| The swift moth Hepialus gonggaensis larvae are predominant hosts of Cordyceps sinensis which parasites H. gonggaensis larvae and forms fungi-insect complex called Cordyceps or Dongchongxiacao, a valuable traditional Chinese medicine and used as a general tonic for protecting and improving lung and kidney functions, enhancing physical performance also. H. gonggaensis larvae live on the roots of weed at meadow of cold and high altitude area. There are six larval instars and it will take three to four years to become adults from eggs. Because of its special residence environment and life inhabit, it is not sufficient to meet people's increasing demand. lots of work has been down to try to rearing the moth at controlling condition, but very low livability obtained.Liu li had detected that the most dominant bacteria group was Carnobacterium in the gut using both traditional culturing and culture-independent methods. Our objective of this study was to evaluate the effect of bacteria Hg4-03 (Carnobacterium sp., a kind of lactic acid bacteria), the most dominant bacteria in the gut of H. gonggaensis larvae, as probiotics supplements in diets for H. gonggaensis larvae, which was a kind of the most valuable insect in China. It was expected to find out the key factors infected the host of Dongchongxiacao's development and antiadversity in larvae breeding, provide theoretical and technical support for the scale artificial process of the Dongchongxiacao.Firstly the probiotics was added to the diet of larvae, Four trials were carried out with H. gonggaensis larvae containing three treatments and one control with three replicates for each group. In Treatment 1 (T-1), the natural diet (Polygonum viviparum L.) was dipped in 1ml liquid culture medium of Hg3-04 bacteria. In Treatment 2 (T-2), the natural diet was dipped in 2ml liquid culture medium of Hg3-04 bacteria. In Treatment 3 (C-1) was accepted as Control group in which the natural diet was dipped in fresh liquid culture medium. Treatment 4 (C-2) the natural diet was dipped in cultivated Hg3-04 bacteria killed by heat sterilization. At the end of the experiment, the survival rate was counted. All collected larvae from each tank was weighted respectively at initial and the end during the experiment, which treated as initial weight and final weight, to calculate growth rate. At the same time, larvae survival was determined by counting the individuals in all tanks. Protease activity was measured using a Folin-phenol reagent. The activities of amylase, trehalase and cellulose were determined with 3, 5-Dinitrosalicylic acid. Enzyme activities were all expressed as specific activity (U mg-1 protein). Secondly Denatured Gradient Gel Electrophoresis (DGGE) method was used to etection the guts microbiota diversity of H. gonggaensis larvae reared by probiotics based on 16S rDNA. Amplified regions by bacterial universal primers with GC clamp were separated on the gradient gel. Each band of gel was cut, stored, and sequenced.The result showed the growth of H. gonggaensis larvae after bacteria supplement Twenty-eight days after feeding the diet of bacteria supplement, the final weights of the larvae group all groups were weigh in 0.442±0.047g for T-2, 0.435±0.060g for T-1, 0.387±0.042g for C-2 and 0.397±0.062g for C-1, respectively. Values of growth rate for each group was calculated as 0.5206±0.0816 for T-2, 0.5153±0.0741 for T-1, 0.3929±0.0657 for C-2 and 0.3634±0.0774 for C-1. There are no significant differences on the final weight in two groups treated with probiotics at different concentrations. And the same result was found in C-2 and C-1 whereas significant differences were found between T-2 and C-1. Moreover, death rates were determined as 15.63%, 18.75%, 32.26% and 28.13% for T-2, T-1, C-2 and C-1, respectively. Compared with the latter two groups, T-2 and T-1 have obviously lower death rates.The mean digestive enzyme activities of the two groups treated with probiotics were significantly different from that of S and control group.In all digestive enzyme, the activity of amylase in the treated groups had been improved most highly compared with C-2 and C-1 at the end of first period. Whereas, there was no significant difference between C-2 and T-1. At the end of the second period, the same phenomenon was observed.Protease activity in the treated groups also was changed compared to the other two groups. After culture for 14 days significant difference had been determined between T-2 and C-1 besides C-2, at the same time no significant difference had been found in the two treated groups. After for 28 days, activity of protease in H-1 showed remarkably higher than that in the first period. But the values of C-2 and control in all two period were not significantly different each other.The highest trehalase specific activity was detected after 28 days in T-2 and T-1 as 11.88(±0.44)U mg-1 protein and 11.82(±0.83)U mg-1 protein. But on day 14, no remarkable difference was found in T-1 compared to C-2 and C-1. There was no difference among four values of two control groups in two periods.The file of DGGE showed that bacterial diversity index of treatment groups with Hg4-03 was higher than control group, and the species of bacteria increased. Comparing the bands of DGGE profiles between three groups, the intensity of band stand for Carnobacterium sp. Hg4-03 have enhanced. Another band with swelled intensity found in profiles come from treatment group2 after 28 d fed, the sequence analysis showed it belongs to Bacillus subtilis. Feeding with Carnobacterium sp. Hg4-03 could balance the bacteria in larvae gut and increase bacteria diversity, that means the bacteria made a positive effect on the bacterial diversity of H. gonggaensis larval gut. Hg4-03 may be used as probiotic bacteria for rearing H. gonggaensis larvae in controlling conditions or the larvae's hemi-wild rearing.
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