Venom gland organogenesis in the common house spider.

Venom is a remarkable innovation found across the animal kingdom, yet the evolutionary origins of venom systems in various groups, including spiders, remain enigmatic. Here, we investigated the organogenesis of the venom apparatus in the common house spider, Parasteatoda tepidariorum. The venom apparatus consists of a pair of secretory glands, each connected to an opening at the fang tip by a duct that runs through the chelicerae. We performed bulk RNA-seq to identify venom gland-specific markers and assayed their expression using RNA in situ hybridisation experiments on whole-mount time-series. These revealed that the gland primordium emerges during embryonic stage 13 at the chelicera tip, progresses proximally by the end of embryonic development and extends into the prosoma post-eclosion. The initiation of expression of an important toxin component in late postembryos marks the activation of venom-secreting cells. Our selected markers also exhibited distinct expression patterns in adult venom glands: sage and the toxin marker were expressed in the secretory epithelium, forkhead and sum-1 in the surrounding muscle layer, while Distal-less was predominantly expressed at the gland extremities. Our study provides the first comprehensive analysis of venom gland morphogenesis in spiders, offering key insights into their evolution and development.

Temperature fluctuations during embryonic development implicated in a naturally occurring instance of abnormal spinnerets in the spider Australomimetus maculosus (Araneae, Mimetidae).

We record developmental abnormalities of the spinnerets in a field-collected adult male specimen of Australomimetus maculosus. These include (1) a supernumerary right posterior lateral spinneret (PLS), (2) ectopic piriform silk gland spigots and tartipores on the left PLS that are normally restricted to anterior lateral spinnerets (ALSs), and (3) what appear to be ectopic ALS sensilla on the left posterior median spinneret (PMS). Published results of teratological experiments and climate data for the collection site indicate that fluctuating sub- and supra-optimal temperatures during embryogenesis may have been responsible for these anomalies. This specimen thus supports the view that spinneret abnormalities, among other aberrations, may be induced when embryos of entelegyne spiders are exposed to fluctuations between high and low temperatures, whether in the laboratory or, as here, in nature. To our knowledge, the ectopic structures seen on the left PLS and left PMS have not been observed previously. Their locations are consistent with a hypothesis by which only the lateral portion of the araneomorph ALS is serially homologous to the PLS, while the remainder of the ALS, along with the colulus/cribellum, is homologous to the PMS.

Developmental expression of doublesex-related transcripts in the common house spider, Parasteatoda tepidariorum.

Sex determination is a rapidly evolving biological process controlled by differential gene expression. One family of transcription factors that initiate sex-specific gene expression and differentiation in many animal species are the Doublesex and Mab-3 (DM) domain proteins. While much is known about Doublesex-related proteins in various insect orders and commonly studied model systems, little is known about their function in basally branching arthropods. Spiders are an emerging model for molecular and evolutionary development that could fill this gap. Arachnids share an ancient whole-genome duplication providing a unique opportunity to study the effect of major genomic rearrangements on the evolution of developmental processes. In this study, we aimed to identify the repertoire of Dsx-related proteins encoded by the genome of the common house spider, Parasteatoda tepidariorum. While insects have four DM domain proteins, the P. tepidariorum genome encodes seven, indicating the possibility of duplicate retention. At least four of the DM protein genes demonstrated sex bias expression in adult spiders. Embryonic expression of these genes suggests roles in development of the spinnerets, nervous system, and appendages.

Candidate gene screen for potential interaction partners and regulatory targets of the Hox gene labial in the spider Parasteatoda tepidariorum.

