| The larvae of Hepialus gonggaensis is one of the predominant host of Cordyceps sinensis. The insect infected by C. sinensis can produce famous herb medicine Dongchongxiacao which has very high price in China and Asia area. The intestinal microbial flora played an important action in larvae's development, nutrient and resistant to the diseases. In the study, intestinal microbial flora of H. gonggaensis larvae, collected from Kangding, Sichuan Province, had been isolated and identified with classic methods. The results showed that the intestinal microbial flora of H. gonggaensis belongs to low-temperature bacteria. The bacteria grew very slowly and the typical colony appeared on cultural plate only after 2 days incubated under the most profitable temperature, 15℃. Totally 12 different isolates of bacteria were obtained from the larvae intestine with 5 different of cultural media. It showed that Staphylococcus. sp was the predominant community, whose numberable colony counted 2.05×109mL-1, and the detection rate as high as 100%. It revealed that Staphylococcus sp. could be the normal flora in the larva's intestine. And the other isolates can only be detected occasionally; they were supposed to be the passer microbes. The biochemical identity showed that the identifying values are relative low for most bacteria, which are consistent with the larvae's abnormal living environment. Because of large sorts of bacteria could be uncultured, molecular technology were also applied to analyze the bacteria diversity in the larva gut of H. gonggaensis. The primers designed by conservative sequences of 16SrRNA gene of E.coli was used to amplificate intestinal bacterial gene group DNA from H. gonggaensis , and then amplificational products were separated directly by DGGE. Twelve bands were obtained with DGGE in the denaturant range of 10%~60%, and 76 bands were separated at last under the much narrow denaturant range of 25%~48%. Every band might present a different microbiological sort, so the number and the brightness of bands in DGGE patterns can reflect the number of bacterium sorts and the predominant bacterium in intestinal flora. All the study indicated that there were great number of bacterium sorts and quantity in the intestine of H. gonggaensis. Repeated experiment can get the same result. It showed that 16SrDNA sequence combined DGGE analysis was effective method for larvae's intestinal bacterial diversity study. The eight most bright bands separated by DGGE were purified, cloned and sequenced. The sequences were compared with the database in Gene Bank and aligned by the software BLAST. The results showed that the 5 of 8 sequences of isolators were different from that of bacteria which were already knew by biologists. This suggested that these bacteria had not be studied yet up to now. The different bacterial flora was obtained by classic culture and DGGE, especialy the predominant species. This inconsistant probably because of each method having limited use area and not perfect for all sorts of bacterial. It also indicated that the predominance bacterial strains got from purified culture were probably not the real predominant flora. They maybe only accounted a few part of flora in the insect intestine.
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