RESUMEN
To survive in terrestrial and aquatic environments, spiders often rely heavily on their silk. The vast majority of silks that have been studied are from orb-web or cob-web weaving species, leaving the silks of water-associated spiders largely undescribed. We characterize transcripts, proteins, and silk fibres from the semi-aquatic spider Dolomedes triton. From silk gland RNAseq libraries, we report 18 silk transcripts representing four categories of known silk protein types: aciniform, ampullate, pyriform, and tubuliform. Proteomic and structural analyses (scanning electron microscopy, energy dispersive X-ray spectrometry, contact angle) of the D. triton submersible egg sac reveal similarities to silks from aquatic caddisfly larvae. We identified two layers in D. triton egg sacs, notably a highly hydrophobic outer layer with a different elemental composition compared to egg sacs of terrestrial spiders. These features may provide D. triton egg sacs with their water repellent properties.
Asunto(s)
Fibroínas/química , Arañas/metabolismo , Animales , Femenino , Fibroínas/genética , Interacciones Hidrofóbicas e Hidrofílicas , Microscopía Electrónica de Rastreo , Caracteres Sexuales , Arañas/genética , Transcriptoma , AguaRESUMEN
Orb-web weaving spiders produce a variety of task-specific silks from specialized silk glands. The genetics underlying the synthesis of specific silk types are largely unknown, and transcriptome analysis could be a powerful approach for identifying candidate genes. However, de novo assembly and expression profiling of silk glands with RNA-sequencing (RNAseq) are problematic because the few known gene transcripts for silk proteins are extremely long and highly repetitive. To identify candidate genes for tubuliform (egg case) silk synthesis by the orb-weaver Argiope argentata (Araneidae), we estimated transcript abundance using two sequencing methods: RNAseq reads from throughout the length of mRNA molecules, and 3' digital gene expression reads from the 3' region of mRNA molecules. Both analyses identified similar sets of genes as differentially expressed when comparing tubuliform and nonsilk gland tissue. However, incompletely assembled silk gene transcripts were identified as differentially expressed because of RNAseq read alignments to highly repetitive regions, confounding interpretation of RNAseq results. Homologues of egg case silk protein (ECP) genes were upregulated in tubuliform glands. This discovery is the first description of ECP homologues in an araneid. We also propose additional candidate genes involved in synthesis of tubuliform or other silk types.
Asunto(s)
Seda/genética , Arañas/genética , Secuencia de Aminoácidos , Animales , ADN Complementario/genética , ADN Complementario/metabolismo , Femenino , Expresión Génica , Filogenia , ARN Mensajero/genética , ARN Mensajero/metabolismo , Alineación de Secuencia , Seda/química , Seda/metabolismoRESUMEN
Spiders spin multiple types of silks that are renowned for their superb mechanical properties. Flagelliform silk, used in the capture spiral of an orb-web, is one of the few silks characterized by both cDNA and genomic DNA data. This fibroin is composed of repeating ensembles of three types of amino acid sequence motifs. The predominant subrepeat, GPGGX, likely forms a beta-turn, and tandem arrays of these turns are thought to create beta-spirals. These spring-like helices may be critical for the exceptional ability of capture silk to stretch and recoil. Each ensemble of motifs was found to correspond to a different exon within the flagelliform gene. The pattern of sequence similarity among exons indicates intragenic concerted evolution. Surprisingly, the introns between the iterated exons are also homogenized with each other. This unusual molecular architecture in the flagelliform silk gene has implications for the evolution and maintenance of spider silk proteins.
Asunto(s)
Proteínas/química , Arañas/química , Secuencia de Aminoácidos , Animales , ADN Complementario/genética , Evolución Molecular , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Proteínas/genética , Arañas/genéticaRESUMEN
Spider flagelliform silk is one of the most elastic natural materials known. Extensive sequencing of spider silk genes has shown that the exons and introns of the flagelliform gene underwent intragenic concerted evolution. The intron sequences are more homogenized within a species than are the exons. This pattern can be explained by extreme mutation and recombination pressures on the internally repetitive exons. The iterated sequences within exons encode protein structures that are critical to the function of silks. Therefore, attributes that make silks exceptional biomaterials may also hinder the fixation of optimally adapted protein sequences.
Asunto(s)
Evolución Molecular , Exones , Genes , Intrones , Proteínas/genética , Arañas/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Intercambio Genético , ADN/genética , Replicación del ADN , Conversión Génica , Datos de Secuencia Molecular , Mutación , Proteínas/química , Recombinación Genética , Secuencias Repetitivas de Ácidos Nucleicos , Selección Genética , Especificidad de la EspecieRESUMEN
Several types of silks and silk protein coding genes have been characterized from orb-web weaving spiders. When the protein sequences of major ampullate, minor ampullate, and flagelliform silks from Nephila clavipes are compared, they can be summarized as sets of shared amino acid motifs. Four of these motifs and their likely secondary structures are described. Each structural element, termed a module, is then associated with its impact on the mechanical properties of a silk fiber. In particular, correlations are drawn between an alanine-rich 'crystalline module' and tensile strength and between a proline-containing 'elasticity module' and extensibility.
Asunto(s)
Proteínas de Insectos/química , Estructura Secundaria de Proteína , Arañas/química , Secuencia de Aminoácidos , Animales , Modelos Químicos , Modelos Moleculares , Datos de Secuencia Molecular , SedaRESUMEN
Orb-web weaving spiders rely on their aerial nets to entrap flying prey. A key mechanical feature of orb-web design is the high elasticity of the capture spiral. We report the cloning of substantial cDNA for flagelliform gland silk protein, which forms the core fiber of the catching spiral. Like all silks, the flagelliform protein is composed largely of iterated sequences. The dominant repeat of this protein is Gly-Pro-Gly-Gly-X, which can appear up to 63 times in tandem arrays. This motif likely forms Pro2-Gly3 type II beta-turns and the resulting series of concatenated beta-turns are thought to form a beta-spiral. We propose that this spring-like helix is the basis for the elasticity of silk. The variable fifth position of the motif (X) is occupied by a small subset of residues (Ala, Ser, Tyr, Val). Moreover, these X amino acids occur in specific patterns throughout the repeats. This ordered variation strongly suggests that with hydration, the beta-spirals form hydrogen-bonded networks that increase the elasticity of flagelliform silk. The self-assembly of flagelliform protein monomers into silk fibers may be promoted by beta-spiral/beta-spiral interactions. Additionally, the other two motifs in the flagelliform protein, Gly-Gly-X and a spacer that disrupts the glycine-rich regions, may contribute to the alignment of monomers into fibers. The flagelliform protein cDNA was compared to the other members of the spider silk gene family. We show that all spider silk proteins can be characterized as sets of shared structural modules. The occurrence of these modules among the proteins is inconsistent with the phylogenetic relationships inferred from the C-terminal regions. This observation, along with the high level of variation among individual flagelliform protein repeats, but striking lack of such variation in the other silk proteins, suggests that unusual homogenization processes are involved in silk protein evolution.