| The Dojo loach Misgurnus anguillicaudatus (Cobitidae, Cypriniformes) is a species endemic to Asia, mainly inhabits streams, ditches and rice paddy fields, preferably with a soft muddy bottom. It is popular in China as a traditional Chinese medicine in folk remedies for treatment of hepatitis, osteomyeitis, carbuncles, inflammations and cancers, as well as for restoration to health in debilities caused by various pathogens and aging. However, the wild populations of M. anguillicaudatus almost collapsed due to the intensive fishing and water pollutions in the recent years, prompting the development of the culture practices of this species. The culture system implying the complete life cycle was developed in 1990s and currently M. anguillicaudatus occupies a significant position in freshwater fish culture in China both for food and commercial purpose. The analysis of blood indices has proved to be a valuable guide in assessing the health status of aquatic organisms as it provides a reliable index of their physiological conditions. However, there is limited data on the comparative heamatological differentiae among different ploidy levels, and also between cultured and wild types of loach that share the same ecological zone which used to assess the ploidy level and living conditions influences on the energy metabolism and fish health. The main objectives of this study include:1. Hematological characterization of loach M. anguillicaudatus:a comparison among diploid, triploid and tetraploid specimensTo determine whether diploid, triploid and tetraploid M. anguillicaudatus differed in terms of main haematological and physiological characteristics. Diploid and tetraploid fish were produced by crossing of natural diploids (2n×2n) and natural tetraploids (4n×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 hemoglobin, mean cell volume, mean cellular hemoglobin content, and mean cell hemoglobin 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 hematological characteristics.2. Comparison of haematology and serum biochemistry of cultured and wild M. anguillicaudatusHeamatological parameters and serum biochemistry differentiae were studied and compared between cultured and wild ecotypes of Dojo loach M. anguillicaudatus during the most important fisheries season i.e. May-to-August. Data were analyzed for the impact of feeding regime and other ecological conditions on the physiology of fish. The results revealed that the concentrations of haemoglobin, cholesterol, total protein, creatinine and uric acid in the two ecotypes were significantly different(n=56, df=54, P< 0.05). Furthermore, the red blood cells, glucose, triglyceride and urea nitrogen levels in cultured individuals were significantly (n=56, df=54, P<0.01) higher than in their wild counterparts. On the contrary, the white blood cells level in the cultured fish was significantly (n=56, df=54, P<0.01) lower as compared to wild ones. These differences can be attributed to physiological acclimatization of the fish to their living conditions and feeding regime, which influences the energy metabolism and subsequently, fish health.3. Genetic diversity and population structure of Chinese M. anguillicaudatusWe investigated twelve loach populations originating from middle-lower mainstream sub-basin and the Hanjiang sub-basin of the Yangtze River. Total thirteen microsatellite loci with varying levels of polymorphism were employed to examine genetic differentiation and population structure of the species within sampling localities. The number of alleles per locus varied between 2 and 8 with an average of 4.6 alleles per locus. Observed heterozygosity varied from below 0.38 up to a maximum of 0.95; over all loci the average observed and expected heterozygosity was 0.57 and 0.56, respectively. Significant deviations from Hardy-Weinberg equilibrium (P<0.01) were observed in about 50% of the total locus-population combination tests with apparent heterozygote deficits. This heterozygote deficit might be attributed to fishing pressure and migration-drift disequilibrium. AMOVA analyses indicated that most of the variance existed among the individuals (90.50%) rather than among populations within groups (9.03%). Based on pairwise Fsr and unbiased genetic distance, significant differentiation was found among the samples from scattered habitats with different connections to the Yangtze River. The UPGMA dendrogram clustered the populations onto two major branches, surprisingly following their ichthyogeographic distribution except ZIG and XGN populations which genetically departed from their geographical origin. The combined analyses showed a significant population genetic structuring at sub-basin level. Overall, the present study revealed that Chinese loach inhabiting the central China is genetically differentiated into two distinct geographical groups. The existing differentiation in loach populations is attributed to the current demographic configuration of the region, nonmigratory behavior of the loach and reduced flooding in the Yangtze River which restricted the gene flow among loach populations in riparian ecosystems.
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