Film formation and structural characterization of silk of the hornet Vespa simillima xanthoptera Cameron

We extracted silk produced by the larva of the hornet Vespa simillima xanthoptera Cameron from its nest. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the extracted hornet silk showed four major components with molecular weights between 35 and 60 kDa. The main amino acid components of the hornet silk protein were Ala (33.5%), Ser (16.9%), Asp (8.5%) and Glu (8.1%). The hornet silk could be dissolved in hexafluoroisopropyl alcohol (HFIP) at 25 degrees C without incurring molecular degradation. A transparent film of hornet silk was obtained readily by the formation of a cast upon drying of the hornet silk in the HFIP solution. Residual HFIP solvent was removed from the film by extraction with pure water. Solid-state 13C NMR and FT-IR measurements revealed that the secondary structures of hornet silk proteins in the native state consisted of coexisting alpha-helix and beta-sheet conformations. The beta-sheet to alpha-helix ratio, which was changed by processing, was mainly responsible for the silk's thermostability.     

Cardioactive effects of hornet venom, Vespa mandarinia

In the experiment of electrocardiogram, the crude venom of giant hornet (Vespa mandarinia) showed cardioactive effects on rat heart. The heart rate was accelerated within 5 min after injection of the venom intraperitoneally, then the heart beat was blocked, resulting in conduction delay. The cardioactive constituent was separated into two components by gel filtration. One which was high molecular species such as protein showed complete atrioventricular block. Another component, having a low molecular weight, was fractionated in 5 peaks, which accelerated heart rate.     

Evidence of α-helical coiled coils and β-sheets in hornet silk

α-Helical coiled coil and β-sheet complexes are essential structural building elements of silk proteins produced by different species of the Hymenoptera. Beside X-ray scattering at wide and small angles we applied cryo-electron diffraction and microscopy to demonstrate the presence and the details of such structures in silk of the giant hornet Vespa mandarinia japonica. Our studies on the assembly of the fibrous silk proteins and their internal organization in relation to the primary chain structure suggest a 172 Å pitch supercoil consisting of four intertwined alanine-rich α-helical strands. The axial periodicity may adopt even multiples of the pitch value. Coiled coil motifs form the largest portion of the hornet silk structure and are aligned nearly parallel to the cocoon fiber axis in the same way as the membrane-like parts of the cocoon are molecularly orientated in the spinning direction. Supercoils were found to be associated with β-crystals, predominantly localized in the l-serine-rich chain sequences terminating each of the four predominant silk proteins. Such β-sheet blocks are considered resulting from transformation of random coil molecular sequences due to the action of elongational forces during the spinning process.     

Identification of four major hornet silk genes with a complex of alanine-rich and serine-rich sequences in Vespa simillima xanthoptera Cameron

Hornet silk, a fibrous protein in the cocoon produced by the larva of the vespa, is composed of four major proteins. In this study, we constructed silk-gland cDNA libraries from larvae of the hornet Vespa simillima xanthoptera Cameron and deduced the full amino acid sequences of the four hornet silk proteins, which were named Vssilk 1-4 in increasing order of molecular size. Portions of the amino acid sequences of the four proteins were confirmed by Matrix-assisted laser desorption/ionization-time of flight/mass spectrometry (MALDI-TOF/MS) and N-terminal protein sequencing. The primary sequences of the four Vssilk proteins (1-4) were highly divergent, but the four proteins had some common properties: (i) the amino acid compositions of all four proteins were similar to each other in that the well-defined and characteristic repetitive patterns present in most of the known silk proteins were absent; and (ii) the characteristics of the amino acid sequences of the four proteins were also similar in that Ser-rich structures such as sericin were localized at both ends of the chains and Ala-rich structures such as fibroin were found in the center. These characteristic primary structures might be responsible for the coexisting alpha-helix and beta-sheet conformations that make up the unique secondary structure of hornet silk proteins in the native state. Because heptad repeat sequences of hydrophobic residue are present in the Ala-rich region, we believe that the Ala-rich region of hornet silk predominantly forms a coiled coil with an alpha-helix conformation.     

