Chinese Loach Research Articles

Re-description of the loach species Homatula variegata (Dabry de Thiersant, 1874) (Pisces: Nemacheilidae) from the middle Huang-He basin in Shaanxi Province of Central China.

Despite the wide recognition of Homatula variegata (Dabry de Thiersant, 1874) as a valid Chinese loach, its type locality, identity and distribution still remain contentious. A molecular phylogenetic analysis based on the mitochondrial cytochrome b (cyt b) gene for samples from all known range of the species showed that three distinct species are involved. Morphological comparisons, coupled with examination on the types, confirmed that H. variegata s. str. is characterized by having a sparsely scaled predorsal body, an adipose crest along the dorsal midline of the caudal peduncle that extends forward to the vertical through the posterior end of the anal-fin base and a broadly rounded caudal fin. It is found merely in the Wei-He of the Huang-He basin. The Jinsha-Jiang population, previously misidentified as H. variegata, represents a distinct species, for which Homatula oxygnathra is the available name. Homatula laxiclathra, endemic to the Wei-He, is also a valid species distinct from H. variegata.

Cloning, characterization and function analysis of DAX1 in Chinese loach (Paramisgurnus dabryanus).

The mechanisms of sex determination and differentiation have not been elucidated in most fish species. In this study, the full-length cDNAs of DAX1 was cloned and characterized in aquaculture fish Chinese loach (Paramisgurnus dabryanus), designated as Pd-DAX1. The cDNA sequence of Pd-DAX1 was 1261bp, including 795bp open reading frame (ORF) encoding 264 amino acids. Pd-DAX1 shares highly identical sequence with DAX1 homologues from different species. The expression profiles of Pd-DAX1 in different developmental stages and diverse adult tissues were analyzed by quantitative real-time RT-PCR and in situ hybridization (ISH). Pd-DAX1 was continuously expressed during embryogenesis, with the extensive distribution in the development of the central nervous system. Tissue distribution analysis revealed that Pd-DAX1 expressed widely in adult tissues, with the highest expression level found in testis, moderate level in ovary, showing a sex-dimorphic expression pattern. Pd-DAX1 mainly located in spermatogonia cells, spermatocytes, primary oocytes and previtellogenic oocyte cells, implying that Pd-DAX1 may involve in gametogenesis. These preliminary findings suggest that Pd-DAX1 gene is highly conserved during vertebrate evolution and involved in a wide range of developmental processes including embryogenesis, central nervous system development and gonad development.

Molecular cloning, characterization, and expression profiles of the Sox3 gene in Chinese loach Paramisgurnus dabryanus.

A number of studies have established that in vertebrates, Sox3 is involved in a wide range of developmental processes, including sex differentiation and neurogenesis. However, the exact functions of the Sox3 gene have not been documented so far in teleosts. Here, we cloned the full length cDNA of Sox3 from the teleost fish, Paramisgurnus dabryanus, which we designated PdSox3. Sequence analysis revealed that PdSox3 encodes a hydrophilic protein, and shares high homology with Sox3 in other species, ranging from mammals to fishes. Quantitative real-time reverse transcription PCR, and in situ hybridization showed that PdSox3 is consistently expressed during embryogenesis, mainly localized in the developing central nervous system. Tissue distribution analyses revealed that PdSox3 is abundant in the adult brain, especially in particle cell layer. Furthermore, PdSox3 expression was higher in gonads, in primary spermatocyte cells, primary oocytes, and previtellogenic oocyte cells. All of these results suggest that PdSox3 plays an important role in early embryonic development, in particular the formation and development of the nervous system, and gonad development, similarly to other vertebrates. This is the first report describing Sox3 gene expression from this species, and the results are necessary to provide fundamental information on both the functional and evolutionary role of Sox3 across different species.

