Expression And Functional Study On Matrix Metalloproteinase Genes In Aethina Tumida

The hive beetle（Aethina tumida）is a class of Coleopteran that mainly feeds on pollen and honey,resulting in honey fermentation and damage to the bee brood,and has become a serious problem for beekeeping production and wild bee colonies.Matrix metalloproteinases（Mmps）are a family of Zn²⁺dependent endopeptidases that degrade most components of the basement membrane（BM）and extracellular matrix（ECM）and play an important role in metamorphosis development and innate immunity of insects.However,there is no study reported on the association of Mmps with A.tumida.In this project,6 AtMmps genes family members（AtMmp1,AtMmp2,AtMmp3,AtMmp4,AtMmp5 and AtMmp6）were identified based on the obtained transcriptome database of A.tumida,analyzed the molecular properties of the 6 proteins encoded by the above mentioned genes by bioinformatics and determined the temporal and spatial expression profiles of AtMmp1,AtMmp2 and AtMmp3,further investigated the biological functions of AtMmp1,AtMmp2 and AtMmp3using RNAi technology,and finally,transcriptome sequencing technology was used to analyze the regulatory role of AtMmp2 in adult eclosion and wing development.The major reserach findings were as follows:（1）A total of 6 AtMmps genes family members were obtained,which were named AtMmp1,AtMmp2,AtMmp3,AtMmp4,AtMmp5 and AtMmp6 according to the conserved nature of the catalytic structural domain,encoding 561,686,691,247,248 and 242 amino acid（aa）,respectively.Among these,AtMmp1,AtMmp2 and AtMmp3 had a typical structural domain of Mmps,while AtMmp4,AtMmp5 and AtMmp6 had no hemopexin-like C-terminal domain.In different tissue parts of larvae of A.tumida,AtMmp1,AtMmp2 and AtMmp3 had the highest expression level in the gut,followed by the fat body.Among the different tissue parts of the adults,AtMmp1 and AtMmp2 had the highest expression in the hindwing,followed by the forewing and integument;AtMmp3 had the highest expression in the integument,followed by the fat body.All 3 genes mentioned above were expressed at different developmental stages of A.tumida,among which AtMmp1 had lower expression in both embryo and larval stages,with higher expression in1-day-old pupae and highest expression in 1-day-old adults;compared to embryo and feeding stage larvae,the expression of AtMmp2 and AtMmp3 increased after 9-day-old larvae,while AtMmp2 had the highest expression in 1-day-old pupae,followed by 1-day-old adults;AtMmp3had the highest expression in 1-day-old adults,followed by 14-day-old larvae.（2）Compared to the dsgfp group,the expression of AtMmp1 was significantly downregulated in the ds AtMmp1 group（P<0.05）,the expression of AtMmp2 was significantly downregulated in the ds AtMmp2 group（P<0.05）and the expression of AtMmp3 was extremely significantly downregulated in the ds AtMmp3 group（P<0.01）;the AtMmps activity was significantly reduced in both the ds AtMmp1and ds AtMmp3 groups（P<0.05）and was highly significantly reduced in the ds AtMmp2 group（P<0.01）.However,only ds AtMmp2-treated insects showed eclosion failure and lethal phenotype.Compared to the dsgfp group,the hindwing surface of the ds AtMmp2 group showed folds,uneven distribution and blunting of hair-like structures,as well as a large empty cavitie between the upper epidermis and lower epidermis and thickening of the chitin layer.The synthesis of new cuticle and the degradation of old cuticle were delayed after injection of ds AtMmp2,and the new cuticle and chitin layer of emerged adult became thinner.Interference with ds AtMmp2 resulted in epidermal permeability increased and significantly reduced（P<0.05）the survival time of A.tumida in both elevated and desiccation environments.Meanwhile,interference with ds AtMmp2 had no effect on the survival rate,Am Mmp2 expression and adult phenotype in Apis mellifera ligustica（A.m.ligustica）.（3）DEGs analysis showed that whole body and hindwing contained 540 and 2396 DEGs,respectively,with 221 were shared DEGs and 319 and 2175 unique DEGs,respectively.These DEGs are mainly involved in metabolic pathways and cellular processes,while significantly enriched in Toll and Imd signaling pathways and steroid biosynthesis.Cluster analysis revealed that interference with AtMmp2may affect the hindwing ecdysone metabolic pathway-related genes（CYP307A1,CYP302A1,CYP315A1 and CYP18A1）,epidermal sclerosis and melanization metabolic pathways-related genes（bursicon,balck,ebony,th,ddc,yellow,tan,laccase-2 and aa NAT）,chitin metabolism pathway-related genes（Treh,Gfat1,Gnpnat,Uap,chs-1,chs-2,idgf4,cht-2,cht-3,cht-4,cht-5 and CDA1）,prophenoloxidase activating system-related genes（PPAF1,PPAF2 and PPAF3）and Toll and Imd signaling pathways-related genes（PGRP-SA,PGRP-SC,PGRP-LA,PGRP-LB,Spatzle,Dorsal,Toll,cactus,Jra,Kenny and DUOX）and other expressions.In summary,this study identified 6 AtMmps genes,clarified the expression patterns of AtMmp1,AtMmp2 and AtMmp3 in different tissue sites and developmental stages,analyzed the functions of AtMmp2 in the developmental process of A.tumida,and revealed the preliminary revealed the regulatory role of AtMmp2 in the development process of A.tumida.The results of this study can provide a theoretical basis and candidate targets for the control of A.tumida.