| Plutella xylostella(L.)is one of the most serious migratory pests that damage cruciferous plants in the world.Gut bacteria play a significant role in insect nutrition,physiology and behavior.Previous studies of gut microbial diversity were primarily based on the sequencing analysis of 16 S rDNA,but this method could not distinguish active microbes in gut bacteria.In order to comprehensively evaluate the composition and changes of the gut bacteria of P.xylostella,especially the composition of active microbes during different growth and development and different physiological states.This study combines the research of 16 S rDNA and16 S rc DNA in order to analyze the diversity,dynamic changes,influencing factors and potential functions of gut bacteria of P.xylostella in development,feeding,sex and mating,which lay the foundation for following basic researches and the development of new pest management strategies.The results are as following:1.Gut bacterial diversity on P.xylostella life cycleProteobacteria,Firmicutes and Actinobacteria are the principal dominant bacteria in the gut microbes of P.xylostella,primarily consisting of Enterobacteriales,Pseudomonadales,Burkholderiales,Rhizobiales,Sphingomonadales,Lactobacillales and Actinomycetales.The gut bacteria of P.xylostella were relatively stable in terms of species composition,but there were dynamic changes in species abundance.Among them,the egg,larval stage,pupal stage,and adult stage showed obvious population specificity associated with development.The composition structure of egg species is relatively simple.As the larvae begin to eat,the diversity of gut microbes becomes more complicated.After entering the pupa,the composition structure of the gut microbesis further simplified due to metamorphosis.At this time,the Enterobacteria of Proteobacteria became the absolute dominant bacterial group.It is worth noting that the gut diversity of the advanced larvae and the pre-pupa is close.However,with the development of the pupa,the gut microbes are simplified.After entering the adult stage,and the species structure is restored.These results indicate that as the growth and development of P.xylostella,the diversity ofgut microbes will be accompanied by changes to adapt to the corresponding physiological alterations and functional requirements.Comparing the results of sequencing methods between 16 S rDNA and16 S rc DNA,the sample microbes based on 16 S rc DNA sequencing was found with more diverse composition and more uniform distribution of active species.Pseudomonadales,Burkholderiales and Actinomycetales are more abundant in 16 S rc DNA-derived samples.Especially,Actinomycetales are high-abundant active microbial groups at various stages of development,indicating that these bacterial groups undertake more roles during the growth and development.Conversely,the relative abundance of Lactobacillales in samples based on 16 S rc DNA is significantly lower than that in 16 S rDNA samples,suggesting that Lactobacillus may not be as important as previous thoughts in actual processes of metabolism.The above results demonstratethat the true active flora in the gut of P.xylostella are easy to be found through comparing the sequencing results of 16 S rDNA and 16 S rc DNA,providing a direction for better functional approach.2.P.xylostella gut bacterial diversity on different gendersAnalysis of the diversity of different gender of gut microbes in P.xylostella(4th instar females,4th instar males,female pupae,male pupae,female adults and male adults)found that there are differences in the diversity of different gender of gut microbes in P.xylostella.However,the change pattern is different at different developmental stages with no consistent rule,indicating that the difference between male and female is also related to the physiological state of a specific development period.The relative abundance of Burkholderia in unmated males is higher than that in unmated females;the relative abundance of Bacillales in unmated females is higher than that of males;Rhodobacterales is relatively abundant in females is higher than that of males after mating,suggesting that these bacterial groups may be related to the regulation of the physiological functions of P.xylostella with different gender,or due to the physiological status of P.xylostella with different genderregulating the diversity of these microbes.In addition,the relative abundance of Pseudomonadales,Burkholderiales,Rhizobiales,Rhodobacterales and Bacillus in the male and female P.xylostella based on 16 S rc DNA samples are higher than that in 16 S rDNA samples.These floras are worthy of attention in the following studies on the male and female differences of P.xylostella.3.P.xylostella gut bacterial diversity on different mating conditionsData analysis indicates that mating does affect the diversity of the gut bacteria of P.xylostella.The analysis based on 16 S rDNA or 16 S rc DNA alpha-diversity both show that gut microbial diversity decreases after mating,and such trend is more significant in 16 S rDNA samples.Interestingly,as the female gut microbial diversity decreases after mating,the microbial diversity of the eggs laid by females after mating is also lower than the parthenogenetic females.After copulation,the relative abundance of Rhodobacterales and Paracoccus in the gut tract of female adults increased,while the relative abundance of Ralstonia in the male decreased.The relative abundance of Lactobacillus in female adults is higher than that in males after mating.Besides,the relative abundance of Pseudomonas,Burkholderia,Rhizobia,and Bacillus were all higher in samples by 16 S rc DNA sequencing.It indicated that these bacteria may also play an important physiological role in reproductive mating process of P.xylostella.4.P.xylostella gut bacterial diversity on different feeding conditionsA research on whether feeding affects the gut microbes of P.xylostella found that feeding can significantly increase the gut microbial diversity of P.xylostella.After feeding,the relative abundance of Cyanobacteria and Burkholderiales increased significantly in the first instar larva and the relative abundance of Pseudomonas,Burkholderiales and Rhizobiales also increased in the adults.While the Actinobacteria is relatively less abundant After feeding.It is speculated that these bacteria may participate in the food metabolic process as active bacteria in the adults after feeding.The gut microbial diversity is more abundant after eating,it may suggest that food not only brings microbes,but also promotes the reproduction of microbes in the intestine of P.xylostella,which may affect the related physiological functions of P.xylostella.In conclusion,there are differences in the gut microbes of P.xylostella under different physiological and performance states.The 16 S rc DNA sequencing-derived bacterial flora has a higher diversity at the classification level below the Order,which indicates that a large number of bacteria are involved in related physiological and metabolic processes at the level of live bacteria.This research investigates the gut bacteria of P.xylostella based on the two dimensions of 16 S rDNA and 16 S rc DNA,and analyzed in detail the biodiversity and changes of gut flora between different ages,feeding,sex,mating,etc.The diverse microecological environments lays the foundation for follow-up basic research and the development of new pest management strategies based on the gut microbes of P.xylostella. |
PDF Full Text Request