Changes to tissue glutamine content, glutamine synthetase, and glu tamate dehydrogenase activities during ammonia and aerial exposure in Chinese loach ( Paramisgurnus dabryanus )

Changes to tissue glutamine content, glutamine synthetase, and glu tamate dehydrogenase activities during ammonia and aerial exposure in Chinese loach ( <i>Paramisgurnus dabryanus</i> )

Changes of ammonia, urea contents and transaminase activity in the body during aerial exposure and ammonia loading in Chinese loach Paramisgurnus dabryanus.

The Paramisgurnus dabryanus was exposed to 30mmolL-1 NH4Cl solution and air to assessing the change of body ammonia and urea contents and the activities of alanine aminotransferase (ALT) and aspartate transaminase (AST). After 48h of ammonia exposure, ammonia concentration in the plasma, brain, liver and muscle were 3.3-fold, 5.6-fold, 3.5-fold and 4.2-fold, respectively, those of the control values. Plasma, brain, liver and muscle ammonia concentrations increased to 2.2-fold, 3.3-fold, 2.5-fold and 2.9-fold, respectively, those of control values in response to 48h of aerial exposure. Within the given treatment (ammonia or aerial exposure), there was no change in plasma, brain and liver urea concentrations between exposure durations. The plasma ALT activity was significantly affected by exposure time during aerial exposure, while the liver ALT activity was not affected by ammonia or aerial exposure. Exposure to NH4Cl or air had no effect on either plasma or liver AST activity. Our results suggested that P. dabryanus could accumulate quite high level of internal ammonia because of the high ammonia tolerance in its cells and tissues, and NH3 volatilization would be a possible ammonia detoxification strategy in P. dabryanus. Urea synthesis was not an effective mechanism to deal with environmental or internal ammonia problem. The significant increase of ALT activity in plasma during aerial exposure, indicating that alanine synthesis through certain amino acid catabolism may be subsistent in P. dabryanus.

Aneuploid progenies of triploid hybrids between diploid and tetraploid loach Misgurnus anguillicaudatus in China.

Triploid Chinese loach, Misgurnus anguillicaudatus, hybrids between tetraploids from Hubei Province and diploids from Liaoning Province were mated with either diploid wild-type or triploid hybrids to analyze viability and ploidy of the resultant progenies. Both triploid males and females generated fertile gametes, but progenies from the crosses using gametes of triploid hybrids did not survive beyond the larval stages. In crosses between wild-type diploid females and triploid hybrid males, embryos ranging from 2.2n to 2.6n were predominant with a mode of either 2.4n (chromosome numbers 59, 60, 61) or 2.5n (chromosome numbers 62, 63). Those from the crosses between triploid hybrid females and diploid males gave a modal ploidy level at approximately 2.5n in one case, but a shift to a higher ploidy level was observed in other embryos. In the progenies between triploid hybrid females and males, the ploidy level at approximately 3.0n (chromosome numbers 74, 75, 76) was most frequent. The cytogenetic results of the progenies suggest the production of aneuploid gametes with a modal ploidy level at approximately 1.5n in triploid hybrids. However, a shift to higher chromosome numbers in gametes was observed in certain cases, suggesting the involvement of mortality selection of gametes and/or zygotes with lower chromosome numbers.

Morphological changes and allometric growth in hatchery-reared Chinese loach Paramisgurnus dabryanus (Dabry de Thiersant, 1872)

The length-weight relationship and allometric growth patterns of hatchery-reared Chinese loach, Paramisgurnus dabryanus (Dabry de Thiersant, 1872), were determined from hatching to 60 days after hatching. A best power model was estimated for the length-weight relationship during the early life stages. Positive allometric growth for the head segment, trunk length, tail length and eye diameter was also found in the early life stages, while body depth, tail depth, tail fin length, pectoral fin length and barbel length displayed a negative coefficient. During the subsequent early developmental stage, the growth coefficients showed a clear and common tendency towards isometry for all measured body ratios. The allometric growth changes in Chinese loach during the early stage are possibly the result of selective organogenesis directed towards survival priorities.

Changes in digestive enzyme activities during larval development of Chinese loach Paramisgurnus dabryanus (Dabry de Thiersant, 1872).

