Research On The Genetic Basis Of Mulberry Silkworm Flight Degeneration

The acquisition of insect flight ability occurred approximately 400 million years ago and is an important milestone in adaptive evolution of insects.Flight capacity greatly expands locomotor ability of insects,which is beneficial for them to find mates,avoid predators,and forage for food,and greatly promotes a wide range of spread of insects.However,in the course of evolution,many flying insects lost their ability to fly.Wollaston et al.recorded that 200 of the 500 beetles on the Madeira Islands lost their flight ability.Almost all insects have no flying ability in some insect groups,such as Blattaria and Siphonaptera.Flight loss is also common in groups such as stick insects,locusts,crickets,and earwigs.Although some insects have wings,they have lost the ability to fly.Roff et al.had estimated that there are about 5%of winged insects cannot to fly,and some insects even lose their wings entirely.In summary,the degradation or loss of flight ability is common in insects.Studying the genetic mechanism of insect flight degradation is of great significance for humans to understand the adaptive evolution of insects and pest control.Silkworm,Bombyx mori(B.mori)is domesticated from wild silkworm,Bombyx mandarina(B.mandarina),and is the only insect that has been fully domesticated by humans.In the field of fundamental research,silkworm is regarded as a model insect of Lepidopteran.In the long-term process of artificial selection,silkworm had lost the flight ability and became a group of flightless insects.The loss of flight ability facilitates the large-scale breeding of silkworms and egg production,which is an important event in the domestication of silkworms.For a long time,researches on the economic traits,genetic mechanism of important genetic mutations and individual development has been the main direction of the basic research of silkworm.Flight loss is regarded as a normal functional degradation and few people pay attention.But as an important morphological change,it is necessary to analyze the genetic mechanism of silkworm flight degeneration.This work can not only enhance people's understanding of the domestication history of silkworm,but also provide reference for the study of insect flight degradation.It is also of great significant for explaining of the genetic mechanism of insect adaptive evolution and of great value for applied research of pest control in Lepidopteran.Compared with B.mandarina,the phenotype of B.mori has changed greatly during the domestication process.Which phenotypic changes lead to the degradation of silkworm flight.Is the loss of silkworm flight a conscious choice of humans,or a gradual degradation(relaxation of purification selection)in the process of consciously improving economic traits?Which genes are related to the flight loss of silkworm.What happened to these genes during the domestication of silkworm?These issues are still unclear.In this study,approaches of morphology,genomic bioinformatics and functional genomics are used to investigate and analyze some morphological traits related to silkworm flight.Comparisions of morphological traits between B.mandarina and B.mori were performed to find some differences.By the detection of the positive selection sites of the whole genome,the selection pressure analysis of related genes,and combined with the expression data,the genes related to the degradation of flight ability were screened out,and the functions of sevral important related genes were analyzed using RNA interference(RNAi)approach.The main results are as follows:1.Investigation and comparative analysis of morphological characters related to flight of silkwormWith the methods of tissue dissection,tissue sectioning and high-speed imaging,the wing area,wing shape,wing mechanical properties(stiffness),wing loading(body weight/wing area),flapping frequency,flapping amplitude,Flight muscle weight,flight muscle microstructure,ratio of flight muscle weight to body weight(flying muscle weight/body weight)and other related parameters were compared and analyzed.We found that the mechanical properties,wing loading,wing flapping ability,flight muscle microstructure and flight muscle ratio are significantly different between B.mori and B.mandarina.Further analysis showed that the degeneration of flight muscles may have played an important role in silkworm flight loss.The mitochondrial number and ATP content of the flight muscles of B.mori and B.mandarina were investigated and compared.It was found that the ATP content in the flight muscles of the wild silkworm was three times that of the domestic silkworm,indicating that insufficient energy supply may be an important reason for the flight loss of silkworm.2.Analysis of selection pressure on the whole genome of silkwormGenome-wide variation detection and analysis were performed on 135 and 20resequencing data of B.mori and B.mandarina,and 37 417 881 SNPs(Single Nucleotide Polymorphism)and 10 183 483 INDELs(Insert and Deletion)were screened.A total of397 genes with positive selection signals were identified in the whole genome.Analyzing the results of the NR(non-redundant database)annotations of these genes,we found 2genes related to muscle development,7 genes related to energy metabolism,1 gene related to neurodevelopment and 2 genes related to apoptosis and autophagy.Paramyosin and tintin are associated with muscle development.Energy metabolism related genes include ATP synthase,fatty acid binding protein and glycogen phosphorylase.3.Transcriptome analysis of silkworm flight musclesThe anatomical observation and analysis of the marker gene(?