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Darwin’s bark spider (Caerostris darwini) produces giant orb webs from dragline silk that can be twice as tough as other silks, making it the toughest biological material. This extreme toughness comes from increased extensibility relative to other draglines. We show C. darwini dragline-producing major ampullate (MA) glands highly express a novel silk gene transcript (MaSp4) encoding a protein that diverges markedly from closely related proteins and contains abundant proline, known to confer silk extensibility, in a unique GPGPQ amino acid motif. This suggests C. darwini evolved distinct proteins that may have increased its dragline’s toughness, enabling giant webs. Caerostris darwini’s MA spinning ducts also appear unusually long, potentially facilitating alignment of silk proteins into extremely tough fibers. Thus, a suite of novel traits from the level of genes to spinning physiology to silk biomechanics are associated with the unique ecology of Darwin’s bark spider, presenting innovative designs for engineering biomaterials., Jessica Garb et al. report the transcriptome of the major ampullate silk gland of Darwin’s bark spider, known for its extraordinarily tough silk. They identify a novel predicted protein, MaSp4, which is highly divergent compared to related proteins in other species and has properties predicted to confer silk extensibility.

et al., [Background]: The duplication of genes can occur through various mechanisms and is thought to make a major contribution to the evolutionary diversification of organisms. There is increasing evidence for a large-scale duplication of genes in some chelicerate lineages including two rounds of whole genome duplication (WGD) in horseshoe crabs. To investigate this further, we sequenced and analyzed the genome of the common house spider Parasteatoda tepidariorum. [Results]: We found pervasive duplication of both coding and non-coding genes in this spider, including two clusters of Hox genes. Analysis of synteny conservation across the P. tepidariorum genome suggests that there has been an ancient WGD in spiders. Comparison with the genomes of other chelicerates, including that of the newly sequenced bark scorpion Centruroides sculpturatus, suggests that this event occurred in the common ancestor of spiders and scorpions, and is probably independent of the WGDs in horseshoe crabs. Furthermore, characterization of the sequence and expression of the Hox paralogs in P. tepidariorum suggests that many have been subject to neo-functionalization and/or sub-functionalization since their duplication. [Conclusions]: Our results reveal that spiders and scorpions are likely the descendants of a polyploid ancestor that lived more than 450 MYA. Given the extensive morphological diversity and ecological adaptations found among these animals, rivaling those of vertebrates, our study of the ancient WGD event in Arachnopulmonata provides a new comparative platform to explore common and divergent evolutionary outcomes of polyploidization events across eukaryotes., This work was supported by NIH grant NHGRI U54 HG003273 to RAG, the National Science Foundation (IOS-0951886 to NAA and DEB-1257053 to JHW), a Leverhulme visiting fellowship for EES (VF-2012-016), funding and PhD studentships (DJL, LG and AS) from Oxford Brookes University, and a Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) scholarship to CLBP. N-MP was funded by the Deutsche Forschungsgemeinschaft (grant numbers PR 1109/ 4-1, PR 1109/7-1 and PR 1109/6-1 to N-MP). Additional financial backing has been received from the Göttingen Graduate School for Neurosciences, Biophysics and Molecular Biosciences (GGNB), the Göttingen Center for Molecular Biosciences (GZMB), and the University of Göttingen (GAU). NT is supported by a Christiane Nüsslein-Volhard-Foundation fellowship and a “Women in Science” Award by L'Oréal Deutschland and the Deutsche UNESCO-Kommission. NP has been funded by the Volkswagen Foundation (project number: 85 983) and the Emmy Noether Programme of the Deutsche Forschungsgemeinschaft (grant number: PO 1648/3-1). Funding to RJ was provided by the Swedish Research Council VR grant 621-2011-4703.

