produce an albino mutant of high ornamental and commercial value, and for the first time showed successful use of the CRISPR/Cas9 system for genome editing in a Cobitidae species.

     

Spatial distribution of a single river endemic allotetraploid spined loach,Cobitis takenoi,in comparison with a co-inhabiting tetraploid congener species

Ichthyological Research - Cobitis takenoi (hereinafter, Tango loach), an endangered species of tetraploid spined loach, is known to inhabit only a limited area of the Noda River in Kyoto...     

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.