-actin)expression suggested that the 6^thday of pupa stage(P6)is an important period of flight muscle development.Our analysis of the transcriptome of silkworm in P6 showed that 6187 genes were differentially expressed in the flight muscles and 3654 genes were down-regulated in the domestic silkworm.KEGG enrichment shows that the functions of these genes are mainly enriched in pathways such as metabolic pathways,MAPK signaling pathway,mitophagy,autophagy and apoptosis pathway.There are 1422 genes that are expressed differently in the flight muscles of M1(the 1^stday of adult)in domestic and wild silkworm,and 723 genes are down-regulated in domestic silkworm.KEGG enrichment analysis shows that these genes are mainly enriched in metabolic pathways,citrate cycle and fatty acid metabolic pathways.The results suggest that genes related to citrate cycle and fatty acid metabolism may be related to the difference in energy supply of flight muscles between the B.mori and B.mandarina.Among these differential expressed genes,Bmtitin2,Bmscu and Bmgs2 harbor positive selection singals.Bmtitin2 is a structural protein related to flight muscle,Bmscu encodes 3-hydroxyacyl-Co A dehydrogenase(HOAD),and Bmgs2 is related to the assembly of neurotransmitters and nerve synapses.We consider these 3 genes as candidates for the flight loss of silkworm.4.Nucleic acid sequence analysis of Bmtitin2,Bmscu and Bmgs2 genesThe protein coding sequences and regulatory sequences of Bmtitin2,Bmscu and Bmgs2 in B.mori and B.mandarina were analyzed respectively,and it was found that the coding regions of these three genes had no variations that could cause gene function changes between wild and domestic silkworm.In the regulatory region,compared with B.mandarina,there are two P-element transposon insertions in B.mori at 341bp and 361bp location of upstream of the Bmscu gene,respectively,and the insertion lengths are 1343bp and 2814bp,respectively.Among them,the insertion of the transposon at 361bp is common in silkworms.Silkworm strains with this transposon insertion accounted for about 83%of the silkworm population,and the transposon insertion was not detected in the wild silkworm population,which suggests that the insertion of the transposon occurred during the domestication of silkworm and was gradually fixed through artificial selection.Several B.mori specific indels were found in the upstream sequence of the Bmgs2 gene in B.mori.These specific indels may affect the expression of Bmgs2 in silkworm.In the downstream of Bmtitin2,20 SNPs with significant differences of allele frequencies between domestic and wild silkworm were identified.The variation of these SNPs may be related to post-transcriptional regulation of Bmtitin2.5.Functional verification of genes related to silkworm flight lossThe upstream 2kb sequences of the Bmscu and Bmgs2 of domestic and wild silkworm were cloned,and the dual luciferase reporter assay was conducted.Results show that the upstream regulatory sequences of B.mandarina has higher promoter activities than that of B.mori.We performed RNA interference(RNAi)of Bmscu and Bmgs2 in F₁hybrids of domestic and wild silkworm and results show that the maximum speed and average speed of silkmoths were significantly reduced,and the numbers of pause were significantly increased.These results indicate that Bmscu and Bmgs2 play an important role in maintaining the flight ability of silkmoths.Bmscu encodes 3-hydroxyacyl-Co A dehydrogenase(HOAD),which has an important catalytic role in fatty acid beta oxidation and can catalyze fatty acid oxidation and release large amounts of ATP.We performed RNAi of Bmscu in the BmE cell line and found that the enzyme activity of HOAD and ATP content were significantly reduced.This indicates that the down-regulated expression of Bmscu has limited the produce of ATP,and the decrease in ATP content in the silkworm flight muscle may be caused by the down-regulated expression of Bmscu.In summary,we draw the following conclusions:Flight loss of silkworm is related to the microstructure and energy supply of flight muscles.The flight loss of silkworm is affected by positive selection and is a result of artificial selection.The expression of Bmtitin2,Bmscu and Bmgs2 in the flight muscles of B.mori was significantly lower than that of B.mandarina.This differential expression pattern was caused by the variation of their regulatory sequences.The down-regulation of Bmscu and Bmgs2 affected the flight ability of silkworm,which may be the reason for the silkworm flight loss.The down-regulation of Bmscu can inhibit the enzyme activity of HOAD and affect the produce of ATP in silkworm,indicating that Bmscu may affect the energy supply of flight muscles by inhibiting fatty acid beta oxidation,thereby affecting the flight of B.mori.