SUMMARY The discovery of genetic mechanisms that can transform a morphological structure from a plesiomorphic (¼primitive) state to an apomorphic (¼derived) one is a cardinal objective of evolutionary developmental biology. However, this objective is often impeded for many lineages of interest by limitations in taxonomic sampling, genomic resources, or functional genetic methods. In order to investigate the evolution of appendage morphology within Chelicerata, the putative sister group of the remaining arthropods, we developed an RNA interference (RNAi) protocol for the harvestmanPhalangiumopilio.Wesilencedtheleggapgenes Distal‐less (Dll) and dachshund (dac) in the harvestman via zygotic injections of double‐stranded RNA (dsRNA), and used in situ hybridization to confirm RNAi efficacy. Consistent with the conserved roles of these genes in patterning the proximo‐ distal axis of arthropod appendages, we observed that embryos injected with Dll dsRNA lacked distal parts of appendages and appendage‐like structures, such as the labrum, the chelicerae, the pedipalps, and the walking legs, whereas embryos injected with dac dsRNA lacked the medial podomeres femur and patella in the pedipalps and walking legs. In addition, we detected a role for these genes in patterning structures that do not occur in well‐established chelicerate models (spiders and mites). Dll RNAi additionally results in loss of the preoral chamber, which is formed from pedipalpal and leg coxapophyses, and the ocularium, a dorsal outgrowth bearing the eyes. In one case, we observed that an embryo injected with dac dsRNA lacked the proximal segment of the chelicera, a plesiomorphic podomere that expresses dac in wild‐type embryos. This may support the hypothesis thatlossofthecheliceraldacdomainunderliesthetransitionto the two‐segmented chelicera of derived arachnids.

Background Black widow spiders are infamous for their neurotoxic venom, which can cause extreme and long-lasting pain. This unusual venom is dominated by latrotoxins and latrodectins, two protein families virtually unknown outside of the black widow genus Latrodectus, that are difficult to study given the paucity of spider genomes. Using tissue-, sex- and stage-specific expression data, we analyzed the recently sequenced genome of the house spider (Parasteatoda tepidariorum), a close relative of black widows, to investigate latrotoxin and latrodectin diversity, expression and evolution. Results We discovered at least 47 latrotoxin genes in the house spider genome, many of which are tandem-arrayed. Latrotoxins vary extensively in predicted structural domains and expression, implying their significant functional diversification. Phylogenetic analyses show latrotoxins have substantially duplicated after the Latrodectus/Parasteatoda split and that they are also related to proteins found in endosymbiotic bacteria. Latrodectin genes are less numerous than latrotoxins, but analyses show their recruitment for venom function from neuropeptide hormone genes following duplication, inversion and domain truncation. While latrodectins and other peptides are highly expressed in house spider and black widow venom glands, latrotoxins account for a far smaller percentage of house spider venom gland expression. Conclusions The house spider genome sequence provides novel insights into the evolution of venom toxins once considered unique to black widows. Our results greatly expand the size of the latrotoxin gene family, reinforce its narrow phylogenetic distribution, and provide additional evidence for the lateral transfer of latrotoxins between spiders and bacterial endosymbionts. Moreover, we strengthen the evidence for the evolution of latrodectin venom genes from the ecdysozoan Ion Transport Peptide (ITP)/Crustacean Hyperglycemic Hormone (CHH) neuropeptide superfamily. The lower expression of latrotoxins in house spiders relative to black widows, along with the absence of a vertebrate-targeting α-latrotoxin gene in the house spider genome, may account for the extreme potency of black widow venom. Electronic supplementary material The online version of this article (doi:10.1186/s12864-017-3551-7) contains supplementary material, which is available to authorized users.

Background Orb-web weaving spiders and their relatives use multiple types of task-specific silks. The majority of spider silk studies have focused on the ultra-tough dragline silk synthesized in major ampullate glands, but other silk types have impressive material properties. For instance, minor ampullate silks of orb-web weaving spiders are as tough as draglines, due to their higher extensibility despite lower strength. Differences in material properties between silk types result from differences in their component proteins, particularly members of the spidroin (spider fibroin) gene family. However, the extent to which variation in material properties within a single silk type can be explained by variation in spidroin sequences is unknown. Here, we compare the minor ampullate spidroins (MiSp) of orb-weavers and cobweb weavers. Orb-web weavers use minor ampullate silk to form the auxiliary spiral of the orb-web while cobweb weavers use it to wrap prey, suggesting that selection pressures on minor ampullate spidroins (MiSp) may differ between the two groups. Results We report complete or nearly complete MiSp sequences from five cobweb weaving spider species and measure material properties of minor ampullate silks in a subset of these species. We also compare MiSp sequences and silk properties of our cobweb weavers to published data for orb-web weavers. We demonstrate that all our cobweb weavers possess multiple MiSp loci and that one locus is more highly expressed in at least two species. We also find that the proportion of β-spiral-forming amino acid motifs in MiSp positively correlates with minor ampullate silk extensibility across orb-web and cobweb weavers. Conclusions MiSp sequences vary dramatically within and among spider species, and have likely been subject to multiple rounds of gene duplication and concerted evolution, which have contributed to the diverse material properties of minor ampullate silks. Our sequences also provide templates for recombinant silk proteins with tailored properties. Electronic supplementary material The online version of this article (doi:10.1186/s12862-017-0927-x) contains supplementary material, which is available to authorized users.