Do battles lead to coexistence? Role of interference competition in structuring the insect community on fermented tree sap

Abstract.  1. In insect communities on fermented tree sap, agonistic interactions occur frequently among the component species. This study examined the effects of such interference competition on community structure and species co-occurrence on a spatio-temporally small scale.
2. Experimental removal of giant hornets ( Vespa mandarinia ), one of the most dominant species, showed no effect on either species richness or total abundance of the remaining insects. Significant increases in abundance were detected for five out of 19 taxa: V. ducalis , V. crabro , Lasius japonicus , Cryptarcha lewisi , and a group of micro sap beetles ( Epuraea spp., Haptoncus spp., and Haptoncurina spp.).
3. Behavioural observations indicated that interference did not alter the staying time at a patch (an exuding spot on a tree) for V. mandarinia , Rhomborrhina japonica , and Neope goschkevitschii . Interference-mediated departure from a patch was less frequent than departure without interference for all nine species observed, except for R. japonica . These behavioural contexts varied yearly for several species.
4. These results suggest that interference competition is a minor determinant of overall community structure, despite its conspicuousness. This weak effect of interference may be attributed to the short stay of dominant species and to individual behavioural responses, such as competitor avoidance. The variable influence of interference might be related to differences in species attributes, such as dominance hierarchies.     

Identification of hornet silk gene with a characteristic repetitive sequence in Vespa simillima xanthoptera

Vssilk 5 is a gene encoding a component protein of the silk produced by the larvae of the yellow hornet (Vespa simillima, Vespinae, Vespidae). In this study, we deduced the complete cDNA sequence of Vssilk 5. It was found that 2 silk proteins, Vssilk 5 N and Vssilk 5 C, in the cocoon of the yellow hornet are both encoded by the Vssilk 5 gene. Vssilk 5 N and 5 C are the N- and C-terminal regions, respectively, of the Vssilk 5 pro-protein (Vssilk 5p). The complete amino acid sequences of Vssilk 5 N and Vssilk 5 C were deduced. Although a non-repetitive amino acid sequence and coiled-coil structure are properties common to the major components of silk proteins produced by the larvae of the social superfamilies Apoidea and Vespoidea of the Apocrita, nearly the entire sequence of Vssilk 5 C consisted of a repeated sequence of amino acids, and the calculated coiled-coil probability for this protein was low. Vssilk 5 N is a protein without a repetitive amino acid sequence and has a low coiled-coil probability. Moreover, we found a water soluble protein, Vssilk 5S that is likely segmented from Vssilk 5 C and contains an N-terminal sequence identical to that of Vssilk 5 C.     

Competition between the native and the introduced hornets Vespa crabro and Vespa velutina: a comparison of potentially relevant life‐history traits

1. Invasive alien species are a major threat to biodiversity. In addition to predation and parasitism, native species might suffer from competition when invasive alien species occupy a similar ecological niche. 2. This study focused on the potential interspecific interaction between two hornets: the Asian yellow‐legged hornet, Vespa velutina, a high‐concern invasive alien species recently arrived in Europe; and the native European hornet, Vespa crabro. The two species share a similar ecological niche and V. velutina is rapidly expanding across Europe, which suggests that V. crabro might suffer from competition. 3. Under laboratory‐controlled conditions, two life‐history traits that might cause the two species to compete were investigated: (i) the ability of workers to find food sources and their flexibility in exploiting them (through individual food item choice tests and exploration assays); and (ii) the worker resistance to pathogens (through immune challenge tests). 4. The results show that trophic preference of both species highly overlaps, with a marked dietary preference for honeybees compared with other insect prey and non‐prey protein items. No differences were observed in the exploratory behaviour of both species. Finally, constitutive antibacterial activity was greater in workers of the native species than in workers of the invasive hornet. 5. This laboratory study provides a first assessment under controlled conditions of the factors affecting competition between workers of two hornet species and proposes a framework to assess, in wild contexts, the magnitude of the competition and the impact of the introduced V. velutina on the native V. crabro.     

Single honeybee silk protein mimics properties of multi-protein silk

Honeybee silk is composed of four fibrous proteins that, unlike other silks, are readily synthesized at full-length and high yield. The four silk genes have been conserved for over 150 million years in all investigated bee, ant and hornet species, implying a distinct functional role for each protein. However, the amino acid composition and molecular architecture of the proteins are similar, suggesting functional redundancy. In this study we compare materials generated from a single honeybee silk protein to materials containing all four recombinant proteins or to natural honeybee silk. We analyse solution conformation by dynamic light scattering and circular dichroism, solid state structure by Fourier Transform Infrared spectroscopy and Raman spectroscopy, and fiber tensile properties by stress-strain analysis. The results demonstrate that fibers artificially generated from a single recombinant silk protein can reproduce the structural and mechanical properties of the natural silk. The importance of the four protein complex found in natural silk may lie in biological silk storage or hierarchical self-assembly. The finding that the functional properties of the mature material can be achieved with a single protein greatly simplifies the route to production for artificial honeybee silk.     

