Different Comparison And Characterization Of Lipase-producing Bacteria In The Silkworm Intestinal Microflora

Insect intestinal system is a changeful environment followed by intaking food, digestion and excretion. The guts harbor abundant microorganisms which play important roles in the host's nutrition, development, reproduction, and resistance to pathogens. Compared with mammals, the functions of microorganisms in insect guts have been studied extensively only in several systems such as termites and desert locusts. Some specific roles of microorganisms in these insect guts have been disclosed, including lignocellulose digestion, methanogenesis, acetogenesis from H2 and CO2, nitrogen fixation, maintenance of a low redox potential and prevention of entry of foreign microorganisms. However, relatively little is known about the roles of microorganisms in other insect groups. Lepidoptera is an important group of insecta, a few studies have indicated that the Lepidopters harbor gut bacteria and the possibility that microorganisms produce some of the digestive enzymes to provide essential nutrients or assist in important biochemical function related to host food ingestion.The silkworm, Bombyx mori L. (Lepidoptera:Bombycidae), as a model for Lepidoptera, is an agriculturally important insect for silk production. The silkworm, an oligophagous insect, mainly feeds on mulberry leaves, but tricuspid cudrania (Moraceae) leaves can also act as the forage of the larvae. Recent studies have found that the silkworm larvae not only grow retarded but also are susceptible to Bombyx mori nucleopolyhedrovirus (BmNPV) when reared with tricuspid cudrania leaves. However, a few studies have pointed out that the lipase from silkworm larval alimentary canal shows strong antiviral activity against BmNPV. Therefore, in this study, we used tricuspid cudrania leaves to feed silkworm to construct a BmNPV susceptible model and adopted the identification technology combined molecular biology with traditional bacteriological method. The objective of this study was to explore the diversities and differences of dominant intestinal bacteria, especially the lipase-producing bacteria between the BmNPV susceptible and non-susceptible silkworm. These understanding of diet-derived intestinal bacterial community may yield insight into the relationship between the gut bacteria and disease resistance of the silkworm. The experimental results are as follows:1. A total of fifty-six dominant isolates were successfully collected from the intestine of the fourth and fifth instar silkworm larvae using pure culture isolation method and classified into twelve phenotypes based on the colony color, size and cellular morphology. Ten and seven phenotypes existed in the intestine of the larvae fed with mulberry leaves and tricuspid cudrania leaves respectively, and four of them were common in the intestine of the two treatment groups.2. Based on morphological and physiological-biochemical characteristics,16S rRNA sequence determination and phylogenetic analysis, twelve dominant bacteria belong to Aeromonas, Brevibacterium, Corynebacterium, Klebsiella, Citrobacter, Agrobacterium, Pseudomonas, Staphylococcus, Enterobacter, Stenotrophomonas, Brevundimonas and Bacillus. Four phylotypes of the isolated dominant bacteria including Staphylococcus, Enterobacter, Stenotrophomonas, Brevundimonas, were common in the intestine of both the silkworm larvae fed on different forage. There were five phylotypes including Aeromonas, Brevibacterium, Corynebacterium, Klebsiella and Citrobacter only existed in the intestine of the larvae reared with mulberry leaves, while the larvae fed with tricuspid cudrania leaves possessed other two different genuses, Agrobacterium and Pseudomonas. Besides, Bacillus was common only in the intestine of the fourth-instar silkworm.3. According to screening fifty-six dominant strains on Rhodamine B agar plate, nine lipase-producing bacteria were obtained and classified into six genera including Bacillus, Brevibacterium, Corynebacterium, Staphylococcus, Klebsiella and Stenotrophomonas. The common bacterium belonged to genus Stenotrophomonas, and the other bacteria only existed in the intestine of the silkworm larvae reared with mulberry leaves. The results showed that the diversity of lipase-producing bacteria had a significant difference when the silkworm larvae were reared on different forage, the diversity of lipase-producing bacteria from the intestine of the tricuspid cudrania leaves feeding silkworm was considerably deficient compared with that of the larvae reared with mulberry leaves.4. The maximum cell density of lipase-producing bacteria was observed at about 48 h of fermentation. However, the maximum bacterial density showed significant difference in which strain SW41 had the maximum yield, whereas strain SW72 had the minimum yield. Besides, lipase activity reached the maximum after 48 h of fermentation in vitro. At their maximum bacterial density, Strain SW41 showed highest lipase activity which was 249.59 U/L, but strain SW54 was the lowest that the activity was only 77.46 U/L.