Study Of The Diversity Of Cuticular Microbiome Of Locusta Migratoria

The cuticle is the first barrier of insects to resist the external environment.There are a large number of microorganisms attached to the surface of the cuticle.In the long-term evolutionary process,insects have formed a complex symbiotic relationship with microorganisms,which makes insects have stronger adaptability to the environment.However,there is still a lack of systematic studies on insect cuticular microbes.Locusta migratoria are important agricultural pests,which mainly harm crops of gramineous plants and cause huge losses in crop yields.However,there are few studies on the structural composition and community diversity of locust cuticular microorganisms.In this paper,L.migratoria is used as the research object,using metagenomic high-throughput sequencing technology,traditional isolation and culture methods to analyze the diversity of bacteria and fungi in the cuticle of locusts,and compare the differences of cuticular microorganisms at different stages of migratory locusts.The results provide a basis for screening microorganisms that have protective effects on the development of locusts.The main findings are as follows:一、Diversity of the bacterial community in the cuticle of L.migratoria.Selecting the fifth-instar 1-day nymphs(N5D1)and 3-days after ecdysis nymphs(N5D3)that have just molted and are not tanned,and using high-throughput sequencing technology to sequence the V3-V4 regions of the16 S r RNA gene fragments of locusts cuticular microorganisms.Analyze the diversity of bacterial community of cuticle.It was found that the identified classification of the cuticular bacteria of locusts belongs to 22 phyla,44 classes,92 orders,145 families,257 genera,and 168 species.Firmicutes(50.62%)is the dominant bacteria in N5D1 nymphs,and Proteobacteria(37.67%)is the dominant bacteria in N5D3 nymphs.At the genus level,Weissella(1.93%-34.94%)is the dominant genus and has a higher abundance in N5D1.At the species level,N5D1 migratory locust cuticular bacteria(168species)are 28 more species than N5D3 cuticular bacteria(140 species).Lactococcus garvieae(7.97%)is the dominant genus and is highly abundant in N5D1.Diversity index analysis showed that N5D1 nymphs of L.migratoria had a higher level of cuticular bacterial diversity,while N5D3 nymphs were lower.二、Community diversity of cuticular fungi in L.migratoria.Using high-throughput sequencing technology to sequence and analyze the ITS1 area of the cuticular microbes of the L.migratoria,it was detected that the annotated main classification ofthe N5D1 and N5D3 samples belong to 5 phyla,15 classes,30 orders,45 families,65 genera and 56 species.Fungus of Ascomycota are the predominant bacteria in all samples(58.07%-99.05%).At the genus level,the dominant bacteria of N5D3 epidermis is Meyerozyma(39.54%),followed by Aspergillus(22.41%-22.89%).The bacterial abundance of the cuticle in N5D1 locusts is higher.At the species level,the dominant strain is M.caribbica distributed in N5D3 nymphs.By comparing the different strains of cuticular fungi in the two periods,it can be seen that the diversity level of cuticular fungi of N5D1 nymphs is not significantly different from that of N5D3 nymphs.Four fungus are found on the cuticle of N5D1 samples(A.levis,M.thermophilus,A.caviariforme,R.thermophila),but not found in N5D3;The cuticle of the N5D1 nymphs does not contain three kinds of fungi: Lactobacillus horakii,L.dimorphum,and M.brevicaulis.三、Traditional isolation,culture and identification of cuticular microbes of L.migratoria.Seven strains of bacteria were isolated and identified from the cuticle of N5D1 nymphs using traditional culture methods,including K.cowanii,S.marcescens,E.hormaechei,O.pseudogrignonense,S.cohnii,C.funkei and M.arborescens.Six strains of bacteria were identified in the cuticle of N5D3 nymphs,including C.testosteroni,S.gallinarum,S.marcescens,and K.pneumoniae K.pneumoniae,D.tsuruhatensis and S.cohnii.Among them,S.marcescens and S.cohnii were identified in the two sets of samples and belonged to the dominant bacteria.The 13 strains of bacteria identified belong to the phylum Proteobacteria and Firmicutes,which is consistent with the results of high-throughput sequencing.Two fungi were isolated from the cuticle of N5D1 nymphs using traditional culture methods,including M.guilliermondii and Fusarium sp.The two fungi identified in the epidermis of N5D3 locusts were C.quercitrusa and M.guilliermondii.Among them,M.guilliermondii was identified in both sets of samples and is dominant bacteria.The cuticular fungus of the locusts were identified by traditional culture methods belong to the phylum Ascomycota,which is consistent with the results of high-throughput sequencing.The identification of bacteria and fungi on locust cuticle through high-throughput sequencing technology and traditional pure culture technology has laid a solid foundation for the analysis of key microbial related genes in the cuticle of L.migratoria and the analysis of microbial functions,which provides more powerful information and theoretical data for the green prevention and control of L.migratoria.