Organizational Diversity And Functional Characterization Of Microbiota In The Midgut Of Diamondback Moth, Plutella Xylostella (L.)

The diamondback moth (DBM), Plutella xylostella (L.), is one of the most destructive pests attacking cruciferous crops in the world. Overuse and misuse of insecticides have caused P. xylostella to rapidly develop resistance to many different classes of insecticides making it difficult to control, thus threatening the production of many food crops. Recently, the insecticide resistance and herbivory of P. xylostella have important fields of research. Insect gut microbiota is important in food digestion, host nutrition, development, immune response and even pathogen protection. Recent studies have indicated the links between insect gut microbiota and herbivory or insecticide resistance. In the present study, we investigated these two fields from with regard to P. xylostella midgut microbiota. Our study provided information on the genetic and molecular basis of P. xylostella midgut microbiota for adaptation to food digestion, nutrition and plant defense compounds, which make the insect a more evolutionarily adaptive herbivore. Our study also revealed the effect and mechanism of the midgut microbiota mediating P. xylostella insecticide resistance. The main results are as follows:1. Diversity of microbiota in the P. xylostella midgutBased on the methods of16S rRNA sequencing, metagenome sequencing, and the method of pure culture of microorganisms, we examined the diversity of the P. xylostella midgut microbiota. The result suggest that the Proteobacteria is the most highest abundant bacteria in P. xylostella midgut, and the second is Firmicutes, these two phylum dominated the midgut microbiota, comprised more than97%of the P. xylostella midgut bacteria. The orders of Enterobacteriales and Lactobacillales dominated the phylum of Proteobacteria and Firmicutes, respectively. The three genus of Enterobacter cloacae (Enterobacteriales), Enterobacter asburiae (Enterobacteriales), and Carnobacterium maltaromaticum (Lactobacillales) comprised more than60%of the P. xylostella larval midgut bacteria at species level. Comparative analysis of larva, pupa and adult of the species diversity in the midgut showed that there are no intense change in the three developmental stages, only a slight abundance increase of Proteobacteria in pupa and adult, corresponding to the decrease of Firmicutes. However, at the classification level of species, relative to the larva and pupa, the abundance of Enterobacter asburiae have a greater increased corresponding to the decrease of Enterobacter cloacae in adult. Since larva feeding obligate cruciferous plants, and adults only with honey, the change of species is likely correlated to the transformation of food, prompt P. xylostella midgut commensal bacteria may play a role in the food digestion and nutrient supply. We compared the difference between susceptible and insecticide P. xylostella strains, and studied the change of bacteria structure when P. xylostella was exposed to insecticide. Our results suggest that the insecticide-resistant strains had more Firmicutes and less Proteobacteria compared to the susceptible strain. Consistently, a second study observed an increase in the proportion of Firmicutes in the midgut of P. xylostella individuals from a generation treated with insecticides. The results indicated a possible relationship between P. xylostella midgut microbiota and insecticide resistance.2. Midgut microbiota enhancing the herbivory of P. xylostellaBased on the metagenome of P. xylostella midgut microbiota combined with the functional identification, we analyzed the food digestion, nutrition and detoxification functions of P. xylostella midgut microbiota. A series of enzymes associated with cellulose, xylan and pectin hydrolysis were identified in P. xylostella midgut microbiota. We isolated the bacteria from P. xylostella midgut, and the functional analysis suggested that the bacterial symbionts can hydrolyze cellulose, xylan and pectin. Base on the P. xylostella genome database, we discovered that the P. xylostella lacked the entire pathway to synthesize histidine (His) and threonine (Thr). Interestingly, the metagenome of P. xylostella midgut microbiota possessed the entire pathway of genes that participate in the synthesis of His and Thr, thus indicating host-symbiont cooperation in the production of amino acids. The P. xylostella midgut microbiota may play an important role in host nutrition. Plants in a large variety of cruciferous crops possess a large variety of secondary metabolites that contain a phenolic group. Some phenolics, such as catechol and phenol, are usually toxic to cells and are harmful to insects as antinutritive compounds. Phenolics with catechol nuclei can stimulate DNA degradation by the induction of reactive oxygen species (ROS). These compounds comprise an important part of the plant defense system against herbivores. We discovered the complete aerobe pathway that degrades catechol in the P. xylostella midgut microbiota metagenome. We "also performed experiments to identify the degradation potential, and the functional analysis suggested that the P. xylostella midgut bacteria possess the activity to degrade phenol. Our study also identified a large amount of superoxide dismutases (SODs), catalases and peroxidases in the P. xylostella midgut microbiota, which may participate in the ROS detoxification. Our results suggest that the midgut microbiota contribute to the P. xylostella to become a more evolutionarily adaptive herbivore.3. Midgut microbiota mediating insecticide resistance of P. xylostellaThis project aimed to explore the effect and mechanism of P. xylostella midgut microbiota mediating insecticide resistance. We isolated bacteria from P. xylostella midgut and analyzed the functional effect of the midgut bacteria on the development of insecticide resistance, and we also tested and verified the molecular insights into how the midgut bacteria mediate insecticide resistance by inducing the P. xylostella detoxification enzyme or immune response. The bacteria of Enterococcus sp.(Firmicutes) and Enterobacter sp.(Proteobacteria), and Serratia sp.(Proteobacteria) were isolated from the P. xylostella midgut. P. xylostella were reared in the presence of these bacteria and antibiotics to detect their effect on insecticide resistance. The results suggested that Enterococus sp. enhanced the insecticide resistance of P. xylostella, Serratia sp. decreased the insecticide resistance, and Enterobacter sp. had no significant effect. Moreover, we found that the heat-killed bacteria had no effect on the P. xylostella insecticide resistance. Interesting, the antibiotics enhanced the insecticide resistance. Our results suggest that the composition and structure of midgut microbiota have a role in P. xylostella insecticide resistance. Based on the study of mechanism of the midgut bacteria mediating insecticide resistance, we discovered that the main mechanism does not include the bacteria participating in the direct detoxification of insecticides. The Enterococus sp. and antibiotics all upregulated the P. xylostella midgut detoxification enzyme genes (glutathione S-transferase (GST) and carboxylesterase (COE)), which may be an important mechanism of the midgut bacteria regulation of insecticide resistance. We also identified that the Enterococcus sp. bacteria and three chemicals (antibiotics, vitamin C and acetylsalicylic acid) that enhance P. xylostella insecticide resistance had a similar mode of regulation on the expression of P. xylostella antimicrobial peptide (AMP), but the bacteria with different effects on P. xylostella insecticide resistance induced a different gene expression profile. These results indicate that there may be a relationship between the insecticide resistance and P. xylostella immunity, which is regulated by midgut bacteria.This is the first report of high-throughput DNA sequencing of the entire microbiota of P. xylostella. Our studies combined with the previous P. xylostella genome research provide insights into the mechanism of P. xylostella herbivory. Our studies also revealed that the composition and structure of midgut microbiota correlated with the P. xylostella insecticide resistance. The results from this new project will dramatically advance understanding in the evolution of and basis for P. xylostella insecticide resistance, and they will open new avenues for the development of environmentally benign, cost-effective and sustainable strategies for monitoring and management of P. xylostella. The understanding of the relevant mechanisms and symbiont taxa may lead to more active forms of co-evolution for other insects, thus leading to the development of strategies for more sustainable agriculture.