Effects Of Gut Bacteria On Phosphine Tolerance Of Tribolium Castaneum

Tribolium castaneum（Herbst）is a significant secondary pest of stored grain,which seriously endangers the safety of stored grain.At present,phosphine fumigation is still an important technology for the control of stored grain pests in China and even around the world.However,the long-term and unreasonable application of phosphine has led to the rapid development of phosphine resistance of stored grain pests worldwide.Gut bacteria can affect insect resistance,which provides a new research direction for the causes and influencing factors of phosphine resistance in stored grain pests.In this study,firstly,through the determination of phosphine resistance of different strains,the phosphine-resistance strain of Guangdong Shantou（GDST）of T.castaneum was screened out,and it was used as the experimental object to isolate and identify gut bacteria.Optimization of removal and reintroduction conditions for gut bacteria,effects of specific gut bacteria on phosphine tolerance,fitness,redox system,and immune system of T.castaneum.The main research findings are as follows:（1）Through the traditional microbial culture and bacterial 16S r DNA molecular identification technology,in vitro culture and screening of gut bacteria of T.castaneum,12strains of gut bacteria were successfully cultivated,namely Bacillus aryabhattai,Brevibacterium sanguinis,Enterococcus faecalis,Enterococcus faecium,Mesobacillus subterraneus,Microbacterium arborescens,Micrococcus lylae,Ornithinibacillus scapharcae,Staphylococcus capitis,Staphylococcus hominis,Staphylococcus kloosii,and Staphylococcus epidermidis.（2）Through the drug susceptibility test,combined with the quantification of the abundance of gut bacteria and fitness test,the removal and reintroduction conditions of gut bacteria in T.castaneum were optimized.In the drug susceptibility test,the antibiotics used to remove gut bacteria were determined to be penicillin,ciprofloxacin,and vancomycin.By feeding different concentrations of antibiotic mixture（1:1:1）,the physiological indicators of the adult worms and the abundance of gut bacteria have significant changes.Based on this,the best way to remove the gut bacteria from the adults was selected as 20μg/g antibiotic mixture was treated for 72 h.By feeding antibiotic mixture feeds with different concentrations,the developmental duration of the larvae and the abundance of gut bacteria in the gut of the larvae changed significantly.Based on this,the best way to remove the gut bacteria from the larvae was selected as 10μg/g antibiotic mixture was treated for 72 h.The following quantitative experiments showed that the selected method can meet the expectations of the experiment effect,and minimize the negative impact of antibiotics on T.castaneum.（3）Through the determination of phosphine tolerance and fitness of T.castaneum,the relationship between gut bacteria and phosphine tolerance of T.castaneum was studied.Under the stress of phosphine,compared with the sterile group,the mortality of the gnotobiotic group of E.faecium and E.faecalis was significantly increased,the tolerance was significantly reduced,and the egg production of adults was significantly decreased.This shows that E.faecium and E.faecalis can reduce the phosphine tolerance of T.castaneum.（4）The effects of E.faecium and E.faecalis on the oxidative balance of T.castaneum under phosphine stress were studied through the determination of oxidative stress indicators,antioxidant enzymes’activities and expressions.Under the stress of phosphine,the content of H₂O₂ and MDA in the sterile group increased significantly,and the content of GSH first increased and then decreased.SOD activity significantly increased,while CAT and POD activity significantly decreased.And compared with the sterile group,the H₂O₂ content in the gnotobiotic group of E.faecium and E.faecalis decreased,the MDA content increased,the GSH consumption decreased,and the activities and expressions of SOD,CAT,and POD were inhibited,which indicated that E.faecium and E.faecalis can aggravate the oxidative stress in T.castaneum under phosphine stress,disrupt the oxidative balance in T.castaneum,and then affect its phosphine tolerance.（5）Through the determination of the abundance of gut bacteria and the expression of immune genes,the effect of gnotobiotic treatment of E.faecium and E.faecalis on the immune stress,its abundance will exceed the normal level at the beginning,but it will also gradually decrease to the normal level and remain stable.In the initial stages of gnotobiotic treatment,the expressions of immune-related genes PLC-β,Can B,ATF2,DUOX,IMD,and Toll were significantly up-regulated,which indicated that the gnotobiotic treatment would cause a strong immune response and affect the oxidative balance and physiological state of T.castaneum.