| Anopheles hyrcanus complex is a complicated and important group in China. There are 19 species in it. This complex is distributed widely. Parts of them were proved that are vectors of malaria and filariasis at their distribution district. Because morphology of the members is similar, distinguishing is often confusable and mistakeble. This study is wanted to take the molecular identification of parts of An. hyrcanus complex, and An. sinensis population molecular genetic polymorphism.First, the ribosomal DNA second internal transcribed spacers ( ITS2) sequences were chosen, which is significative in distinguishing cryptic anopheline. An. sinensis collected from Danfeng Shaanxi (DF), Wuxi Jiangsu (WX), Faku Shengyang ( FK) , Pujiang Sichuan (SC), An. anthropophagus from Xuyi Jiangsu, An. yatsushiroensis from Rongcheng Shandong were sequenced and found to be 468bp (An. sinensis DF, WX, PJ), 462bp (An. sinensis FK) , 452bp (An. anthropophagus), 459bp (An. yatsushiroensis) in length. The range of GC content is from 53.63% to 55.19%. The results of sequence alignment are as following: The sequences of An. from DF, WX, PJ are the same. It shows that the ITS2sequences are intraspecific consensus. The level of difference from An. sinensis and An. anthropophagus, An. yatsushiroensis, An.anthropophagus and An. yatsushiroensis is 28.8%, 17.13%, 15. 18%, respectivly. It shows that interspecific variation in ITS2 is higher. The level of difference from An. sinensis ( FK ) and the above An. iinensis is 20. 97%. This is the interspecific variation. But the difference from An. sinensis ( FK ) and An. anthropophagus is only 3.83%. It is belong to intraspecific variation. It is proved that An. sinensis ( FK) is confusable, should be An. anthropophagus.The primers of PCR assay were designed by sequence difference in ITS2 of ribosomal DNA of An. sinensis and An. anthropophagus, Specific lengths of fragments were amplified 425bp in the An. sinensis and 253bp in the An. anthropophagus. Total 70 specimens of An. sinensis and An. anthropophagus of 3 laboratory lines were detected, all samples gaves correct species- specific amplified fragment. And 440 field collection specimens of An. sinensis from 12 locations in 10 provinces were detected, with correct rate of 66. 1%, it shows that the field routine identification samples was confused with some allied mosquito species. 20 specimens of An. anthropophagus (identified by eggs) collected from 2 locations of Sichuan Province, all amplified with correct species-specific fragment. The control groups of 9 specimens, including An. kweiyangensis, An. liangshanensis and An. pattoni were non-amplified.Samples of Anopheles sinensis from 10 location sites in 9 provinces of China were also analyzed for genetic polymorphisms using RAPD-PCR. 19 RAPD loci were chosen to estimate. The results are as following: The ranges of percentage of polymorphic loci are 78.9% -100%, expected mean heterozygosity are 0.288-0,409. It shows that there are extensive geneticpolymorphisms in natural populations of Anopheles sii>ensis. The Fst and 9 were estimated using three measures. Mean values are 0.069-0.111, corresponding to the range of migrating rate is 2, 0 -3.4. It shows that the level of gene flow is lower. The genetic identity between natural populations is 0. 8524- 0. 9966, mean genetic distance is 0.0511 ±0.0416. These are belong to the interspecies variation. The cluster analysis shows that the genetic distance is not related to the location sites.This study is well to support that the ITS2 sequence is a useful character for distinguishing cryptic anopheline. The PCR assay is established for distinguishing An. sinensis and An. anthropophagus, which is simple, sensitive and stable. Some confusable questions of An. hyrcanus complex are cleared up. Through molecular genetic polymorphism analysis of An. sinensispopulation, it shows that genetic structure of natural populations is similar. Genetic variation is belong to intrapopulation. All of these will give foundation materials for practice.
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