The Hox gene labial (lab) governs the formation of the tritocerebral head segment in insects and spiders. However, the morphology that results from lab action is very different in the two groups. In insects, the tritocerebral segment (intercalary segment) is reduced and lacks appendages, whereas in spiders the corresponding segment (pedipalpal segment) is a proper segment including a pair of appendages (pedipalps). It is likely that this difference between lab action in insects and spiders is mediated by regulatory targets or interacting partners of lab. However, only a few such genes are known in insects and none in spiders. We have conducted a candidate gene screen in the spider Parasteatoda tepidariorum using as candidates Drosophila melanogaster genes known to (potentially) interact with lab or to be expressed in the intercalary segment. We have studied 75 P. tepidariorum genes (including previously published and duplicated genes). Only 3 of these (proboscipedia-A (pb-A) and two paralogs of extradenticle (exd)) showed differential expression between leg and pedipalp. The low success rate points to a weakness of the candidate gene approach when it is applied to lineage specific organs. The spider pedipalp has no counterpart in insects, and therefore relying on insect data apparently cannot identify larger numbers of factors implicated in its specification and formation. We argue that in these cases a de novo approach to gene discovery might be superior to the candidate gene approach.

The forkhead box containing transcription factor FoxB is a potential component of dorsal-ventral body axis formation in the spider Parasteatoda tepidariorum.

In the spider, determination of the dorsal-ventral body (DV) axis depends on the interplay of the dorsal morphogen encoding gene decapentaplegic (Dpp) and its antagonist, short gastrulation (sog), a gene that is involved in the correct establishment of ventral tissues. Recent work demonstrated that the forkhead domain encoding gene FoxB is involved in dorsal-ventral axis formation in spider limbs. Here, Dpp likely acts as a dorsal morphogen, and FoxB is likely in control of ventral tissues as RNAi-mediated knockdown of FoxB causes dorsalization of the limbs. In this study, we present phenotypes of FoxB knockdown that demonstrate a function in the establishment of the DV body axis. Knockdown of FoxB function leads to embryos with partially duplicated median germ bands (Duplicitas media) that are possibly the result of ectopic activation of Dpp signalling. Another class of phenotypes is characterized by unnaturally slim (dorsal-ventrally compressed) germ bands in which ventral tissue is either not formed, or is specified incorrectly, likely a result of Dpp over-activity. These results suggest that FoxB functions as an antagonist of Dpp signalling during body axis patterning, similarly as it is the case in limb development. FoxB thus represents a general player in the establishment of dorsal-ventral structures during spider ontogeny.

Embryonic development and secondary axis induction in the Brazilian white knee tarantula Acanthoscurria geniculata, C. L. Koch, 1841 (Araneae; Mygalomorphae; Theraphosidae).

Tarantulas represent some of the heaviest and most famous spiders. However, there is little information about the embryonic development of these spiders or their relatives (infraorder Mygalomorphae) and time-lapse recording of the embryonic development is entirely missing. I here describe the complete development of the Brazilian white knee tarantula, Acanthoscurria geniculata, in fixed and live embryos. The establishment of the blastoderm, the formation, migration and signalling of the cumulus and the shape changes that occur in the segment addition zone are analysed in detail. In addition, I show that there might be differences in the contraction process of early embryos of different theraphosid spider species. A new embryonic reference transcriptome was generated for this study and was used to clone and analyse the expression of several important developmental genes. Finally, I show that embryos of A. geniculata are amenable to tissue transplantation and bead insertion experiments. Using these functional approaches, I induced axis duplication in embryos via cumulus transplantation and ectopic activation of BMP signalling. Overall, the mygalomorph spider A. geniculata is a useful laboratory system to analyse evolutionary developmental questions, and the availability of such a system will help understanding conserved and divergent aspects of spider/chelicerate development.

Developmental gene expression as a phylogenetic data class: support for the monophyly of Arachnopulmonata.

