In this study, 207 strains of aerobic and facultatively anaerobic cellulolytic bacteria were isolated from the gut of Holotrichia parallela larvae. These bacterial isolates were assigned to 21 genotypes by amplified ribosomal DNA restriction analysis (ARDRA). A partial 16S rDNA sequence analysis and standard biochemical and physiological tests were used for the assignment of the 21 representative isolates. Our results show that the cellulolytic bacterial community is dominated by the Proteobacteria (70.05%), followed by the Actinobacteria (24.15%), the Firmicutes (4.35%), and the Bacteroidetes (1.45%). At the genus level, Gram-negative bacteria including Pseudomonas, Ochrobactrum, Rhizobium, Cellulosimicrobium, and Microbacterium were the predominant groups, but members of Bacillus, Dyadobacter, Siphonobacter, Paracoccus, Kaistia, Devosia, Labrys, Ensifer, Variovorax, Shinella, Citrobacter, and Stenotrophomonas were also found. Furthermore, our results suggest that a significant amount of bacterial diversity exists among the cellulolytic bacteria, and that Siphonobacter aquaeclarae, Cellulosimicrobium funkei, Paracoccus sulfuroxidans, Ochrobactrum cytisi, Ochrobactrum haematophilum, Kaistia adipata, Devosia riboflavina, Labrys neptuniae, Ensifer adhaerens, Shinella zoogloeoides, Citrobacter freundii, and Pseudomonas nitroreducens are reported to be cellulolytic for the first time in this study. Our results indicate that the scarab gut is an attractive source for the study of novel cellulolytic microorganisms and enzymes useful for cellulose degradation.

RNA interference (RNAi) is a powerful method to inhibit gene expression in a sequence specific manner. Recently silencing the target gene through feeding has been successfully carried out in many insect species. Escherichia coli strain HT115 was genetically engineered to express dsRNA targeting genes that encode ribosomal protein Rpl19, V type ATPase D subunit, the fatty acid elongase Noa and a small GTPase Rab11. qRT-PCR showed that mRNA level of four target genes was reduced compared to ds-egfp control by feeding either engineered bacteria or dsRNAs. The maximum down-regulation of each gene varied from 35% to 100%. Tissue specific examination indicated that RNAi could be observed not only in midgut but also in other tissues like the ovary, nervous system and fat body. Silencing of rab11 through ingestion of dsRNA killed 20% of adult flies. Egg production was affected through feeding ds-noa and ds-rab11 compared to ds-egfp group. Adult flies were continuously fed with dsRNA and bacteria expressing dsRNA for 14 days and up-regulations of target genes were observed during this process. The transcripts of noa showed up-regulation compared to ds-egfp control group in four tissues on day 7 after continuous feeding either dsRNA or engineered bacteria. The maximum over-expression is 21 times compared to ds-egfp control group. Up-regulation of rab11 mRNA level could be observed in testes on day 7 after continuous bacteria treatment and in midgut on day 2 after ds-rab11 treatment. This phenomenon could also be observed in rpl19 groups. Our results suggested that it is feasible to silence genes by feeding dsRNA and bacteria expressing dsRNA in Bactrocera dorsalis. Additionally the over-expression of the target gene after continuously feeding dsRNA or bacteria was observed.

Gut symbiotic bacteria have a substantial impact on host physiology and ecology. However, the contribution of gut microbes to host fitness during long-term low-temperature stress is still unclear. This study examined the role of gut microbiota in host low-temperature stress resistance at molecular and biochemical levels in the oriental fruit fly Bactrocera dorsalis. The results showed that after the gut bacteria of flies were removed via antibiotic treatment, the median survival time was significantly decreased to approximately 68% of that in conventional flies following exposure to a temperature stress of 10°C. Furthermore, we found that Klebsiella michiganensis BD177 is a key symbiotic bacterium, whose recolonization in antibiotic treated (ABX) flies significantly extended the median survival time to 160% of that in the ABX control, and restored their lifespan to the level of conventional flies. Notably, the relative levels of proline and arginine metabolites were significantly downregulated by 34- and 10-fold, respectively, in ABX flies compared with those in the hemolymph of conventional flies after exposure to a temperature stress of 10°C whereas recolonization of ABX flies by K. michiganensis BD177 significantly upregulated the levels of proline and arginine by 13- and 10- fold, respectively, compared with those found in the hemolymph of ABX flies. qPCR analysis also confirmed that K. michiganensis-recolonized flies significantly stimulated the expression of transcripts from the arginine and proline metabolism pathway compared with the ABX controls, and RNAi mediated silencing of two key genes Pro-C and ASS significantly reduced the survival time of conventional flies, postexposure low-temperature stress. We show that microinjection of L-arginine and L-proline into ABX flies significantly increased their survival time following exposure to temperature stress of 10°C. Transmission electron microscopy (TEM) analysis further revealed that low-temperature stress caused severe destruction in cristae structures and thus resulted in abnormal circular shapes of mitochondria in ABX flies gut, while the recolonization of live K. michiganensis helped the ABX flies to maintain mitochondrial functionality to a normal status, which is important for the arginine and proline induction. Our results suggest that gut microbiota plays a vital role in promoting the host resistance to low-temperature stress in B. dorsalis by stimulating its arginine and proline metabolism pathway. The physiological mechanisms by which insects survive at low temperatures have not yet been discovered. In addition, there are no reports to verify the role of gut symbionts in the B. dorsalis biology during low-temperature stress. By combining transcriptomic and metabolomic approaches, we found that the presence of gut symbionts, especially K. michiganensis BD177, helps the host B. dorsalis to elevate the levels of particular cryoprotectant transcripts and metabolites, which suggests that the gut symbiont K. michiganensis BD177 stimulates the host arginine and proline metabolism pathway to promote its resistance to long-term low-temperature stress via influencing the mitochondrial functionality. Finally, the methods of this study are relevant for investigating the underpinnings of other host-microbe interactions during environmental stress.