The digestive physiology of Chinese loach (Paramisgurnus dabryanus) was studied by assessing the specific and total activities of different pancreatic (trypsin, chymotrypsin, amylase and lipase), gastric (pepsin) and intestinal (alkaline phosphatase and leucine-aminopeptidase) enzymes from hatching to 40 days after hatching (DAH). Larvae were reared at 24.4 ± 0.4 °C and fed with rotifers from mouth opening (4 DAH) to 15 DAH, from 10 to 35 DAH with Cladocera and from 30 to 40 DAH with compound diet. Enzyme activities for trypsin, chymotrypsin, amylase and lipase were detected before the onset of exogenous feeding, indicating that these enzymes were genetically pre-programmed. Most of the pancreatic enzyme specific activities increased until 20 DAH and decreased thereafter. The pepsin activity of Chinese loach was firstly detected at 30 DAH, indicating the appearance of functional gastric gland. Alkaline phosphatase specific activity was detected from hatching onward, showed marked increase and reached the second peak at 20 DAH, while a gradual increase in specific leucine-aminopeptidase activity was observed until the end of the experiment. Accordingly, the larvae of Chinese loach possess a functional digestive system before the onset of exogenous feeding and the digestive capacity gradually increases as development progresses. The abrupt increase in intestinal enzyme activities between 10 and 20 DAH demonstrates onset of juvenile-like digestive mode in Chinese loach larvae. The increase in pepsin activity after 30 DAH indicates the shift from alkaline to acidic digestion in Chinese loach larvae, which may be considered as the onset of weaning.

Ontogenetic changes in RNA, DNA and protein contents of Chinese loach, Paramisgurnus dabryanus (Dabry de Thiersant, 1872), larvae and juveniles

The changes in nucleic acid-based indices and protein variables of Chinese loach, Paramisgurnus dabryanus, larvae and juveniles from hatching to 60 days after hatching (DAH) were conducted to assess its growth potential. The nucleic acid contents were analysed using a UV-based method (n = 3, rearing temperature 24.4 ± 0.4°C, dissolve oxygen 7.1 ± 0.5 mg L−1, pH 7.9 ± 0.4). Ribonucleic acid (RNA) concentration significantly decreased from 2 to 5 DAH, then increased rapidly until 10 DAH, declining slightly thereafter. Deoxyribonucleic acid (DNA) concentration increased 2–5 DAH, decreased until 9 DAH, slightly increased again around 26 DAH, and then declined to a relatively stable level. Both RNA-DNA and protein-DNA ratios showed a statistically obvious relationship with growth rates. A significantly positive relationship was found between RNA-DNA ratio and growth rates during the early life stage of Chinese loach. According to the results, growth of Chinese loach is characterized by rapid hyperplasia from hatching through completion of the yolk-sac stage followed by continued rapid hyperplasia combined with increasing hypertrophy after feeding commences. The stage preceding 17 DAH of Chinese loach P. dabryanus is presumed to be critical for its survival and growth at 24°C.

Identification and molecular characterization of an Akirin2 homolog in Chinese loach (Paramisgurnus dabryanus)

Akirin is a nuclear factor involved in innate immune responses of arthropods and mammals. In this study we have cloned an Akirin2 gene, pdakirin2, from freshwater Chinese loach (Paramisgurnus dabryanus) and characterized its biological functions. Phylogenetic analysis revealed deduced PdAkirin2 had high sequence identities to Akirin2 homologs from fish and mammals (70−91%), it contained two conserved nuclear localization signals (NLSs) with verified sub-cellular localization. Quantitative real-time (qRT)-PCR analysis indicated that PdAkirin2 was present in a wide range of loach tissues and showed up-regulation with challenges of Aeromonas hydrophila NJ-1, LPS and poly I:C. PdAkirin2 as an immune factor had significant effects on the expression of cytokines (TNFα, IFN-α, IFN-γ, IL-4 and IL-1β) and transcription factor NF-κB. This study provides insights into the potential role of PdAkirin2 in the innate immune system.