Despite application of genome-scale datasets, the phylogenetic placement of scorpions within arachnids remains contentious between two different phylogenetic data classes. Paleontologists continue to recover scorpions in a basally branching position, partly owing to their morphological similarity to extinct marine orders like Eurypterida (sea scorpions). Phylogenomic datasets consistently recover scorpions in a derived position, as the sister group of Tetrapulmonata (a clade of arachnids that includes spiders). To adjudicate between these hypotheses using a rare genomic change (RGC), we leveraged the recent discovery of ancient paralogy in spiders and scorpions to assess phylogenetic placement. We identified homologs of four transcription factors required for appendage patterning (dachshund, homothorax, extradenticle, and optomotor blind) in arthropods that are known to be duplicated in spiders. Using genomic resources for a spider, a scorpion, and a harvestman, we conducted gene tree analyses and assayed expression patterns of scorpion gene duplicates. Here we show that scorpions, like spiders, retain two copies of all four transcription factors, whereas arachnid orders like mites and harvestmen bear a single copy. A survey of embryonic expression patterns of the scorpion paralogs closely matches those of their spider counterparts, with one paralog consistently retaining the putatively ancestral pattern found in the harvestman, as well as the mite, and/or other outgroups. These data comprise a rare genomic change in chelicerate phylogeny supporting the inference of a distal placement of scorpions. Beyond demonstrating the diagnostic power of developmental genetic data as a phylogenetic data class, a derived placement of scorpions within the arachnids, together with an array of stem-group Paleozoic scorpions that occupied marine habitats, effectively rules out a scenario of a single colonization of terrestrial habitat within Chelicerata, even in tree topologies contrived to recover the monophyly of Arachnida.

Experimental duplication of bilaterian body axes in spider embryos: Holm's organizer and self-regulation of embryonic fields.

Bilaterally symmetric body plans of vertebrates and arthropods are defined by a single set of two orthogonal axes, the anterior-posterior (or head-tail) and dorsal-ventral axes. In vertebrates, and especially amphibians, complete or partial doubling of the bilaterian body axes can be induced by two different types of embryological manipulations: transplantation of an organizer region or bi-sectioning of an embryo. Such axis doubling relies on the ability of embryonic fields to flexibly respond to the situation and self-regulate toward forming a whole body. This phenomenon has facilitated experimental efforts to investigate the mechanisms of vertebrate body axes formation. However, few studies have addressed the self-regulatory capabilities of embryonic fields associated with body axes formation in non-vertebrate bilaterians. The pioneer spider embryologist Åke Holm reported twinning of spider embryos induced by both types of embryological manipulations in 1952; yet, his experiments have not been replicated by other investigators, and access to spider or non-vertebrate twins has been limited. In this review, we provide a historical background on twinning experiments in spiders, and an overview of current twinning approaches in familiar spider species and related molecular studies. Moreover, we discuss the benefits of the spider model system for a deeper understanding of the ancestral mechanisms of body axes formation in arthropods, as well as in bilaterians.

Formation and development of the male copulatory organ in the spider Parasteatoda tepidariorum involves a metamorphosis-like process.

Spiders have evolved a unique male copulatory organ, the pedipalp bulb. The morphology of the bulb is species specific and plays an important role in species recognition and prezygotic reproductive isolation. Despite its importance for spider biodiversity, the mechanisms that control bulb development are virtually unknown. We have used confocal laser scanning microscopy (CLSM) and diffusible iodine-based contrast-enhanced micro computed tomography (dice-µCT) to study bulb development in the spider Parasteatoda tepidariorum. These imaging technologies enabled us to study bulb development in situ, without the use of destructive procedures for the first time. We show here that the inflated pedipalp tip in the subadult stage is filled with haemolymph that rapidly coagulates. Coagulation indicates histolytic processes that disintegrate tibia and tarsus, similar to histolytic processes during metamorphosis in holometabolous insects. The coagulated material contains cell inclusions that likely represent the cell source for the re-establishment of tarsus and tibia after histolysis, comparable to the histoblasts in insect metamorphosis. The shape of the coagulated mass prefigures the shape of the adult tarsus (cymbium) like a blueprint for the histoblasts. This suggests a unique role for controlled coagulation after histolysis in the metamorphosis-like morphogenesis of the male pedipalp.

Sterile Capsule-Egg Cocoon Covering Constitutes an Antibacterial Barrier for Spider Parasteatoda tepidariorum Embryos.

