The Effect Of Gut Microbiota In Chemical Communication Of Tribolium Castaneum

Tribolium castaneum is an important grain storage pest,and its complex feeding has caused serious economic losses to China's grain warehouse.In this paper,the mating behavior of T.castaneum was first observed to clarify the mating rythm of T.castaneum adults Then,during the mating peak,the selection behavior between male and female T.castaneum from different geographical population was determined to clarify the release source of pheromone Geographical populations with different selection behavior were screened out,and the difference of pheromone composition was further confirmed.Then,composition of gut microbiota was analyzed in geographical populations with different chemical communication behavior with 16 SrDNA high-throughput sequencing,combined with microbial culture and physiological and biochemical determination.The relationship between gut microbiota and the pheromone composition was clarified.Finally,the effects of gut microbiota on T.castaneum chemical communication were verified by the removal and transplantation of specific gut bacteria.The main results are as follows1.The results of mating behavior study showed that the mating behavior of T.castaneum had significant daily rhythm and hourly rhythm.Mating behavior occurred on the first day after emergence.The mating frequency was the highest on the fourth day after emergence,and at 16:00-18:00 of a day.During the mating peak period,the mating selection behavior between males and females from six different geographical populations was measured by Y-type olfactometer.The results showed that only the male had significant attraction to the female,and there were significant differences in attraction among males from different geographical populations(P < 0.05).The volatiles from the male of T.castaneum were analyzed and identified by dynamic gas collection and GC-MS.The main chemical components of the volatiles were methyl-p-benzoquinone,4,8-dimethyldecanal,1-tetradecene and 1-pentadecene.The results of behavioral determination showed that 4,8-dimethyldecanol had the strongest attractive activity.Quantitative anlysis results showed that the contents of 4,8-dimethyldecanal and other components in the volatiles of T.castaneum changed significantly in different geographical populations.2.16 SrDNA V3-V4 mutation region sequencing method was used to analyze the composition of gut microbiota pf three geographical populations,viz.ZJJX,HBWH and HNZZ,which has different chemical communication behavior.The results showed that the composition of gut microbiota in different geographical populations was similar,and the abundance of gut microbiota was significantly different.The dominant genera in T.castaneum are unclassified-f-Enterobacteriaceae,Pseudomonas,Acinetobacter.It was found that the abundance of unclassified-f-Enterobacteriaceae in three geographical populations was: HBWH > HNZZ > ZJJX,which was consistent with the releasing rate of 4,8-dimethyldecanal.It suggests that the change of chemical communication might be related to gut microbiota.Six strains with different morphological characteristics were isolated and purified from gut microbiota of T.castaneum by microbial culture method.They were Escherichia coli,Citrobacter freundii,Klebsiella pneumonia,Bacillus cereus,Staphylococcus aureus,Enterococcus faecalis when they were further identified with 16 SrDNA amplification sequencing.3.The susceptibility of six culturable microbial to ten antibiotics was determined by K-B paper test.The results showed that six culturable microbial were sensitive to various antibiotics,and they most sensitive to ciprofloxacin.qPCR was used to detect the abundance of culturable gut microbotal.The results showed that after feeding ciprofloxacin,the abundance of culturable gut microbiota decreased significantly;After feeding culturable gut microbiota,the abundance of corresponding gut microbiota increased significantly,It suggests that ciprofloxacin and gut bacteria feeding can effectively regulate the abundance of specific gut microbiota.Combined with the functional verification experiments,it was found that the attraction of male to female and the releasing rate of 4,8-dimethyldecanal increased significantly after E.coli and C.freundii were implanted,which suggest that gut microbiota can affect the chemical communication of T.castaneum,and the key microbiota might be E.coli and C.freundii.