Chitin Binding Proteins Affect The Flexural Stiffness Of Wing In Bombyx Mandarina And B. Mori

Insects are the first animals on the earth to have the ability to fly,which makes them extremely competitive in many aspects such as finding mates,searching for food,avoiding predators,and expanding their distribution.Therefore,they have become the most prosperous animal group on earth.About 5,000 years ago,the silkworm(Bombyx mori)was domesticated from the wild silkworm(B.mandarina).Long-term domestication and artificial breeding made it impossible to fly.Wild silkworms living in the wild still rely heavily on their flight ability in terms of finding mates,laying eggs,and avoiding natural enemies.Wings are an important organ for insects to produce flight behavior,and their morphological structure and mechanical properties are important factors that determine flight efficiency.When the rigidity of the insect's wings is low,it is insufficient to resist the air stress during flight and excessive deformation occurs,which will cause the insect to fail to fly normally.Therefore,the comparative study of B.mori and wild silkworm is a good model for exploring flight-related mechanisms.In this study,B.mandarina and B.mori were used as the study models.The hardness of vein and the thickness of epidermis were compared.At the same time,based on the identified differentially expressed genes between the wings of B.mandarina and B.mori,we have screened out the genes related to wing firmness formation through phylogenetic analysis and temporal and spatial expression patterns.We used prokaryotic expression,immunohistochemistry,RNAi and other techniques to study the gene functions.The role of the target gene in wing formation was elucidated,and the possible molecular mechanism of the loss of flight ability was revealed.The results are as follows:(1)In this study,the wing disc of B.mandarina and B.mori were dissected at different developmental stages.In the whole process of wing development,wing morphology obviously changed twice during pupation and eclosion.From larval to pupal stage,the wing disc develops from vesicle to wing shape,and its area increases about 10-15 times.During the pupal stage,the size of wings changed small and the most significant feature was that the veins of wings became thicker and hardened gradually.During eclosion,the veins are tanned and the wing is completely expanded and hardened.The size of the wing is about twice that of the wings at the late pupal stage.Generally speaking,wing development is a process from small to large and from soft to hard.The wing discs of B.mori and B.mandarina showed the same tendency during the whole development process.However,the hardness and thickness of the wing in B.mori were significantly lower than those of B.mandarina.The results showed that the flexural stiffness of the wing vein of B.mandarina was significantly higher than that of Bombyx mori(P < 0.0001).The thickness of the skin layer of the vein was observed under the scanning electron microscope.The results showed that the thickness of wing vein in B.mandarina was significantly larger than that in Bombyx mori(P < 0.001).These results quantitatively proved that the hardness and thickness of the wings in B.mandarina were significantly greater than those in Bombyx mori.(2)Fifteen differentially expressed genes have been identified between the wing transcriptomes of B.mandarina and B.mori,Among them,8 genes belong to RR-2subfamily and 7 genes belong to RR-1 subfamily.BmCPR59 is the gene with the highest expression level in the adult wing of wild mulberry silkworm,and its expression level is tens to hundreds of times different from that in the wing of B.mori.The results showed that BmCPR59 was highly expressed in the wings of pupal 1 day and adult.Phylogenetic analysis showed that BmCPR59 had high homology with Tc CPR27,which was highly expressed in the elytra of Tribolium castaneum.These results indicate that BmCPR59 might play an important role in wing formation.(3)The function of BmCPR59 was further verified.The prokaryotic expression vector p Cold ?-BmCPR59 was constructed,and the protein in the form of inclusion body was induced and expressed in E.coli.High purity protein was obtained by nickel column and molecular sieve,and polyclonal antibody was prepared.The distribution of BmCPR59 protein in wing tissue was localized by polyclonal antibody.At the same time,the chitin in wing tissue was localized by Fluorescent Brightener 28.Under the confocal microscope,the localization of BmCPR59 protein and chitin almost coincided with each other,and they almost existed in the whole wing vein and wing membrane.The expression level of BmCPR59 in B.mandarina was decreased by RNA interference.Compared with the control group,the individual gene expression level after interference was significantly decreased(P < 0.01).The flexural stiffness and the thickness of the vein in the interference group and the control group were measured.The results showed that the flexural stiffness of the vein in the interference group was significantly lower than that in the control group(P1,P < 0.01;P2,P < 0.05).The correlation between the expression level of BmCPR59 and the flexural stiffness of the vein was analyzed,showing a moderate positive correlation(P1,r = 0.7574123;P = 0.0004295;P2,r = 0.6874889;P =0.002291)The results showed that the BmCPR59 could affect the vein stiffness of silkworm through the comparison of the vein flexural stiffness between B.mandarina and B.mori,and the study of the function of BmCPR59.The low expression of BmCPR59 gene may be one of the main reasons for the lower stiffness of the silkworm's wings,thereby affecting its flight ability.The results of this study provide an important basis for exploring the mechanism of insect flight loss.