Coexistence of organisms and pathogens has resulted in the evolution of efficient antimicrobial defense, especially at the embryonic stage. This investigation aimed to substantiate the hypothesis that the layers of silk in a spider cocoon play a role in the immunity of the embryos against microorganisms present in the external environment. A two-step interdisciplinary attempt has been made. First, the eggs and empty cocoons of the spider Parasteatoda tepidariorum were incubated on lysogeny broth agar media for 3 d. In the samples of eggs, no growth of bacteria was detected. This indicated that the eggs inside cocoons were sterile. Therefore, in the second step, the cocoons and egg surface were analyzed using SEM, TEM, and LM. The obtained images demonstrated that both inner and outer layers of the silk are built of threads of the same diameter, set in an irregular manner, and randomly clustered into groups. The threads in the outer layer were packed more densely than in the inner one. TEM analysis revealed threads of two types of fibrils and their arrangement. The resultant thread tangle of the cocoon, possibly correlated with the ultrastructure of the fibers, seems to be an example of a structure-function relationship playing a crucial ecoimmunological role in spider embryonic development.

Duplication and expression of Sox genes in spiders.

BACKGROUND: The Sox family of transcription factors is an important part of the genetic 'toolbox' of all metazoans examined to date and is known to play important developmental roles in vertebrates and insects. However, outside the commonly studied Drosophila model little is known about the repertoire of Sox family transcription factors in other arthropod species. Here we characterise the Sox family in two chelicerate species, the spiders Parasteatoda tepidariorum and Stegodyphus mimosarum, which have experienced a whole genome duplication (WGD) in their evolutionary history. RESULTS: We find that virtually all of the duplicate Sox genes have been retained in these spiders after the WGD. Analysis of the expression of Sox genes in P. tepidariorum embryos suggests that it is likely that some of these genes have neofunctionalised after duplication. Our expression analysis also strengthens the view that an orthologue of vertebrate Group B1 genes, SoxNeuro, is implicated in the earliest events of CNS specification in both vertebrates and invertebrates. In addition, a gene in the Dichaete/Sox21b class is dynamically expressed in the spider segment addition zone, suggestive of an ancient regulatory mechanism controlling arthropod segmentation as recently suggested for flies and beetles. Together with the recent analysis of Sox gene expression in the embryos of other arthropods, our findings support the idea of conserved functions for some of these genes, including a potential role for SoxC and SoxD genes in CNS development and SoxF in limb development. CONCLUSIONS: Our study provides a new chelicerate perspective to understanding the evolution and function of Sox genes and how the retention of duplicates of such important tool-box genes after WGD has contributed to different aspects of spider embryogenesis. Future characterisation of the function of these genes in spiders will help us to better understand the evolution of the regulation of important developmental processes in arthropods and other metazoans including neurogenesis and segmentation.

A quantitative study of the diversity of stripe-forming processes in an arthropod cell-based field undergoing axis formation and growth.

One of the conserved traits of arthropod embryonic development is striped expression of homologs of Drosophila segment polarity genes, including hedgehog (hh). Although a diversity of stripe-forming processes is recognized among arthropod embryos, such varied stripe-forming processes have not been well characterized from cellular and quantitative perspectives. The spider Parasteatoda tepidariorum embryo, which has a hh-dependent mechanism of axis formation, offers a cell-based field where the stripes of Pt-hh (a hh homolog) expression dynamically develop in accordance with axis formation and growth, with the patterning processes varying among the regions of the field. In this study, using cell labeling, we mapped the future body subdivisions to the germ disc in the spider embryo and provided substantial evidence for the occurrence of kinetic waves of Pt-hh expression in the presumptive head and opisthosomal (or abdominal) regions of the embryonic field. Notably, combined with cell tracking, we showed that surface cells at and near the center of the germ disc persist in the posterior portion of the field from where Pt-hh stripes sequentially arise, suggesting the operation of ordered oscillations of Pt-hh expression. We then conducted a quantitative analysis of forming/formed Pt-hh stripes using serially timed fixation of sibling embryos. By utilizing length measurements that reflect the axis growth of the embryonic field, we reconstructed the pattern dynamics, which captured repeated splitting of Pt-hh stripes and oscillations of Pt-hh expression in the presumptive head and opisthosomal regions, respectively. In the intermediate thoracic region, three stripes of Pt-hh expression showed a late appearance, with the segmental units specified much earlier by another mechanism. Analyses provided quantitative estimates related to axis growth and stripe-splitting and oscillation events, including the periods of the patterning cycles. This work characterizes the diversity of stripe-forming processes in a cell-based field in a common spatiotemporal framework and highlights the contrasting dynamics of splitting versus oscillation. The cellular and quantitative data presented here provide the foundation for experimental, theoretical and evolutionary studies of cell-based pattern formation, especially body axis segmentation in arthropods.