Conformation change of hornet silk proteins in the solid phase in response to external stimulation

Hornet silks adopt a variety of morphology such as fibers, sponge, films, and gels depending on the preparation methods. We have studied the conformation change of hornet silk proteins (Vespa mandarina) as regenerated films, using chiroptical spectrophotometer universal chiroptical spectrophotometer 1, which can measure true circular dichroism spectra without artifact signals that are intrinsic to solid‐state samples. The spectra showed that the proteins in films alter the conformation rapidly from the α‐helix to the coiled coil and then to a β‐sheet structure in response to heat/moisture treatment, but the transformation stopped at the coiled coil state when the sample was soaked in EtOH/water solution. Water is required for the α‐helix to the coiled coil transition, and extra energy is required for the further transition to a β‐sheet structure. This is the first successful circular dichroism study of fibril silk proteins to follow the conformation changes in the solid state. This work shows that proteins can undergo conformational changes easily even in the solid phase in response to external stimuli, and this can be traced by solid‐phase‐feasible chiroptical spectrophotometers. Application of unnatural stress to proteins gives valuable insights into their structure and characteristics.     

Molecular and mechanical properties of major ampullate silk of the black widow spider, Latrodectus hesperus

Molecular and material properties of major ampullate silk were studied for the cobweb-building black widow spider Latrodectus hesperus. Material properties were measured by stretching the silk to breaking. The strength was 1.0 +/- 0.2 GPa, and the extensibility was 34 +/- 8%. The secondary structure of the major ampullate silk protein was studied using carbon-13 NMR spectroscopy. Alanine undergoes a transition from a coiled structure in pre-spun silk to a beta sheet structure in post-spun silk. We have also isolated two distinct cDNAs (both about 500 bp) which encode proteins similar to major ampullate spidroin 1 and 2 (MaSp1 and MaSp2). The MaSp1-like silk contains polyalanine runs of 5-10 residues as well as GA and GGX motifs. The MaSp2-like silk contains polyalanine runs of varying length as well as GPG(X)(n) motifs. L. hesperus major ampullate silk is more like major ampullate silk from other species than other L. hesperus silks.     

In-situ observation of tryptophan in larval cocoon silk of the hornet Vespa simillima xanthoptera cameron by ultraviolet resonance raman spectroscopy

We performed in-situ ultraviolet resonance Raman (UVRR) spectroscopy of the larval cocoon silk of the hornet, Vespa simillima xanthoptera Cameron, and compared the result with that of the silkworm, Bombix mori. The UVRR spectrum of the hornet cocoon differed markedly from that of the B. mori cocoon: peaks attributable to tyrosine (Tyr) were observed strongly, and tryptophan (Trp) peaks weakly, in the spectrum of the B. mori cocoon, whereas peaks attributable to Trp exclusively appeared in the spectrum of the hornet cocoon.     

Microsatellite loci for genetic research in the hornet Vespa mandarinia and related species

We developed 5 microsatelitte loci in the hornet Vespa mandarinia from RAPD fragments. The 5 loci showed 3–7 alleles in V. mandarinia and many were also polymorphic in the related species, V. ducalis, V. analis, Dolichovespula norvegicoides and Vespula schrenckii. These results suggested the loci will be useful for analyzing genetic structure of Vespinae, at both the colony and population levels.     

Molecular characterization and evolutionary study of spider tubuliform (eggcase) silk protein

As a result of hundreds of millions of years of evolution, orb-web-weaving spiders have developed the use of seven different silks produced by different abdominal glands for various functions. Tubuliform silk (eggcase silk) is unique among these spider silks due to its high serine and very low glycine content. In addition, tubuliform silk is the only silk produced just during a short period of time, the reproductive season, in the spider's life. To understand the molecular characteristics of the proteins composing this silk, we constructed tubuliform-gland-specific cDNA libraries from three different spider families, Nephila clavipes, Argiope aurantia, and Araneus gemmoides. Sequencing of tubuliform silk cDNAs reveals the repetitive architecture of its coding sequence and novel amino acid motifs. The inferred protein, tubuliform spidroin 1 (TuSp1), contains highly homogenized repeats in all three spiders. Amino acid composition comparison of the predicted tubuliform silk protein sequence to tubuliform silk indicates that TuSp1 is the major component of tubuliform silk. Repeat unit alignment of TuSp1 among three spider species shows high sequence conservation among tubuliform silk protein orthologue groups. Sequence comparison among TuSp1 repetitive units within species suggests intragenic concerted evolution, presumably through gene conversion and unequal crossover events. Comparative analysis demonstrates that TuSp1 represents a new orthologue in the spider silk gene family.     