Identification, expression and bioactivity of Paramisgurnus dabryanus β-defensin that might be involved in immune defense against bacterial infection

β-defensins are a large family of multi-disulfide-bonded peptides with broad-spectrum antimicrobial activities that contribute to innate host defense in many organisms, but little information is available about β-defensins produced by freshwater fish lacking scales. We therefore cloned and identified a β-defensin gene from Chinese loach (Paramisgurnus dabryanus) by designing degenerate primers and using thermal asymmetric interlaced PCR. This gene is the first defensin gene ever identified in a non-scaled freshwater fish. Annotation of the protein domain architecture showed that the putative Chinese loach β-defensin contains the signature motif of six conserved cysteines within the mature peptide, an aspect similar to β-defensins of other marine fish. We also used quantitative real-time PCR to investigate the expression pattern of the Chinese loach β-defensin gene, mRNA of which could be observed in various tissues. After challenge with the pathogenic bacterium Aeromonas hydrophila, β-defensin expression was induced in the eye, gill, skin, and spleen of the adult loach. The bioactivity of the recombinant P. dabryanus β-defensin was examined against pathogenic bacteria, and the results suggest that this class 2 β-defensin has potential applications for treatment of bacterial infections.

Determination and comparison of two complete mitochondrial genomes of golden Chinese loach, Sinibotia superciliaris (Teleostei, Cypriniformes)

The golden Chinese loach, Sinibotia superciliaris (Günther, 1892) is an endemic fish in China, which is only distributed in the main river and tributaries of the upper and middle Yangtze River. In this study, we sequenced two mitochondrial genomes simultaneously from two samples of S. superciliaris collected from two different locations. Both mitogenomes were 16,572 bp in length, showing a typical vertebrate mitogenome feature with 13 protein-coding genes, 22 transfer RNAs, 2 ribosomal RNAs, and 1 major non-coding control region (CR). Alignment and comparative analyses showed that the two mitogenome sequences possessed 22 mutation sites, all of which were transitions and mainly occurred in protein-coding genes (13/22) and CR (7/22). Among the 13 mutation sites that occurred in protein-coding genes, three mutation sites occurred in the first codon position and the remaining 10 sites occurred in the third codon position. However, only one mutation that occurred in the first codon position resulted in non-synonymous substitution in COI gene. The molecular data presented in this study will contribute to further phylogenetic analyses for this group of fishes.

Induction of viable gynogenetic progeny using eggs and UV-irradiated sperm from the Chinese tetraploid loach, Misgurnus anguillicaudatus

When eggs from the Chinese tetraploid loach that had 100 chromosomes were fertilized with UV-irradiated sperm, we obtained viable gynogenetic progeny without any additional treatment for the duplication of maternal chromosomes, which survived beyond first feeding towards adult stage of development. Gynogenetic progeny were determined to be diploid since they possessed 50 chromosomes, along with two chromosomes bearing nucleolar organizing regions (NORs), detected by silver nitrate staining (Ag-NORs), chromomycin-A3 (CMA3)-positive sites and fluorescence in situ hybridization (FISH) signals for rDNA loci. In contrast, when gynogens were induced using eggs from diploid loach fertilized by UV-irradiated sperm, but without chromosome doubling, we found that all resultant progeny were non-viable haploid gynogens with 25 chromosomes, along with one NOR-bearing chromosome detected by Ag-NORs, CMA3 and FISH. These observations demonstrate the true genetic tetraploid nature of the Chinese loach possessing 100 chromosomes, and the potential use of this tetraploid as a source of functional diploid gametes for further ploidy manipulation experiments.