Summaryoskar is the only gene in the animal kingdom necessary and sufficient for specifying functional germ cells [1, 2]. However, oskar has only been identified in holometabolous (“higher”) insects that specify their germline using specialized cytoplasm called germ plasm [3]. Here we show that oskar evolved before the divergence of higher insects and provide evidence that its germline role is a recent evolutionary innovation. We identify an oskar ortholog in a basally branching insect, the cricket Gryllus bimaculatus. In contrast to Drosophila oskar, Gb-oskar is not required for germ cell formation or axial patterning. Instead, Gb-oskar is expressed in neuroblasts of the brain and CNS and is required for neural development. Taken together with reports of a neural role for Drosophila oskar [4], our data demonstrate that oskar arose nearly 50 million years earlier in insect evolution than previously thought, where it may have played an ancestral neural role, and was co-opted to its well-known essential germline role in holometabolous insects.

The proximo-distal axis of the arthropod leg is patterned by mutually antagonistic developmental expression domains of the genes extradenticle, homothorax, dachshund, and Distal-less. In the deutocerebral appendages (the antennae) of insects and crustaceans, the expression domain of dachshund is frequently either absent or, if present, is not required to pattern medial segments. By contrast, the dachshund domain is entirely absent in the deutocerebral appendages of spiders, the chelicerae. It is unknown whether absence of dachshund expression in the spider chelicera is associated with the two-segmented morphology of this appendage, or whether all chelicerates lack the dachshund domain in their chelicerae. We investigated gene expression in the harvestman Phalangium opilio, which bears the plesiomorphic three-segmented chelicera observed in "primitive" chelicerate orders. Consistent with patterns reported in spiders, in the harvestman chelicera homothorax, extradenticle, and Distal-less have broadly overlapping developmental domains, in contrast with mutually exclusive domains in the legs and pedipalps. However, unlike in spiders, the harvestman chelicera bears a distinct expression domain of dachshund in the proximal segment, the podomere that is putatively lost in derived arachnids. These data suggest that a tripartite proximo-distal domain structure is ancestral to all arthropod appendages, including deutocerebral appendages. As a corollary, these data also provide an intriguing putative genetic mechanism for the diversity of arachnid chelicerae: loss of developmental domains along the proximo-distal axis.

Among chelicerates, Hox gene expression has only been investigated in representatives of two arachnid orders to date: Acari (mites and ticks) and Araneae (spiders). Limited data are available for the "primitive" arachnid orders, such as Scorpiones (scorpions) and Opiliones (harvestmen). Here, we present the first data on Hox gene expression in the harvestman Phalangium opilio. Ten Hox genes of this species were obtained from a de novo assembled developmental transcriptome using the Illumina GAII platform. All 10 genes are expressed in characteristic Hox-like expression patterns, and the expression of the anterior and central Hox genes is similar to those of other chelicerates. However, intriguingly, the three posteriormost genes-Ultrabithorax, abdominal-A, and Abdominal-B-share an identical anterior expression boundary in the second opisthosomal segment, and their expression domains extend through the opisthosoma to the posterior growth zone. The overlap in expression domains of the posterior Hox genes is correlated with the absence of opisthosomal organs posterior to the tubular tracheae, which occur on the second opisthosomal segment. Together with the staggered profile of posterior Hox genes in spiders, these data suggest the involvement of abdominal-A and Abdominal-B in the evolution of heteronomous patterning of the chelicerate opisthosoma, providing a mechanism that helps explain the morphological diversity of chelicerates.

