| Blattella germanica(German cockroach)belong to arthropods door blatt,is a common worldwide health pests.German cockroach relatively short life cycle,with a strong reproduction and adaptability,has gradually become indoor cockroach dominant species it can carry a variety of bacteria,viruses and parasitic ovum,lead to the spread of the disease,the insect body secretions or death can result in serious body allergy use of chemical pesticides,the B.germanica resistance problem increasingly prominent,it is particularly important to research efficient environmental protection biological control pesticide.Metarhizium anisopliae as a common fungus preparation,due to the strong specificity high security on the prevention and control of cockroaches and other pests are widely used,its are main the feature is insects by penetrating skin infection,through the mouth infection study is less,in only a few reports in insects,but due to pests commonly for chewing mouthparts,complicated diets,edible glue bait in the roaches prevention drug application is more,M.anisopliae for cockroaches control play a role in biological agents into the host of the digestive tract of chance is very high,so the mode of transoral infection of B.germanica with M.anisopliae is worth investigating.B.germanica has a large number of microorganisms in its gut,which play an important role in protecting the host against the invasion of insect pathogens and enhancing immunity.Therefore,the study on the interaction mechanism of B.germanica’s gut microbiota and its fungi suppression has important theoretical significance and application value for the biological control of cockroaches.In this paper from the cultivation of the German cockroach in the laboratory and high virulence beetle bassiana fungus(M.anisopliae)as the research object,through determined the toxicity of B.germanica to M.anisopliae transoral infection,tissue slice observation,composition of the gut microbiota,and reveal the host immune response and so on several levels of insect gut bacteria to M.anisopliae resistant bacteria infection mechanism,explore new pesticide effect targets or way,for the development of new high efficient environmental protection after the match fungus insecticidal baits to lay the foundation.This experimental study mainly obtained the following experimental results:1.Toxicity of periplaneta germanica after oral feeding of metarhiziumFirst measured the bacteria through the mouth feeding different concentration of M.anisopliae conidium(1×10~5,1×10~6,1×10~7,1×10~8and 1×10~9cfu/m L)and the inside of the virulence of German cockroach adult male individuals after 15 d,found that mortality is proportional to the concentration of size,along with the increase of the concentration of spores of the mortality of the German cockroach also will increase,which in high concentrations of 1×10~9cfu/m L concentration can reach 50±2.89%mortality rate,preliminary confirmed bassiana fungus infection can pass through the mouth the blattela germanica.At the same time,compared with the infection mode of surface infection with metarhizium,it was found that the efficiency of infection of surface infection with M.anisopliae through digestive tract to blattella germanica was also significant,although far lower than the mortality rate of surface infection 15d after inoculation(75±5.47%,P<0.05).2.Histopathological studyHistopathological sections were used to observe the pathological development of B.germanica oral infection with M.anisopliae(1×10~9cfu/m L)within 7 d.The results showed that spore germination was inhibited during the survival period of B.germanica infected with M.anisopliae,and no intestinal infection and malpighian tube and other surrounding tissues in the spore germination and mycelium was found before host insect dead,fungus infected host insects success after dying insect body organization began to appear in the walls of the intestines of hyphae,try insect mycelium after death spread to various organizations,intestinal parts of the hyphae are mainly concentrated in the outer wall of the gut,spore propagation through the intestinal walls from the blood cavity and dissolve the gut,which results in the digestive tract is full of hyphae;Mycelium was found in other tissues,such as muscle,testis and martensite canal,and the tissues were dissolved to some extent.The germination and penetration of M.anisopliae spores in the gut of B.germanica were not observed within 7 d.3.Effect of gut microbiota of German cockroach on M.anisopliae oral infectionMore than 41%of the spores of metarium suspension were excreted in the feces24 h after oral feeding to B.germanica,and the spore germination rate was significantly increased by comparing the spore containing feces suspension with the unwashed culture after cleaning and separation(P<0.05),indicating that there were indeed substances inhibiting the fungal spore germination in B.germanica’s intestines.The death rate of B.germanica was significantly lower than that of B.germanica(P<0.05).The results showed that the gut microbiota of B.germanica played a certain role in resisting pathogen fungus infection.Then,Mi Seq sequencing was performed on the gut microbiota of B.germanica after 4-day oral infection of metarhizium,and the changes of gut microbiota of B.germanica before and after oral infection of metarhizium were compared.OUT cluster analysis showed that the cockroaches in Germany were mainly Firmicutes,Bacteroidetes,Proteobacteria and Fusobacteria,but the relative abundance of Bacteroidetes decreased significantly after infection with M.anisopliae(29.82%<40.86%,P<0.001).At the genus level,the relative abundance of Alistipes decreased significantly after infection with metarium(4.92%<7.31%,P<0.05),while the relative abundance of Weissella also increased(4.99%>1.41%,P>0.05).Through further analysis of species composition,it was found that among the unique 14 OUT contained in the experimental group,including Proteobacteria,Firmicutes,Bacteroidetes and Actinobacteria,the bacteria contained in the experimental group,such as Perlucidibaca,Ralstonia,Flavobacterium and Granulicatella,were mostly opportunistic pathogens,although the abundance of these pathogens was still very low.However,there will be a potential promotion effect for metarhizium infection in the later stage.In addition,a total of nine strains of Pseudomonas and Bacillus with disease-resistant fungal activity were screened and identified.4.Dynamic changes of immune genesAfter oral infection with M.anisopliae,the immune system of B.germanica was challenged.By means of q RT-PCR,it was detected that the expression of related immune genes was up-regulated or down-regulated within 7 d of infection compared with the normal control.When M.anisopliae infected B.germanica by oral infection,the expression level of Akirin gene was significantly up-regulated at 24-72 h(P<0.05),and gradually up-regulated after the down-regulated expression level in the middle and late stage of infection.The expression level of Bg TPS gene was significantly down-regulated at 48 h after host intake of M.anisopliae(P<0.05)and increased at the later stage of infection.The expression level of Bg Po began to be significantly up-regulated after metarhizium stimulated the host immune system for24-72 h(P<0.05),and was significantly up-regulated at the later stage of infection(P<0.05).The expression of Bg Chi gene was significantly increased when a large amount of M.anisopliae entered the intestinal wall of the digestive tract for 48 h in the early stage of infection(P<0.05),and significantly increased in the middle and late stage of infection(P<0.05).The expression level of Bg Fas 1 gene in the process of infection was generally stable,and the expression level in response to M.anisopliae infection was significantly up-regulated only at the first 48 h(P<0.05).CYP5G19 gene was significantly upregulated during antifungal infection(P<0.05),and significantly upregulated in the early stage of infection compared with the later stage.The immune system of blattella germanica stimulates and competes with M.anisopliae.M.anisopliae has a great influence on the immunity of B.germanica in the early and late stages of infection.5.Control of B.germanica by biocombination of M.anisopliae and boric acidThe lab by oral feeding the M.anisopliae and boric acid treatment of B.germanica,found that the different concentration of boric acid(0.4-1.6%)with different concentration of M.anisopliae(1×10~7,1×10~8and 1×10~9cfu/m L)of synergy12 compound with each one in the experimental group with the German cockroach mortality rates were greater than 93%,with 11 distribution group of poison factor>20,belong to the synergistic effect,1 group for additive effect(total poison factor=10),each group has not been found antagonism,and concentration of boric acid in the highest(1.6%)and metarhizium anisopliae bacteria distribution can achieve 5 d of the lethal time experiments showed that metarhizium and boric acid had a good synergistic effect,and the preparation of compound bait had a good application prospect in the control of German cockroach. |
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