Comparative Research On Silk And Silk Protein Of Butterflies And Moths

Spiders forming webs,silkworms spinning silk,wasps building nests...Animal secretion of silk is widespread in nature.For hunting,for protection,or for reproduction...A variety of silk secreting behaviors have become spiders,insects and other millions of animals are important means for survival and reproduction.In nature,there are more than850,000 species of animal silk,more than 200,000 species of Lepidoptera insects and30,000 species of spiders can secrete silk.Among them,lepidopteran insects are composed of butterfly insects,Rhopalocera,and moth insects,Heterocera.There is evidence that butterfly insects have the same silk secretion ability as other lepidopteran insects.However,the current research on lepidopteran insect silk is only focused on some insects of Bombycidae and Saturniidae,which are closely related to the silkworm.But the butterfly silk secretion and butterfly silk has not been reported yet.The researches of materials science have revealed the excellent comprehensive mechanical properties of animal silk.As a natural protein fiber,it has both toughness and strength.Even the best comprehensive mechanical properties of artificial fibers are still difficult to compare with animal silk.At present,researchers have analyzed the unique structure of animal silk on the macro and micro scale,that is,a semi-crystalline fiber material formed by the crystal region embedded in the amorphous protein matrix region.Among them,the crystal region is mainly an antiparallel?-sheet structure,which ensures that the silk fiber provides strength and elastic modulus.the amorphous protein matrix region is mainly a random crimp and helical structure,which provides the ductility of the silk fiber.The crystal region and the amorphous region are further assembled to form nanofiber bundles,and finally constitute the entire silk fiber.This progressive multi-level structure is the reason for the excellent mechanical properties of animal silk.Although there is a mass of factors that affect the performance of silk formation,the most important determinant is the sequence of silk protein itself,that is,the composition and sequence of amino acids.The excessively high mutation rate of insect silk protein coding genes leads to the rapid differentiation of their sequence composition,resulting in a rich diversity of lepidopteran insect silk protein sequences.So far,the lepidopteran insects with the silk protein sequence have been reported to include only a few species with high spinning volume such as Bombyx mori,Antheraea pernyi,Attacus cynthia ricini Boisduval,etc.However,as far as we know,most of the lepidopteran insects can spin silks.Therefore,based on the above background analysis,we collected 7 kinds of butterfly silk and nearly 30 kinds of moth silk,and carried out comparative studies on silk structure and silk properties.In order to explain the effect of silk protein sequence on the mechanical properties of silk,this study simplified the research model,using silk protein sequences of different lepidopteran insects for eukaryotic expression and artificial spinning.In this way,we investigate the influence of pure sequence differences on the final filament structure and performance.The main findings are as follows:1.The phenomenon of butterfly silk secretion and butterfly silkFirstly,we collected Kallima inachus Swinhoe,Cethosia biblis,Tirumala limniace,Danaus chrysippus,Idea leuconoe,Papilio demoleus Linnaeus and Papilio polytes,a total of 7 species of butterfly larvae and pupae,from 2 superfamily and 7 families,together with their host plants.According to its different pupation methods,it can be divided into two types:pupa adheraena and pupa contigua.The pupa adheraena are more common in Nymphalidae,Satyridae,Danaidae,etc.,while the pupa contigua are more common in Papilionidae,Pieridae,Riodininae,etc.The pupa adheraena and the pupa contigua use silk in the same way but some differences.The same is that they both have cremaster on their tails,which can be hooked with silk pads secreted by the larvae to fix the pupae.The barbs on the cremaster and silk pads are hooked and fixed to each other in a way like Velcro.The special feature of constriction pupa lies in the constriction filament.Constriction filament is a structure like a wire rope formed by winding 8-12monofilaments secreted by larvae.It is connected to a silk pad and passes through the pupa shell to fix the upper part of the pupa body.There is no essential difference in composition between the constriction filaments and the silk pad.However,after multiple strands of constriction filaments are helically wound and assembled,the comprehensive mechanical properties of the constriction are greatly improved.2.Comparison of differences between cocoons and silks of lepidopteran insectsIn addition to the 7 species of butterflies mentioned above,we also collected cocoons and silks of various lepidopteran insects.5 insect cocoons were selected from them,and the morphological comparison of various cocoons and silks was carried out by means of cameras,microscopes,and scanning electron microscopes.It is found that the cocoon layer morphology of lepidopteran moths can be divided