Biogeographic Patterns Of Polyploid Misgurnus Anguillicaudatus In Hubei Province And Basic Studies On Its Breeding

The loach Misgurnus anguillicaudatus is a small freshwater fish blonging to the family Cobitidae (Cypriniformes). Cytogenetic studies on M. anguillicaudatus have revealed an extensive karyotype polymorphism of this species in both China and Japan. Among Japanese populations, bisexual diploid individuals are most common, but natural triploids have been recorded in some localities. Experimental crosses including induced gynogenesis and inter-specific hybridization showed that triploid loaches arose from unreduced eggs produced by sympatric diploids. In China, populations of both diploid (2n=50) and tetraploid (4n=100) loach are common to be recorded. Due to such natural karyotype polymorphism, this species is considered as an ideal model not only for studying cytogeography, chromosome evolution and developmental biology, but also can be used as valuable material for genetic and breeding studies in aquaculture. Unfortunately, despite the existence of distinct ploidy levels in natural populations having been recognized for a long time, advancement in the genetic studies on different ploidy levels of the Chinese loach M. anguillicaudatus have been insignificant. To buildup detailed cytogenetic information for Chinese M. anguillicaudatus, we carried out serial studies to investigate the biogeographic patterns of polyploid M. anguillicaudatus in Hubei province. Furthermore, to maximize the fish productivity of this species, a 15-month rearing experiments were conducted to determine the cytotype with better traits for the aquaculture practices of this species. The main objectives of this study include:1. Investigation of the biogeographic patterns of polyploid M. anguillicaudatus in Hubei province(1) The morphometric parameters of diploid and tetraploid M. anguillicaudatus were compared for ploidy discrimination. The results revealed no significant differences in external appearance, i.e. shape and colour between diploid and tetraploid fish, except for the relatively larger body size of tetraploids. Analysis of discriminant-function revealed a discrimination accuracy of 93.4% with all of the most significant variables while the same was found to be 91.3% if we use only HL/CPD and HL/SL as parameters for discrimination. So the latter can be used for the preliminary discrimination of the either ploidy level loach from a mixed collection of the fish.(2) In search for an easy, rapid and cost-effective method to determine the ploidy levels of diploid and tetraploid M. anguillicaudatus distributed naturally in China, the method of karyotyping, flow cytometry, erythrocyte nuclear measurements and morphometric analysis were compared. It was discovered that the flow cytometry is accurate, rapid and can be applied for extensive sampling analysis. Using this method, ploidy status of 1138 M. anguillicaudatus specimens from 27 populations was examined. The results showed that, most of the samples from Hubei province were diploid; tetraploids were distributed mainly in the populations along Yangtze River basin. Along the Yunshui water system, coexistence of diploids and tetraploids was observed in some populations, but no triploid individual has been detected in these populations, In Shenlongjia area, populations from Dajiuhu with the altitude of 2500 meters were all diploid, while populations from Songbo Town with the altitude of 1200 meters and Fangxian County with the altitude of 600 meters were all tetraploids. No geographic line of demarcation could be drawn on the distribution patterns of polyploids, it was rather uneven.(3) In Diaochahu area (113°43'18.5"E, 30°39'44.6"N), natural hexaploidy in rare individuals of M. anguillicaudatus was discovered. The hexaploid individual share the external appearance with its diploid and tetraploid counterparts while the chromosome were found to be 6n= 150 (24m+18sm+108t) .2. The growth performance and reproduction of different cytotypes of M. anguillicaudatusTo determine the cytotype with better traits for the aquaculture practices of the M. anguillicaudatus from the viewpoint of fish farming improvement, factorial crosses (2n♀×2n♂, 2n♀×4n♂, 4n♀×2n♂, 4n♀×4n♂) were made between natural diploids (D) and tetraploids (T), producing DD, DT, TD and TT groups (first letter denotes female). The potential benefits of the different cytotypes regarding aquaculture were evaluated by comparing growth performance and survival rate for a 15 months rearing trail under the same production conditions.(1) The average levels of fertilization were 83.8% for DD. 68.5% for DT, 79.3% for TD and 50.6% for TT. The eggs from the tetraploids exhibited about the same average fertilization rate as eggs from the normal diploids (e.g.83.8% for DD vs. 79.3% for TD), suggesting that eggs from tetraploids were as capable as diploid eggs in terms of fertilization and subsequent cell divisions. On the other hand, the fertilization level of sperm from tetraploid males (e.g. 68.5% for DT) was lower than those of sperm from diploids (e.g. 83.8% for DD), indicating that sperms from the tetraploids might be less potent in fertilizing eggs.(2) DNA content, microstructure and Ultrastructure of spermatozoa from tetraploid M. anguillicaudatus were examined and compared to those of diploids. The result revealed that Ultrastructure of sperm from tetraploids was compositionally similar to that of diploid except for overall size and the number of mitochondria. The DNA content (2.56±0.08pg N^-1) and head size (11.37±0.55μm³) of the tetraploids was about 2 times of the diploids (1.33±0.05pg N^-1 for DNA content and 5.65±0.38μm³ for head size), but the number of mitochondria was just 41.7% more than the diploids, which may render the tetraploid sperm less potent in fertilizing egg.(3) Under exactly the same rearing conditions, the TT group displayed obviously poor survival rate, possibly indicating that the increased cell surface of tetraploids have limited cellular metabolism and, thus, poor anti-adversity in certain environmental conditions. Tetraploid females produced remarkably larger eggs than diploids, subsequently leading to significantly increased initial body length of TT followed by TD >DD>DT fry. The TD group exhibited better growth performance than that of the DT group, though both were triploids. It indicates the possible existence of significant maternal effects on growth performance of M. anguillicaudatus, since natural tetraploids always show obviously lager body size than diploids.(4) Comparison of ovarian development among different cytotypes of M. anguillicaudatus was conducted on 7 and 10-month-old females. The results revealed that all genotypes in the present study were able to reach sexual maturity. However, ovaries among different groups did not develop synchronously. Tetraploid progenies, especially TD, showed relatively retarded gonadal development even though the gonads appeared similar under the microscope. The normal gonadal development of TD and DT M. anguillicaudatus limited the application of the triploid individuals.3. Comparison of the heterozygosity among different cytotypes of M. anguillicaudatus using microsatellite markersWith the allele frequencies, the genetic heterozygosity of four cytotypes of M. anguillicaudatus was analyzed. The results showed that, a high level of variability among the cytotypes. The average genetic heterozygosity of four cytotypes (DD, DT, TD and TT) was 0.4779, 0.6287, 0.5547 and 0.4448, respectively. The large number of homozygotes in TT group might be responsible for the higher mortality in this cytotype.