The abuse of chemical pesticides has led to strong drug resistance of Blattella germanica,and even had a negative impact on the ecological environment,so there is an urgent need to explore alternative strategies for chemical control of cockroaches.In contrast,biological insecticide made from microorganisms have the advantage of target touch and pests are less likely to develop resistance,making them a highly promising research direction.S.marcescens is a gram-negative bacteria that produce toxins to control pests,which can infect some Lepidoptera and Orthoptera pests,and has control effect on B.germanica.M.anisopliae is an entomopathogenic fungus with a wide parasitic spectrum,it mainly infects insects through the body surface,deprives the host of nutrition or produces toxins,which has been widely used in the control of various pests.Compared with traditional chemical insecticides,single entomopathogenic bacteria have the disadvantages of slow onset,long cycle time and short duration of control effect,in order to shorten the insecticide time and improve the insecticide efficiency.This study attempts to use S.marcescens in combination with M.anisopliae,exploring their insecticidal effects and mechanisms,which may provide a good experimental basis for developing an efficient and environmentally friendly biological insecticide.1.Insecticidal effect of the compound on B.germanicaWhen the combination of S.marcescens and M.anisopliae was used to treat B.germanica,the mortality rate significantly increased compared to the single treatment group at the same time and concentration.For instance,when the B.germanica was treated with 2μl of S.marcescens and M.anisopliae at the concentration of 1×10~9cfu/m L,it killed all of them at 9 d.The mortality was 5.00±0.00 in S.marcescens group and 85.00±7.64 in M.anisopliae group(P<0.05).The co-toxicity factor was 54(According to the co-toxic factor method of Mansour,higher than 20 is synergistic effect,lower than-20 is antagonistic effect,and there is additive effect between them),indicating a synergistic effect.2.Effect of compound on the histopathology of B.germanicaThe compound treatment caused more damage to the tissue of B.germanica.After 4 d of infection with S.marcescens,only part of the peritrophic membrane was destroyed,while after 4d of infection with M.anisopliae,the gut was still intact,and only a few spores of M.anisopliae were found in the gut.After 4 d of compound treatment,the gut was seriously damaged,large faults appeared,microvilli fell off,a large number of M.anisopliae spores were found in the gut and peritrophic membrane,the septum of fat body was disintegrated completely,a large number of M.anisopliae spores were found at the edges,and the damage to the spermary was obvious and the arrangement of testicular rudiment became loose.3.Effect of compound on the infection process of pathogenic microorganismDetection of the infection process of pathogenic microorganisms against B.germanica in vivo and vitro.After being treated with S.marcescens,a small amount of S.marcescens was found on the gut culture medium on the 6 d;while after compound treatment,S.marcescens grew on the gut medium on the 4 d.After treated with M.anisopliae,the dry weight of M.anisopliae on the 6 d of hemolymph culture in vitro was lower than that on the 4th day of the compound group(49.00±1.45mg vs 57.87±1.73 mg).The results of q RT-PCR showed that on the 2nd,4th and 6th day after infection,the relative abundance of S.marcescens in the gut and hemolymph of the compound group was 2.07,2.87,5.05 times and 1.96,3.7,4.02 times higher than that of the S.marcescens group(P<0.05),respectively.The relative abundance of M.anisopliae in the gut and hemolymph of the compound group was 3.37,3.76,2.74 times and 4.26,3.56,2.35 times higher than that of the M.anisopliae group(P<0.05),respectively.4.Effect of compound on gut microbiota of B.germanicaThe results of high-throughput sequencing of gut microbiota showed that compound had a significant effect on gut microbiota.Compared with the control group,After compound,the relative abundance of Firmicutes increased by 10.39%.At the genus level,the highest relative abundance is Parabacteroides,(C:8.87%,S:10.91%,M:8.76%,SM:12.92%,P<0.05),Weissella(C:0.98%,S:3.70%,M:1.22%,SM:3.80%,P<0.05)were significantly increased in different experimental groups.Verified by q RT-PCR method,it was found that the change of relative abundance was consistent with that of high-throughput sequencing.When it was fed to B.germanica with S.marcescens,it was found that it could significantly enhance the insecticidal effect of S.marcescens,increased the mortality rate by 61.54%within 20 days(P<0.05);when the gut microbiota of B.germanica were removed with antibiotics,the pathogenic effect of S.marcescens was more obvious,and the mortality rate was 46.15%higher than that the gut microbiota were present,while when B.germanica recovered,there was no significant difference in mortality between each treatment group.5.Effect of compound on immune genes of B.germanicaIn this thesis,we studied the effects of compound on five immune genes of B.germanica(drosomycin_g3 and termicin_g3 are anti-fungal infection;Akirin,Imd and Attacin-like_g1 are anti-Gram-negative bacteria).When B.germanica was infected with S.marcescens and M.anisopliae,the expression of two antifungal infection genes increased continuously,and tended to be highly and stably expressed on the 3rd day to fight the pathogen,which was significantly different from that of M.anisopliae(P<0.05),the expression trend of upstream immune genes Akirin and Imd in Imd pathway was similar,both increased at first and then decreased,and a large number of antimicrobial peptides were produced through cascade reaction,and the expression multiple was significantly different from that of S.marcescens(P<0.05).The combination of S.marcescens and M.anisopliae showed obvious synergistic effect,the co-toxicity factor was 54,probably by destroying important tissues such as fat body and may weaken the antibacterial ability by disturbing gut microbiota and inhibiting the expression of immune genes.In this study,we describe the different susceptibilities of the B.germanica to S.marcescens,M.anisopliae and the combination of the two.The traditional chemical insecticidal method was abandoned and a completely biological approach was used to kill the cockroach.These findings will help us to better utilize microorganisms for control of the B.germanica. |