Evidence for diet effects on the composition of silk proteins produced by spiders

Silks are highly expressed, secreted proteins that represent a substantial metabolic cost to the insects and spiders that produce them. Female spiders in the superfamily Araneoidea (the orb-spinning spiders and their close relatives) spin six different kinds of silk (three fibroins and three fibrous protein glues) that differ in amino acid content and protein structure. In addition to this diversity in silks produced by different glands, we found that individual spiders of the same species can spin dragline silks (drawn from the spider's ampullate gland) that vary in content as well. Freely foraging ARGIOPE: argentata (Araneae: Araneoidea), collected from 13 Caribbean islands, produced dragline silk that showed an inverse relationship between the amount of serine and glycine they contained. X-ray microdiffraction of the silks localized these differences to the amorphous regions of the protein that are thought to lend silks their elasticity. The crystalline regions of the proteins, which lend silks their strength, were unaffected. Laboratory experiments with ARGIOPE: keyserlingi suggested that variation in silk composition reflects the type of prey the spiders were fed but not the total amount of prey they received. Hence, it may be that the amino acid content (and perhaps the mechanical properties) of dragline silk spun by ARGIOPE: directly reflect the spiders' diet. The ability to vary silk composition and, possibly, function is particularly important for organisms that disperse broadly, such as Argiope, and that occupy diverse habitats with diverse populations of prey.     

Variation in protein intake induces variation in spider silk expression

Background

It is energetically expensive to synthesize certain amino acids. The proteins (spidroins) of spider major ampullate (MA) silk, MaSp1 and MaSp2, differ in amino acid composition. Glutamine and proline are prevalent in MaSp2 and are expensive to synthesize. Since most orb web spiders express high proline silk they might preferentially attain the amino acids needed for silk from food and shift toward expressing more MaSp1 in their MA silk when starved.

Methodology/Principal Findings

We fed three spiders; Argiope aetherea, Cyrtophora moluccensis and Leucauge blanda, high protein, low protein or no protein solutions. A. aetherea and L. blanda MA silks are high in proline, while C. moluccesnsis MA silks are low in proline. After 10 days of feeding we determined the amino acid compositions and mechanical properties of each species'' MA silk and compared them between species and treatments with pre-treatment samples, accounting for ancestry. We found that the proline and glutamine of A. aetherea and L. blanda silks were affected by protein intake; significantly decreasing under the low and no protein intake treatments. Glutmaine composition in C. moluccensis silk was likewise affected by protein intake. However, the composition of proline in their MA silk was not significantly affected by protein intake.

Conclusions

Our results suggest that protein limitation induces a shift toward different silk proteins with lower glutamine and/or proline content. Contradictions to the MaSp model lie in the findings that C. moluccensis MA silks did not experience a significant reduction in proline and A. aetherea did not experience a significant reduction in serine on low/no protein. The mechanical properties of the silks could not be explained by a MaSp1 expressional shift. Factors other than MaSp expression, such as the expression of spidroin-like orthologues, may impact on silk amino acid composition and spinning and glandular processes may impact mechanics.     

Conservation of essential design features in coiled coil silks

Silks are strong protein fibers produced by a broad array of spiders and insects. The vast majority of known silks are large, repetitive proteins assembled into extended beta-sheet structures. Honeybees, however, have found a radically different evolutionary solution to the need for a building material. The 4 fibrous proteins of honeybee silk are small ( approximately 30 kDa each) and nonrepetitive and adopt a coiled coil structure. We examined silks from the 3 superfamilies of the Aculeata (Hymenoptera: Apocrita) by infrared spectroscopy and found coiled coil structure in bees (Apoidea) and in ants (Vespoidea) but not in parasitic wasps of the Chrysidoidea. We subsequently identified and sequenced the silk genes of bumblebees, bulldog ants, and weaver ants and compared these with honeybee silk genes. Each species produced orthologues of the 4 small fibroin proteins identified in honeybee silk. Each fibroin contained a continuous predicted coiled coil region of around 210 residues, flanked by 23-160 residue length N- and C-termini. The cores of the coiled coils were unusually rich in alanine. There was extensive sequence divergence among the bee and ant silk genes (<50% similarity between the alignable regions of bee and ant sequences), consistent with constant and equivalent divergence since the bee/ant split (estimated to be 155 Myr). Despite a high background level of sequence diversity, we have identified conserved design elements that we propose are essential to the assembly and function of coiled coil silks.     