The effect of temperature and light on embryogenesis and post-embryogenesis of the spider Eratigena atrica (Araneae, Agelenidae).

Embryogenesis and post-embryogenesis of spiders depend on several environmental factors including light and temperature. This study was aimed at evaluating the impact of different thermal and lighting conditions on embryonic and early post-embryonic development of Eratigena atrica. Embryos, larvae, nymphs I and II were incubated at constant temperatures of 12, 22, 25 and 32°C under three different light regimes: light, dark, light/dark. Extreme temperatures (12 and 32°C) significantly increased mortality of embryos (to 100%) and nymphs II, whereas larvae and nymphs I suffered reduced survival only at the lowest temperature. Moreover, the lowest temperature reduced the development rate of all stages. The impact of light conditions was less pronounced and more variable: constant light reduced the survival of nymphs I at lower temperatures, but increased that of larvae. Moreover, light increased the time of embryonic development and duration of nymphal stages, particularly at lower temperatures (12-22°C). Thus, the most optimal locations for spiders seem to be dark (though except larval stage) and warm (25°C) sites, where their development is fastest and mortality lowest.

Embryonic and post-embryonic development inside wolf spiders' egg sac with special emphasis on the vitellus.

The development of Pardosa saltans wolf spiders inside an egg sac includes two periods: an embryonic period and a post-embryonic period after hatching. We investigated spiderlings' energy expenditure to assess energetic costs during the different embryonic and post-embryonic developmental stages during which they are confined within their egg sac. We focused on the following developmental stages: egg, embryonic stages 1 and 2, and two stages, separated by a moult, during post-embryogenesis inside the egg sac: "juvenile instars 1 and 2" until emergence of 2 instar juveniles from their egg sac. We present the first biochemical characterization of the vitellus of wolf spiders' eggs, embryos and juveniles. Lipovitellins (LV) are composed of four apolipoproteins of 116, 87, 70 and 42 kDa, respectively, and LV represent 35-45% of total protein during development. The principal LV lipids are triglycerides, phospholipids, free fatty acids and sterols. Egg caloric content averaged 127 cal/g (proteins: 91 cal/g, lipids: 33 cal/g, carbohydrates: 3 cal/g). During development from undivided egg to emerged "juvenile 2", 67% of proteins, 51% of carbohydrates and 49% of triglycerides stocks were depleted. At the end of the post-embryonic period, at emergence from egg sac, body energy stock of "juveniles 2" was 38% of the initial calorie stocks in the eggs.

Morphology and the central nervous system of Eratigena atrica affected by a complex anomaly in the anterior part of the prosoma.

Spider embryogenesis is affected by a range of environmental factors. Any sudden, drastic change in the environment may impair spider development, leading to various body deformities. In the present study, we analyze changes in the morphology and structure of the central nervous system of an Eratigena atrica larva, obtained in a teratological experiment in which embryos were exposed to alternating temperatures of 14 and 32 °C for the first 10 days. The studied larva had three pedipalps on the right side of the prosoma (polymely), two of which were fused along their entire length (total heterosymely). In addition, there was a short, club-shaped stump between the pedipalps. Histological analysis confirmed major changes in the structure of the subesophageal ganglion, i.e., the fusion of all three ganglia of pedipalps.

A novel role for Ets4 in axis specification and cell migration in the spider Parasteatoda tepidariorum.