The guts of metazoans are in permanent contact with the microbial realm that includes beneficial symbionts, nonsymbionts, food-borne microbes and life-threatening pathogens. However, little is known concerning how host immunity affects gut bacterial community. Here, we analyze the role of a dual oxidase gene (BdDuox) in regulating the intestinal bacterial community homeostasis of the oriental fruit fly Bactrocera dorsalis. The results showed that knockdown of BdDuox led to an increased bacterial load, and to a decrease in the relative abundance of Enterobacteriaceae and Leuconostocaceae bacterial symbionts in the gut. The resulting dysbiosis, in turn, stimulates an immune response by activating BdDuox and promoting reactive oxygen species (ROS) production that regulates the composition and structure of the gut bacterial community to normal status by repressing the overgrowth of minor pathobionts. Our results suggest that BdDuox plays a pivotal role in regulating the homeostasis of the gut bacterial community in B. dorsalis.

The sterile insect technique (SIT) as an eco‐friendly and reliable strategy has been used to control populations of insect pests of agricultural, veterinary and human health importance. Successful applications of SIT rely on the high‐level ecological fitness of sterile males. A suitable and stable gut microbiome can contribute to the ecological fitness of insect by influencing their physiology, biochemistry and development processes. Here, we show that a shift in the gut bacterial composition and structure by sterilizing irradiation, characterized by a decrease in the major gut microbiota community Enterobacteriaceae, an expansion of the minor members (e.g., Bacillaceae) and a higher richness and diversity, is tightly linked to radiation‐induced ecological fitness (male mating competitiveness, flight capacity, survival rate and life span) decline in Bactrocera dorsalis (Hendel) sterile males. Function prediction of gut microbiota indicated that changes in microbiome taxonomy tend to drive microbiome functional shifts. A higher nutrient consumption of the flourishing minor gut microbiota may cause a decline in nutrients and energy metabolic activity of host and then result in the reduced ecological fitness of irradiated flies. Furthermore, we found that a gut bacterial strain Klebsiella oxytoca (BD177) can restore ecological fitness by improving food intake and increasing haemolymph sugar and amino acid levels of irradiated B. dorsalis flies. Our findings suggest that gut symbiont‐based probiotics can be used as agents for reversing radiation‐induced ecological fitness decrease.

The citrus fruit fly Bactrocera minax is associated with diverse bacterial communities. We used a 454 pyrosequencing technology to study in depth the microbial communities associated with gut and reproductive organs of Bactrocera minax. Our dataset consisted of 100,749 reads with an average length of 400 bp. The saturated rarefaction curves and species richness indices indicate that the sampling was comprehensive. We found highly diverse bacterial communities, with individual sample containing approximately 361 microbial operational taxonomic units (OTUs). A total of 17 bacterial phyla were obtained from the flies. A phylogenetic analysis of 16S rDNA revealed that Proteobacteria was dominant in all samples (75%–95%). Actinobacteria and Firmicutes were also commonly found in the total clones. Klebsiella, Citrobacter, Enterobacter, and Serratia were the major genera. However, bacterial diversity (Chao1, Shannon and Simpson indices) and community structure (PCA analysis) varied across samples. Female ovary has the most diverse bacteria, followed by male testis, and the bacteria diversity of reproductive organs is richer than that of the gut. The observed variation can be caused by sex and tissue, possibly to meet the host's physiological demands.