The Interaction Mechanism Research Between Melanotus Cribricollis Larvae And Metarhzium Sp.

Melanotus cribricollis larvae is one of the most rampant forestry pests caused serious damage during shoot period in southern China.It is hard to monitor and control this pest.Metarhizium spp.is a great choice of wireworm biocontrol.Previously,an entomopathogenic fungal strain,Metarhizium pingshaense WP08,was isolated from the beetle cadavers indicating that the fungus could be used as a biocontrol measure against M.cirbricollis.However,prior to building on such measures,it is essential to advance our understanding of the molecular mechanisms concerned on the insect-fungi interaction.The purpose of this paper is to screen high pathogenicity Metarhizium strain and explore the immune response of protective enzyme system in wireworm after fungal infection.And the high throughout sequencing was used for the first genetic background report of M.cirbricollis larvae and M.pingshaense WP08 at the transcriptome level,in order to excavate insect immune related genes and Metarhizium spp.virulence related gene.The results showed as below:(1)The pathogenicity test of three Metarhizium spp.isolates against M.cribricollis larvae under different spore concentrations was performed.The dynamic activity changes of three protective enzymes,superoxide dismutase(SOD),peroxidase(POD),and catalase(CAT),during different infect time were also measured.The results showed that those three Metarhzium spp.isolates possessed lethal property to infected M.cirbricollis larvae,M.pingshaense WP08 strain was the best,the second was M.anisopliae WTKH strain and M.anisopliae var.acridum strain was the worst.The pathogenicity was enhanced with the increased spore concentration.Under 1.3×108 spore.g-1 soil concentration condition,M.pingshaense WP08 strain exhibited the highest toxicity and all of the experimental larvae died at 16 d,it had no significant difference with 1.3×107 spore.g-1 soil concentration but 1.3×106 spore.g-1 soil concentration.The dynamic change trends were varied of three protective enzyme activities.SOD activity presented unobvious change trend,however POD and CAT activities were firstly down-regulated then up-regulated and declined again subsequently.Those results indicated that M.pingshaense WP08 strain was the best entomopathogenic fungi to invade M.cribricollis larvae compared with M.anisopliae WTKH strain and M.anisopliae var.acridum strain,thus could be the first choice for wireworm biocontrol in forestry.It was speculated that fungal infection probably had influence to those three enzyme activities at least during our sampling time,especially as to POD and CAT activities.(2)The transcriptome information of M.cribricollis larvae was obtained at different inoculation times(CK,1d,3d,4d,5d,7d)by RNA-Seq.In total,800 M reads with high quality were acquired,64000 Unigenes were resembled,the mapping ration between various samples and the reference Unigene library was ideal(>80%),and the ratio of functional annotation was about 40% based on NCBI-Nr,Swiss-Prot,KEGG,COG,KOG,GO and Pfam databases.At the same time,the blastx result indicated that four insects(M.cribricollis,Drosophila melanogaster,Tribolium castaneum and Dendroctonus ponderosae)possessed 9512 homologous gene cluster,4145 single copy gene cluster.At the same time,the transcriptome information of M.pingshaense WP08 was also obtained by RNA-Seq,with 134 M high quality reads,28107 Unigenes,and 70% ratio of functional annotation.Amino acid sequence alignment was conducted between M.pingshaense WP08 and other different five fungi strains,included Aspergillus fumigatus,Ustilago maydis,Magnaporthe grisea,Fusarium oxysporum and human pathogen Cryptococcus neoformans,9189 homologous gene cluster and 1703 single copy gene cluster were acquired.Besides,the genomic structure analysis of both organisms was performed,involved in the CDs,AS,SSR and SNP.(3)The differently expressed genes(DEGs)number of M.cribricollis larvae was analyzed as well as functional annotation,GO enrichment,Pathway enrichment,and the insect immune related genes.The number of DEGs was increased as whole which was consistent with the infection process,also the variety and number of genes in the immune related pathways and the GO term “biological process”.At different infectious stages,Unigenes in some pathways/GO terms were specific to certain infectious stages while others were expressed ubiquitously.Some essential genes whose products have known functions in immune defenses or stress responses were found among the Unigenes and include gram negative binding proteins,?-1,3-glucan recognition protein,peptidoglycan receptor,lysozyme,glutathione S transferase,cytochrome P450,dehydrogenase/reductase SDR family,et al.On the other side,PHI database alignment was carried out to dig out more pathogenicity related gene of entomopathogenic fungi.Finally,1529 Unigenes acquired functional annotation of homologous genes from other microorganisms,included 35 annotation results resulted from Metarhzium spp.and Beauveria spp.Those genes were Bbbqr A,BbCHIT1,Chi2,CYP52X1,Kre2,Mnt1,Ktr1,Ktr4,MaSnf1,Pr1,OpS1,mrGAT,MrpacC,Ohmm,Fkh2,MBZ1,mas5,mhk1,Bck1,Fus3,Mkk1/2,Slt2,Ste11,Ste7,Hog1,Pbs2,Ssk2,ras3,vosA,wetA,et al.,acted on insect integument degradation,oxidation-reduction,carbon source utilization,phosphorylation,transcription factors regulation,cell cycle,osmotic regulation,growth and development,and other aspects to regulate the fungal multiple-stress resistance and pathogenicity.The insect-fungi interaction mechanism was explored through physiological and molecular levels in this study.It would be the theoretical basis and supportance for the intensive study of insect immune mechanism,also could benefit for the construction of higher efficient genetically engineered strain,and promote the development of pest biological control technology.