Effects of cadmium, 17β-estradiol and their interaction in the male Chinese loach (Misgurnus anguillicaudatus)

To assess the interaction effect between cadmium (Cd) and 17β-estradiol (E2), male Chinese loaches (Misgurnus anguillicaudatus) were exposed to E2 (1 μg/L) and Cd (50 or 500 μg/L) alone and in combination using a semi-static waterborne exposure system. The effects of E2 on the accumulation and distribution of Cd, as well as the effects of Cd on vitellogenin (Vtg) synthesis induced by E2, were investigated. Cd mainly accumulated in the kidneys, liver, intestines, and gills, with little amounts in muscles, bones, and gonads. Co-exposure with E2 did not change the main targets for Cd. E2 could induce Vtg synthesis in male Chinese loaches, and co-exposure with 50 or 500 μg/L Cd could inhibit the Vtg induced by 1 μg/L E2. Compared with the results reported in the literature, it can be concluded that factors such as fish species, Cd dosage, and manner of exposure might make contributions to the interaction between Cd and E2. Our results also suggested that male Chinese loaches are susceptible to Cd, and can be recommended as a potential sentinel species to study the ecotoxicology of heavy metals.

Ecotoxicological effects of mixed pollutants resulted from e-wastes recycling and bioaccumulation of polybrominated diphenyl ethers in Chinese loach (Misgurnus anguillicaudatus)

To understand potential ecotoxicological effects of electrical and electronic equipment waste (e-waste) recycling and polybrominated diphenyl ethers (PBDEs) bioaccumulation in loaches, a semi-field experiment using Chinese loach (Misgurnus anguillicaudatus) as experimental fish was performed. Larval loaches were kept in net-cage for three months in an e-wastes recycling site and a reference site in Southeastern China. There was significant difference of the survival rate between the loaches from the e-wastes recycling site (27%, 19/70) and from reference site (70%, 49/70). Histopathological responses were also found in all the livers examined in loaches from the e-wastes recycling site. These results showed that mixed pollutants resulted from e-wastes recycling led to ecotoxicological effects on loaches. The bioaccumulation of polybrominated diphenyl ethers (PBDEs), the main pollutants in e-waste, in loaches was also studied, the mean concentration of total PBDEs in sediment was 6726.17 ng/g wet weight and in water samples was 4.08 ng/L (dissolved phase). BDE 209 was the dominant congener in sediment and with relatively high concentration in water. Relatively low concentration of BDE 209 (less than 0.01% of total PBDEs) and high concentration of BDE47 (up to 39.34% of total PBDEs) were detected in loaches.

Interacting effects of tributyltin and 17β‐estradiol in male Chinese loach (Misgurnus anguillicaudatus)

In this study, male Chinese loaches in a semistatic waterborne exposure system were used to study the effects of tributyltin (TBT) on vitellogenin (Vtg) production induced by 17beta-estradiol (E2), TBT accumulation and distribution in tissues, and the effects of E2 on the distribution of TBT. The results demonstrate that TBT does not induce the synthesis of Vtg in male Chinese loach and that TBT could significantly inhibit Vtg production induced by E2, after exposure to binary mixtures of E2 and TBT for 14 days. TBT was found to accumulate in the liver, kidney, gills, intestines, and muscles of male Chinese loach, wherein the liver, kidney, gills, and intestines were found to have the most TBT accumulation. The existence of E2 did not significantly affect tissue distribution of TBT.

Circannual vitellogenin levels in Chinese loach (Misgurnus anguillicaudatus)

To improve the use of the Chinese loach, Misgurnus anguillicaudatus, as a sentinel species for environmental investigations, normal ranges of plasma vitellogenin (Vtg) was studied. Male and female loaches were collected every 2 months from November 2004 to September 2005, and a specific and sensitive competitive enzyme-linked immunosorbent assay (ELISA) was used to determine plasma Vtg levels. Our data indicated that the average maximum Vtg level of female loaches (757 ± 356 µg mL−1) appeared in March, approximately 1 month before spawning. Female plasma Vtg significantly correlated with gonadosomatic index (GSI) which indicated that female plasma Vtg can be used as an indicator of the reproductive stages of the ovary. Low detectable plasma Vtg levels was detected in a number of male loaches, and the highest average plasma Vtg concentration attained to the level of µg mL−1; after exposure to estrogenic compounds by waterborne, injection or oral food, Vtg can be induced in male fish, so the presence of Vtg in male loaches might be attributed to the ingestion of estrogenic substances in food, the contamination of estrogenic compounds from the living environment, or the estrogen used by the local fish breeders to accelerate the rate of growth during artificial culture. The results indicate that Vtg levels in male and female Chinese loaches will be very helpful in field studies which use Chinese loach as a sentinel species.