Web building and silk properties functionally covary among species of wolf spider

Although phylogenetic studies have shown covariation between the properties of spider major ampullate (MA) silk and web building, both spider webs and silks are highly plastic so we cannot be sure whether these traits functionally covary or just vary across environments that the spiders occupy. As MaSp2‐like proteins provide MA silk with greater extensibility, their presence is considered necessary for spider webs to effectively capture prey. Wolf spiders (Lycosidae) are predominantly non‐web building, but a select few species build webs. We accordingly collected MA silk from two web‐building and six non‐web‐building species found in semirural ecosystems in Uruguay to test whether the presence of MaSp2‐like proteins (indicated by amino acid composition, silk mechanical properties and silk nanostructures) was associated with web building across the group. The web‐building and non‐web‐building species were from disparate subfamilies so we estimated a genetic phylogeny to perform appropriate comparisons. For all of the properties measured, we found differences between web‐building and non‐web‐building species. A phylogenetic regression model confirmed that web building and not phylogenetic inertia influences silk properties. Our study definitively showed an ecological influence over spider silk properties. We expect that the presence of the MaSp2‐like proteins and the subsequent nanostructures improves the mechanical performance of silks within the webs. Our study furthers our understanding of spider web and silk co‐evolution and the ecological implications of spider silk properties.     

Cocoon silk chemistry in parasitic wasps (Hymenoptera, Ichneumonoidea) and their hosts

Bulk amino acid compositions of larval cocoon silks of 24 species of ichneumonoid parasitic wasps, representing 13 subfamilies that kill the host in a larval or prepupal stage, are compared with those of their hosts to test the hypothesis that amino acid compositions of major protein products should, in certain cases, be similar on energetic grounds. Although substantial variation in amino acid composition was found among both parasitoids and hosts, suggesting the production of different types of silks, no significant general matching was detected. However, the trend in the degree of similarity observed was in the direction predicted by a priori consideration of the nature of the parasitoid – host association. Lack of a general association may be explained by the very simple silk glands of the parasitic wasps and by the fact that, in most cases, their hosts are not completely consumed at a time when they are likely to contain any large reserves of silk proteins. The three species of Cotesia (Braconidae: Microgastrinae) investigated stood out in that their silks showed considerable interspecific variation in molar percentage amino acid composition, and this might be associated with their apparent utilization of α-helical silks rather than fibroins.  © 2004 The Linnean Society of London, Biological Journal of the Linnean Society , 2004, 81 , 161–170.     

三种蜘蛛丝蛋白组成分析

本文应用高压液相色谱（ＨＰＬＣ）法分析了岳麓山的大腹园蛛Ａｒａｎｅｕｓ ｖｅｎｔｒｉｃｏｓｕｓ（Ｃ．Ｋｏｃｈ,１８７８）,机敏漏头蛛Ａｇｅｌｅｎａ ｄｉｆｆｉｃｌｉｓ （Ｆｏｘ,１９３７）,白额巨蟹蛛Ｈｅｔｅｒｏｐｏｄａ ｖｅｎａｔｏｒｉａ （Ｌｉｎｎａｅｕｓ,１７５７）的丝蛋白的氨基酸组成,以ＳＤＳ－ＰＡＧＥ法测定了大腹园蛛不同丝腺体的未成丝的可溶性丝蛋白的分子量。实验结果表明蛛丝蛋白中占优势的     

Spider silks: recombinant synthesis,assembly, spinning,and engineering of synthetic proteins

Since thousands of years humans have utilized insect silks for their own benefit and comfort. The most famous example is the use of reeled silkworm silk from Bombyx mori to produce textiles. In contrast, despite the more promising properties of their silk, spiders have not been domesticated for large-scale or even industrial applications, since farming the spiders is not commercially viable due to their highly territorial and cannibalistic nature. Before spider silks can be copied or mimicked, not only the sequence of the underlying proteins but also their functions have to be resolved. Several attempts to recombinantly produce spider silks or spider silk mimics in various expression hosts have been reported previously. A new protein engineering approach, which combines synthetic repetitive silk sequences with authentic silk domains, reveals proteins that closely resemble silk proteins and that can be produced at high yields, which provides a basis for cost-efficient large scale production of spider silk-like proteins.