Gut Bacterial Diversity Of The Diamondback Moth And Insecticidal Activity To Larvae By Two Cultivable Bacterial Strains

The larvae of the diamondback moth, Plutella xylostella L.(Lepidoptera: Plutellidae),have a rich microbial communities inhabiting in the gut, and those bacteria contribute to thefitness of the pest. Microbial abundance and diversity of different life stages (fourth instarlarvae, pupae and adults) of P. xylostella, collected from field and reared in laboratory, wereinvestigated using bacterial culture-dependent method and denaturing gradient gelelectrophoresis (PCR-DGGE) analysis based on the sequence of bacteria16S rRNA V3region gene.A large quantity of bacteria was found in all life stages of P. xylostella. Field populationshad higher quantity of bacteria than laboratory populations, and larval gut had higher quantitythan pupae and adults. Culturable bacteria differed in different life stages of P. xylostella. Atotal of25bacterial strains were identified, and of these,20strains were presented in larvalgut,8strains in pupae, and14strains in adults. Firmicutes bacteria, Bacillus spp., were themost dominant species in every life stage.Fifteen distinct bands were obtained from DGGE electrophoresis gel. The sequencesblasted in GenBank database showed that these bacteria belong to six different genera andthree unculturable bacteria. Phylogenetic analysis showed that the sequences of the bacteriabelong to Actinobacteri, Proteobacteria and Firmicutes. Serratia sp. in Proteobacteria was themost abundant species in larval gut. Unculturable bacteria were the most dominant species inpupae, and unculturable bacteria and Serratia sp. were the most dominant species in adults.Our study suggested that a combination of molecular and traditional culturing methods can beeffectively used to analyze and to determine the diversity of gut microflora. These knownbacteria may play important roles in development of P. xylostella.In this study we also evaluated the effects of five antibiotics (rifampicin, ampicillin,tetracycline, streptomycin sulfate and chloramphenicol) on the gut bacterial diversity of P.xylostella larvae. We screened five different concentrations for each antibiotic in a leaf discassay, and found that rifampicin and streptomycin sulfate at3mg ml-1significantly reducedthe diversity of bacterial community, and some bacteria species could be rapidly eliminated.The number of gut bacteria in rifampicin group and streptomycin sulfate group decreased more rapidly than the others. With the increase of antibiotic concentrations, the removalefficiency were improved, whereas toxic effects became more apparent.All antibiotics negetively affected larval growth and development in the form ofmalformation of the prepupae, and hindered pupation and adult emergence. Among the fiveantibiotics, tetracycline was the most toxic, and streptomycin sulfate was a relative mild one.Some dominant bacteria were not affected just by feeding antibiotics. DGGE graph showedthat the most abundant and diverse bacteria in P. xylostella larval gut appeared in the cabbagefeeding group, and diet change and antibiotics intake influenced the gut flora abundance.Bacterial species diversity was significantly reduced in artificial diet and antibiotics treatmentgroups. After feeding the larvae with an artificial diet with rifampicin, streptomycin sulfateand their mixture for10days, agarose gel electrophoresis detection results showed larval gutbacteria could not be completely removed.We cultivated two bacterial strains from the died pupae collected from the field. The P1strain was identified as Pseudomonas cedrina, and the P2sequence was the most similar toBacillus pumilus. The P1strain grew the fastest in LB fluid medium, and after inoculating of4-6h, the P1bacteria entered the logarithmic phase, while Bacillus thuringiensis (BT) wentinto the logarithmic phase after inoculation for6-8h. The P2strain’s growth speed was theslowest among the three strains/species. With the increase of the microbial solution OD value,the mortality of larvae increased especially in the P1treated group. Larval mortality differredafter feeding different OD values of bacteria cells. The insecticidal activity of the P1bacteriato P. xylostella larvae was better than that of BT. After removing bacteria cell, larval mortalitydecreased, implying that a less toxic substance existed in the liquid supernatant of the P1bacteria.