The Study On Synergistic Mechanism Of Boric Acid Enhancing The Infection Of Blattella Germanica By Metarhizium Anisopliae

Blattella germanica,as a vector insect,widely exists all over the world.It can carry and spread a variety of pathogens,cholera,plague,leprosy,typhoid and other diseases transmission of the important carrier.Due to the extensive use of chemical insecticides,B.germanica has developed resistance to many kinds of insecticides.Therefore,it is particularly important to develop new efficient and environmentally friendly biological insecticides.As a common pathogenic fungus,Metarhizium anisopliae has the advantages of strong specificity,high safety and sustainable control as the main biologically active preparation,but the disadvantages of slow action and unstable action limit its production and application.Based on the literature reports and the previous results of our laboratory,it was found that the combination of M.anisopliae and inorganic insecticide boric acid had obvious synergistic effect,but the synergistic mechanism was not clear at present.Based on this,a combination with significant synergistic effect（1.2%boric acid vs.1×10⁹cfu/m L M.anisopliae）was used to treat B.germanica in this study and intended to reveal the synergistic mechanism between boric acid and M.anisopliae from the aspects of midgut histopathology,gut microbiota structure and function,insect immune defense,which would lay a foundation for the development of efficient and environmentally friendly compound fungal insecticidal agents.The experimental results obtained in this study are as follows:1.Effects of boric acid and M.anisopliae on midgut histopathology of B.germanicaThe effects of 1.2%boric acid and 1×10⁹cfu/m L M.anisopliae on the midgut tissues of B.germanica were observed by paraffin section on day 4（the death peak was reached in the combination group）.The results showed that the midgut structure of B.germanica in the control group was complete,and the microvilli were arranged neatly and closely;complete and ordered basement membrane and muscular-conjunctive system separated the midgut epithelium from hemolymph.In contrast,the ingestion of boric acid baits resulted in substantial destruction of midgut epithelium,loose microvilli in the midgut lumen,vacuolation between epithelial cells,disordered nuclear arrangement,and destruction of the integrity of basal layer.The single ingestion of M.anisopliae had no significant effect on midgut.The effect of combination treatment on midgut of B.germanica was similar to that of boric acid treatment alone,but the colonization of M.anisopliae conidium was found on the microvilli,suggesting that the destruction of midgut structure by boric acid could assist M.anisopliae to pass through physical barriers such as peritrophic membrane.2.Effects of boric acid on the infection process of B.germanica by M.anisopliaeMicroscopic examination of the hemolymph of the mixed-treated cockroaches showed that M.anisopliae invaded the hemolymph of B.germanica on the 1st day of the combination,while spores could be detected in the hemolymph of the only M.anisopliae group on the 2nd day.q RT-PCR was used to characterize the relative abundance of M.anisopliae in the gut and hemolymph of B.germanica at day 3,4 and 5 after compound treatment.It was found that the relative abundance of M.anisopliae in the gut and hemolymph of B.germanica in the combination group was significantly higher than that in the M.anisopliae group（P<0.05）,which further confirmed that M.anisopliae in the combination group could invade the hemolymph more quickly and accelerate its proliferation.3.Effects of boric acid and M.anisopliae on gut microbiota of B.germanicaHigh-throughput sequencing based on 16S r RNA was used to characterize the changes of gut microbiota of B.germanica after combined treatment at day 4.Boric acid,M.anisopliae and their combination had significant effects on the gut microbiota of B.germanica,and the optimal bacteria changed from Bacteroidetes in the control group to Firmicutes.In addition,the number of bacteria in the four treatment groups was similar,but there were significant differences in relative abundance.The relative abundance of Parabacteroides（9.40%vs.4.32%,P<0.01）and Enterococcus（3.54%vs.1.74%,P<0.05）was significantly decreased in the gut of the boric acid-M.anisopliae group.The relative abundance of Weissella（12.05%）was significant difference compared with the control group（1.41%,P<0.01）and M.anisopliae group（3.54%,P<0.05）.The results showed that the combination could cause the imbalance of gut microbiota of B.germanica,which may be an important reason for the synergistic effect of boric acid and M.anisopliae.4.Functional validation of differential gut microbiota WeissellaWeissella was successfully isolated from the gut by in vitro culture,and the relative abundance of Weissella in the gut and hemolymph of B.germanica at day 4 was detected by q RT-PCR.It had found that the relative abundance of Weissella in the gut and hemolymph of the compound group was higher than that of the other three groups,in which,the relative abundance in hemolymph was 2.35 times that of the control group,2.37 times that of M.anisopliae group and 1.54 times that of boric acid group.The virulence of Weissella was found to be very small when it was returned into B.germanica by oral feeding.When Weissella was injected into the body wall,a large number of worms died（51.67±5.77%,P<0.001）in the high concentration group（1×10⁹cfu/m L）.In addition,the LT₅₀of removing the gut microbiota and returing Weissella were significantly lower than those of the single compound group（P<0.05）.5.Effects of boric acid and M.anisopliae on immune genes expression of B.germanicaq RT-PCR was used to characterize the expression of six antifungal-related immune genes in B.germanica in the combined treatment at 12 h,24 h,48 h,72 h and 96 h,it was found that after M.anisopliae infection,except Bg Fas1,the expression of the other five genes were up-regulated in different degrees compared with the control group.The expression of Bg Fas1gene did not increase significantly at other time points except that it increased slightly at 72 h（1.62 times of control group）.After boric acid infection,the expressions of Bgchi,CYP4G19and Akirin genes were up-regulated,while the expressions of Bg Po,Bg Fas 1 and DRS-7 genes were down-regulated.It was noteworthy that the expression of these antifungus-related immune genes in B.germanica treated with boric acid and M.anisopliae was almost all significantly lower than that in M.anisopliae alone at the same period,and the expression of Bg Po and CYP4G19 in M.anisopliae group was 15.83 times and 11.14 times as high as that in the compound group at 48 h.6.Effects of boric acid and M.anisopliae on polyphenol oxidase and catalase activity of B.germanicaThe dynamic changes of polyphenol oxidase（PO）activity in the gut and hemolymph of B.germanica in the combined treatment at 12 h,24 h,48 h,72 h and 96 h were characterized by q RT-PCR.It was found that the PO activity in the gut of B.germanica in the compound group was lower than that in the other two groups,and there was significant difference at 72 h and 96h（P<0.05）,which was 0.27 times of that in the boric acid group and 0.03 times of that in the M.anisopliae group at 72 h.The results of catalase（CAT）activity showed that CAT activity in gut and hemolymph of B.germanica in the compound group was significantly lower than that in the other two treatment groups（P<0.01）,and also significantly lower than that in the control group（P<0.001）.At 72 h,the activity of CAT in the compound group was only 0.23times that in the boric acid group,0.22 times that in the M.anisopliae group,and 0.30 times of the control group.The results showed that the combined treatment of boric acid and M.anisopliae could reduce the PO activity and significantly inhibit the CAT activity.In conclusion,the combination of boric acid and M.anisopliae can damage the midgut structure of B.germanica,accelerate the entry of M.anisopliae into the hemolymph and facilitate its colonization in the gut and hemolymph.At the same time,it can disturb the ecological balance of gut microbiota of B.germanica,resulting in excessive proliferation of the opportunistic pathogenic bacteria in the gut,and then Weissella can enter the hemolymph of B.germanica and accelerate the death of test insects.In addition,the combination can also reduce the expression of antifungal-related immune genes and the activities of PO and CAT in insects to weaken the immune defense,and accelerated the infection of M.anisopliae,resulting in a significant synergistic effect.