Research On Cultivable Bacteria Diversity And Ultrastructure Of The Fermentation Chamber Of Holotrichia Parallela (Coleoptera: Scarabaeidae)

Holotrichia parallela, with a large number and wide distribution, are important agricultural pests in China. The larvae and adults both can cause serious damage of crops. The chemical controlling methods are now doing little effect to them while serious damage to the environment. In this paper, we studied the cultivative bacteria community and ultrastructure of fermention chamber of the larvae. The results provided theoretical basis for clarifying the structure of the cultivative bacterial community, the ultrastructure of fermention chamber and the co-evolution between host and whose symbionts. We hoped this work can provide a new idea for bacterial control method to the pest.In this study, we employed traditional culture and PCR-DGGE molecular methods to study the cultivative bacterial community in the fermention chamber of the 3rd larvae, and then studied the ultrastructure variation of the fermention chamber in different developmental stages using slicing and microscopic methods. The main results are shown as follows:From the viable counts, we know the number of cultivative bacteria in fermention chamber is about 33.4×10⁷cfu/ml, while the direct count show the number is 1.67×10¹⁰/ml. Thus, a large number of bacteria in fermention chamber are can not be cultured. The results shown 38 different genotypes of cultivative bacteria in fermentation chamber, including aerobes and anaerobes, belongded to the genus of Enterobacter, Grimontella, Citrobacter, Salmonella, Klebsiella, Acinetobacter, Haemophillus, Leptothrix, Clostridium and the CFB bacteria (Cytophaga-flexibacter-bacteroides) group.The aerobes in chamber wall were classified into 11 genotypes and divided to four genuses: Enterobacter, Grimontella, Citrobacter and Salmonella. These bacteria were commonly found in environment. Seven genotypes of bacteria belonged to genus Enterobacter, which occupied 84.62% of aerobes on chamber wall; Tow genotypes belong to Grimontella, with proportion of 12.31%; one belongs to Citrobacter, with proportion of 3.08%; one belongs to Salmonella, with proportion of 1.54%.11 genotypes of aerobes in intestinal contents were divided into genus Klebsiella, Enterobacter, Salmonella and Acinetobacter. Looking from the distribution of proportions, five kinds of bacteria belonged to genus Klebsiella, with proportion of 20.26%; four kinds belonged to Enterobacter, with proportion of 75.82 % as the main dominant; one belonged to Salmonella, with proportion of 1.96%; one belonged to Acinetobacter, with proportion of 1.96%.8 genotypes of anaerobes on chamber wall were divided into Citrobacter, Enterobacter, Haemophilus, Leptothrix and Clostridium. There genotypes belonged to genus Citrobacter, with proportion of 64.39%; one belonged to Enterobacter, with proportion of 12.12%; one belonged to the case of Haemophilus, with proportion of 6.06%; one belonged to Leptothrix, with proportion of 3.79%; one belonged to Clostridium, with proportion of 5.30%.8 genotypes of anaerobes belonged to Klebsiella and CFB bacteria. Four genotypes belonged to Klebsiella, with proportion of 52.63%; and another four belonged to CFB (Cytophaga-flexibacter-bacteroides) bacteria group, with proportion of 47.37%.The results of ultrastructural studies showed the fermentation chamber was by composition of 3 main layers: the muscle layer (including the transverse and longitudinal muscle actin), epithelial cell layer and cuticular intima. The ultrastructure of different developmental stages, especially the bacteria attached to cuticular intima changed significantly: 1st instar larvae are lack of cuticular intima, a small number of bacteria directly attached to the epithelial cells membrane; The cuticular intima begin to emerge in 2nd instar larvae, and the bacteria attached on which increase significantly; A special and complex structure which composited by bacteria and cuticular intima, definited temporarily as "composite structure", emerged in 3rd larvae. The SEM photos showed there were massive rod-shaped intracellular symbionts in epithelial cells, the role that the intracellular symbiont play need further study.