Parasteatoda tepidariorum is an increasingly popular model for the study of spider development and the evolution of development more broadly. However, fully understanding the regulation and evolution of P. tepidariorum development in comparison to other animals requires a genomic perspective. Although research on P. tepidariorum has provided major new insights, gene analysis to date has been limited to candidate gene approaches. Furthermore, the few available EST collections are based on embryonic transcripts, which have not been systematically annotated and are unlikely to contain transcripts specific to post-embryonic stages of development. We therefore generated cDNA from pooled embryos representing all described embryonic stages, as well as post-embryonic stages including nymphs, larvae and adults, and using Illumina HiSeq technology obtained a total of 625,076,514 100-bp paired end reads. We combined these data with 24,360 ESTs available in GenBank, and 1,040,006 reads newly generated from 454 pyrosequencing of a mixed-stage embryo cDNA library. The combined sequence data were assembled using a custom de novo assembly strategy designed to optimize assembly product length, number of predicted transcripts, and proportion of raw reads incorporated into the assembly. The de novo assembly generated 446,427 contigs with an N50 of 1,875 bp. These sequences obtained 62,799 unique BLAST hits against the NCBI non-redundant protein data base, including putative orthologs to 8,917 Drosophila melanogaster genes based on best reciprocal BLAST hit identity compared with the D. melanogaster proteome. Finally, we explored the utility of the transcriptome for RNA-Seq studies, and showed that this resource can be used as a mapping scaffold to detect differential gene expression in different cDNA libraries. This resource will therefore provide a platform for future genomic, gene expression and functional approaches using P. tepidariorum.

Summary Background In insects, the gap gene hunchback ( hb ) is required for the formation of a set of adjacent segments through the regulation of downstream target genes of the pair rule and segment-polarity classes. In addition, hb is a major regulator of Hox genes and it has been suggested that this is the ancestral role of hb in insects or perhaps even arthropods. To date, however, hb function has been analyzed only in insects. Results Here we show that hb acts as a segmentation gene during anterior patterning of a noninsect arthropod, the spider Achaearanea tepidariorum . The leg-bearing segments L1, L2, and L4 are missing after downregulation of At-hb via RNAi. At-hb is required for the correct organization of target genes in this region of the embryo, suggesting that At-hb acts as a gap gene in the spider. In contrast to insects, hb does not control Hox gene expression in the spider. Furthermore, analysis of twist expression in At-hb knockdown embryos demonstrates that hb is not required for initiating the segmental organization of the mesoderm in the affected region, but only for its maintenance. Conclusions Our findings suggest that hb might have had a segmentation gene function in the arthropod ancestor and contradicts the suggestion that the control of Hox genes is the ancestral role of hb . Anterior spider segmentation thus utilizes a Drosophila -like genetic mode, whereas a vertebrate-like mechanism involving Wnt8 and Notch/Delta signaling is used to pattern posterior segments. These data support the hypothesis that short-germ arthropods employ two distinct mechanisms to segment their anterior and posterior body parts.

A number of recent studies have investigated posterior development in several different arthropods. As previously found in spiders, it has been discovered that Delta-Notch signaling is required for the development of posterior segments in an insect, the cockroach Periplaneta americana. Furthermore analysis of Wnt8 function in the spider Achaearanea tepidariorum and the beetle Tribolium castaneum demonstrates that this Wnt ligand is required for the establishment of the growth zone and development of posterior segments in both these arthropods. Taken together these studies provide an interesting insight into the architecture of the genetic network that regulated posterior development in the common ancestor of the arthropods.

The segmental architecture of the arthropod head is one of the most controversial topics in the evolutionary developmental biology of arthropods. The deutocerebral (second) segment of the head is putatively homologous across Arthropoda, as inferred from the segmental distribution of the tripartite brain and the absence of Hox gene expression of this anterior-most, appendage-bearing segment. While this homology statement implies a putative common mechanism for differentiation of deutocerebral appendages across arthropods, experimental data for deutocerebral appendage fate specification are limited to winged insects. Mandibulates (hexapods, crustaceans and myriapods) bear a characteristic pair of antennae on the deutocerebral segment, whereas chelicerates (e.g. spiders, scorpions, harvestmen) bear the eponymous chelicerae. In such hexapods as the fruit fly,Drosophila melanogaster, and the cricket,Gryllus bimaculatus, cephalic appendages are differentiated from the thoracic appendages (legs) by the activity of the appendage patterning genehomothorax(hth). Here we show that embryonic RNA interference againsththin the harvestmanPhalangium opilioresults in homeonotic chelicera-to-leg transformations, and also in some cases pedipalp-to-leg transformations. In more strongly affected embryos, adjacent appendages undergo fusion and/or truncation, and legs display proximal defects, suggesting conservation of additional functions ofhthin patterning the antero-posterior and proximo-distal appendage axes. Expression signal of anterior Hox geneslabial,proboscipediaandDeformedis diminished, but not absent, inhthRNAi embryos, consistent with results previously obtained with the insectG. bimaculatus. Our results substantiate a deep homology across arthropods of the mechanism whereby cephalic appendages are differentiated from locomotory appendages.