Vitellogenic responses of male Chinese loach (Misgurnus anguillicaudatus) exposed to the individual or binary mixtures of 17β-estradiol and nonylphenol

Male Chinese loaches were exposed to 17β-estradiol (E2) and nonylphenol (NP) both singly and in combination for 42 days using semi-static waterborne exposure system. Plasma vitellogenin (Vtg) was chosen as determining endpoint. The results demonstrated that 0.5 μg/L E2 could induce the enhancement of Vtg contents in male Chinese loaches after exposure for 21 days, which showed a time-related increasing manner; NP was also estrogenic to male Chines loach, and the vitellogenic responses showed in a time-and dose-related manner, which was less potent than that of E2. The binary mixtures of E2 and NP can significantly elicit the production of Vtg in male Chinese loaches, which was more potent than that of individual compounds, and Vtg induced in the binary mixture groups was higher than the summation of Vtg induced in the corresponding single-compound groups at the same concentration.

Vitellogenic effects of 17β-estradiol in male Chinese loach ( Misgurnus anguillicaudatus)

Male Chinese loaches ( Misgurnus anguillicaudatus) were exposed to 17β-estradiol (E 2) in the ambient water at nominal concentrations of 0.5, 1, 5 and 10 μg/L continuously for 8 weeks using a semi-static system. The gonadosomatic index (GSI), total plasma protein, total plasma calcium, magnesium and zinc, and plasma vitellogenin (Vtg) were determined as test endpoints. Our results indicate no significant changes in the GSI between exposed and control groups ( P < 0.05). All exposure concentrations of E 2 dramatically induced the production of Vtg within 7 days. The vitellogenic response of male loach was time- and dose-dependent. At higher concentrations of E 2 (5 and 10 μg/L), total plasma protein, total plasma calcium, and total plasma magnesium presented a significant time- and dose-dependent increase, and the three parameters were significantly correlated with plasma Vtg. We conclude that Chinese loaches are sensitive to estrogenic compounds and may be chosen as potential sentinel species in field and laboratory studies. In addition, total plasma protein, total plasma calcium, and total plasma magnesium can be used as indirect indicators to predict Vtg levels in estrogenized Chinese loach.

Development and validation of an enzyme-linked immunosorbent assay for vitellogenin in Chinese loach ( Misgurnus angaillicaudatus)

A competitive enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of vitellogenin (Vtg) in the plasma of Chinese loach ( Misgurnus angaillicaudatus). Vtg was isolated by anion exchange membrane chromatography from the plasma of 17β-estradiol (E 2)-treated loach. Polyclonal antibody against Vtg was raised in rabbits, and the specificity of the antibody was assessed by Western blotting analysis. No cross-reactivity was observed with plasma from male loach. Plasma samples from vitellogenic females and E 2-treated males were diluted parallel with the purified Vtg standard curve in the ELISA. The detection limit of the assay was 5.7 ng ml − 1 and a working range of the standard curve was between 15.6 and 1000 ng ml − 1. The intra- and inter-assay coefficients of variations were 6.9% and 10.4%, respectively. The recovery was 104.4%. The assay was applied for measuring vitellogenin concentration in the plasma from 24 wild Chinese loach from Lianyungang, Jiangsu Province, in October 2003. It was found that, in addition to females (plasma Vtg concentration ranging from 518.4 μg ml − 1 to 1922.3 μg ml − 1), male loach showed low plasma Vtg—from undetectable (two fish) to 2.9 ± 0.7 μg ml − 1 (mean ± S.D.). The results indicate that this method provides a valuable tool for the study of estrogenic effect in Chinese loach.