Virulence Of Metarhizium Anisopliae Extracellular Proteinases In Midgut Of Locusta Migratoria Manilensis Meyen

Metarhizium anisopliae is one of the entomopathogenic fungi which is widely existed in the world. It has already been widely used in the biological control of many kinds of pests and obtained a good effect. The two major ways of M. anisopliae’s infection are through cuticle and intestinal tract. There are more researchs on the cuticle infection and virulence factors and its mechanism to the host is also clear, but the study on infection through intestinal tract is rare. In the previous study, we have found that the virulence of Metarhizium infection through digestive tract is higher than through cuticle. In this paper, the data of midgut transcriptome of the Locust migratory which was infected by with M. anisopliae, effect of M. anisopliae mixed with different inhibitor on the virulence to L. migratory and on the migut-damaging situation were analyzed. Meanwhile, the dynamic changes of the related enzyme activity of M. anisopliae-fed L. migratory were tested. It indicated the mechanisms of M. anisopliae’s extracellular protoeases and made a further theoretical supply for the control efficiency of M. anisopliae on the L. migratory. The main conclusions are introduced as follows:1. Effects of different inhibitors on virulence of Metarhizium anisopliae 4 kinds of extracellular proteinases in midgut of Locusta migratoriaThe L. migratory were fed with M. anisopliae that mixed with 4 inhibitors. 4 inhibitors had no effects on the survival rate of the L. migratory. While TPCK and CII inhibited the virulence of M. anisopliae significantly and prolonged the LT50 of M. anisopliae to L. migratory, EDTA and APMSF did not affect the virulence of M. anisopliae. Their survival rates have no difference to that treated with M. anisopliae but significantly lower than the control.2.Destructive of different extracellular protease of Metarhizium anisopliae IPPM202 on Locust midgutThe ultrastructure of the L. migratory was obviously destroyed by M. anisopliae, with microvilli falling off edoplasmic reticulum expansion and mitochondrial matrix and nuclear membrane degradation. And the breaking situation appeared differently after the use of different mixture. In the treatment of TPCK and CII’s mixture, the midgut’s ultrastructure of L. migratory keeped relatively complete, while damaging situation of treatment of EDTA and APMSF’s mixture was more serious and similar to the M. anisopliae treatment. According to the results of bioassay, TEM and target of the protease inhibitor, it inferred that that protease Pr1 in the extracellular protease of M. anisopliae was the main virulence factor and then Pr4. Pr2 and Pr3 were not involved in the infecting process of M. anisopliae.3. Effect of M. anisopliae on the midgut related enzymes of L. migratoryWhen L. migratory was fed with the mixture of APMSF and TPCK(two kinds of inhibitors of serine protease inhibitor) and M. anisopliae, it’s midgut protease changed with "U" shape with the time tested. The activity of trypsin increased first and then decreased. The activity of the chymotrypsin-like enzyme increased. APMSF and TPCK had no significant effect on those 3 digestive enzymes. APMSF had no effect on the two kinds of detoxifying enzymes. Situation that TPCK affected those two kinds of detoxifying enzymes was similar to the M. anisopliae treatment, but it’s degree was lower. Compared the enzyme activity of the mixed treatment and the separated treatment, these enzymes changed in different degree.4. Analysis on midgut transcriptome of Metarhizium anisopliae-fed L. migratoryThis study determined the differential expressed gene in the midgut between the 3rd instar nymphs of M. anisopliae-fed L. migratory and the control, screened the differentially expressed genes(DEG) using the new sequencing techniques. Meanwhile, the bioinformatics tools were used for function annotation, classification and signaling pathways of the genes which were obtained by transcriptome sequencing. The research resembles non-redundant unigenes of which a total number is 159,278, in which N50 and N90 are 1313 bp and 252 bp, with an average length of 942 bp. The differential expression analysis of all the genes shows that 194 differentially expressed genes are screened by P<0.05, among which 53 genes are up-regulated and 141 genes are down-regulated. The results of down-regulated expression genes’ GO enrichment analysis reveals that 17 of GO terms has the enrichment phenomena which involves 15 biological processes and 2 molecular functions. Similarly, the results of the up-regulated one involves 4 biological processes, 1 cellular component and 2 molecular functions. KEGG metabolic pathway analysis matched 77 differentially expressed genes of the midgut transcriptomes, in which 4 patheways were enriched. The study supplied information for exploring the mechanism of M. anisopliae infection in midgut of locust and was beneficial for increasing the efficiency of M. anisopliae against target pest.