into two types:dense porous structure and continuous sericin film.The specific difference of the cocoon layer is reflected in the continuity of sericin,the pore size of the cocoon layer,the presence or absence of non-silk components,and oxalate content.The comparative study on the cocoons of 5 species of moths showed that the cocoons of B.mori and Bombyx mandarin are dense and porous structures with small pores,and the composition of the cocoon is relatively uniform,with no non-silk protein components.The Andraca biupunctata Walkeris an insect of Bombycidae.It is closely related to B.mori in taxonomy.Its cocoon layer forms a continuous sericin film.The cocoon layer has a uniform composition and a small amount of calcium oxalate crystals can be seen on the surface.The Eriogyna pyretorum,Saturniidae,moth insect with a large cocoon shape.The cocoon layer forms a continuous sericin film.Many calcium oxalate crystals can be seen on the surface of the cocoon silk.The Clania variegata Snellen is an insect of Psychidae.It is characterized by cocoon covering the whole period of its larvae.Its cocoon layer is a dense and porous structure with large pores.The cocoon layer contains branches and leaves,soil,and other environmental components.Furthermore,this study used static tensile experiments and Fourier Transform Infrared Spectroscopy(FTIR)to compare the mechanical properties and the differences in the secondary structure of silk proteins of three silkworm insect silks and three butterfly silks.The analysis found that the comprehensive mechanical properties of the three butterfly silks are all lower than that of the three silkworm silks.The breaking strength of the silkworm silks are more than 400 MPa,but the breaking strength of butterfly silks are only about half.In terms of extensibility,the extensibility of three silkworm insect is 18-20%,while the extensibility of three butterfly silks is only 10-13%.In terms of tenacity,silkworm silks are far superior to butterfly silks,with a difference of 6-10 times.The results of the secondary structure detection of silk protein showed that the infrared spectra of the 6 kinds of lepidopteran insect silks all showed the material characteristics of the protein matrix.There were obvious absorption peaks in the amide I,amide II,and amide III regions.The deconvolution analysis of the amide III region showed that relative to the35-38%?-sheet content of the three silkworm silks,the?-sheet content in the butterfly silks was significantly lower than that in silkworm silks.This result is consistent with the law of fracture strength in the mechanical performance test results,and the two show an obvious positive correlation.Therefore,we speculate that because butterfly silks contain less?-sheet structure,its breaking strength is lower than silkworm silks.3.The eukaryotic expression and artificial spinning of lepidopteran insect silk fibroin heavy chain proteinIn order to analyze the influence of silk protein sequence on silk formation,we constructed a simple experimental method model.According to the evolutionary degree of the species,from the reported lepidopteran insect silk fibroin heavy chain protein sequences,five species were selected as candidates.A single repeating unit was intercepted in the silk fibroin repeat region,and the design passed the corresponding number of repetitions.The molecular weight of the obtained recombinant protein is about50 k Da.Then,according to the designed protein sequence,with the rare codon library of Pichia pastoris,using the p PICZ?A vector,four protein expression strains were obtained,and large amounts of expression and purification were performed.In the end,two types of lepidopteran insect silk fibroin recombinant proteins were successfully obtained:E.variegate Fb H-RP based on the silk fibroin heavy chain repeat sequence of the Eumeta variegate,and P.machaon Fb H-RP based on the silk fibroin heavy chain repeat sequence of Papilio machaon Linnaeus.Dissolve the two recombinant proteins in HFIP to produce about 10-20%(w/v)spinning dope.Use wet spinning technology to control the same spinning conditions and perform artificial After spinning,the obtained nascent silk fiber is stretched 4 times to form two uniform wet-spun yarns:E.variegate RP-WF and P.machaon RP-WF.And,the mechanical properties and secondary structure of the obtained silk fiber were tested.The experimental results show that the comprehensive mechanical properties of the two wet spinning yarns are obviously different:the breaking strength of E.variegate RP-WF is 67.66 MPa and the elongation is 2.87%;the breaking strength of P.machaon RP-WF is 36.14 MPa and the elongation is 3.26%.Combined with the results of infrared spectroscopy,the content of?-sheet structure in wet-spun silk of the two recombinant proteins was 10.3%and 6.6%,respectively,confirming that the silk properties are indeed related to the content of?-sheet structure.By comparing the analysis results of the secondary structure content of the protein predicted by the software based on the amino acid sequence,it is speculated that the polyalanine block(poly alanine block)and(GA)_n structure in the sequence are the main reason affecting the?-sheet content and silk mechanical properties.