Organizers play important roles during the embryonic development of many animals. The most famous example is the Spemann organizer that sets up embryonic axes in amphibian embryos. In spiders, a group of BMP secreting mesenchymal cells (the cumulus) functions as an organizer of the dorsoventral axis. Similar to experiments performed with the Spemann organizer, transplantation of the cumulus is able to induce a secondary axis in spiders. Despite the importance of this structure, it is unknown which factors are needed to activate cumulus specific gene expression. To address this question, we performed a transcriptomic analysis of early embryonic development in the spider Parasteatoda tepidariorum. Through this work, we found that the transcription factor Pt-Ets4 is needed for cumulus integrity, dorsoventral patterning and for the activation of Pt-hunchback and Pt-twist expression. Furthermore, ectopic expression of Pt-Ets4 is sufficient to induce cell delamination and migration by inducing a mesoderm-like cell fate.

Molecular control of gut formation in the spider Parasteatoda tepidariorum.

The development of a digestive system is an essential feature of bilaterians. Studies of the molecular control of gut formation in arthropods have been studied in detail in the fruit fly Drosophila melanogaster. However, little is known in other arthropods, especially in noninsect arthropods. To better understand the evolution of arthropod alimentary system, we investigate the molecular control of gut development in the spider Parasteatoda tepidariorum (Pt), the primary chelicerate model species for developmental studies. Orthologs of the ectodermal genes Pt-wingless (Pt-wg) and Pt-hedgehog (Pt-hh), of the endodermal genes, Pt-serpent (Pt-srp) and Pt-hepatocyte-nuclear factor-4 (Pt-hnf4) and of the mesodermal gene Pt-twist (Pt-twi) are expressed in the same germ layers during spider gut development as in D. melanogaster. Thus, our expression data suggest that the downstream molecular components involved in gut development in arthropods are conserved. However, Pt-forkhead (Pt-fkh) expression and function in spiders is considerably different from its D. melanogaster ortholog. Pt-fkh is expressed before gastrulation in a cell population that gives rise to endodermal and mesodermal precursors, suggesting a possible role for this factor in specification of both germ layers. To test this hypothesis, we knocked down Pt-fkh via RNA interference. Pt-fkh RNAi embryos not only fail to develop a proper gut, but also lack the mesodermal Pt-twi expressing cells. Thus, in spiders Pt-fkh specifies endodermal and mesodermal germ layers. We discuss the implications of these findings for the evolution and development of gut formation in Ecdysozoans.

Book lung development in embryos of the cobweb spider, Parasteatoda tepidariorum C. L. Koch, 1841 (Araneomorphae, Theridiidae).

Light and transmission electron microscopy were used to study the development of book lungs in embryos of the spider Parasteatoda tepidariorum. There is a bilateral cluster of temporary lamellae that form just posterior to the second opisthosomal (O2) limb buds. These lamellae are replaced by advanced embryo (AE) book lungs that continue into postembryonic stages. Results herein agree with earlier suggestions that the O2 limb buds become the AE book lungs. Each O2 limb bud merges with the ventral surface of the O2 segment, where the limb bud/book lung is internalized by covering with epidermis. A strand of tissue (entapophysis) from the epidermis at the posterior opisthosoma provides precursor cells for the book lung lamellae, and possibly entapophysis cells induce limb bud cells to align and produce lamellae. Electron micrographs show the different modes (I-III) of lumen formation. The result is a spiracle, atrium and alternating air and hemolymph channels. A hypothesis is presented for the role of precursor cell polarity in producing the planar tissue polarity of the channels. Some type of apical/apical affinity results in air channels, while basal/basal affinity results in hemolymph channels. Strong basal/basal affinity is likely as opposed cells in hemolymph channels extend basal processes that span the channel and start pillar trabeculae that continue in postembryonic stages.

Bicephality, a seldom occurring developmental deformity in Tegenaria atrica caused by alternating temperatures.

The experiment was aimed at demonstrating the relationship between deformities of the front part of the prosoma accompanied by changes in the brain structure in bicephalous Tegenaria atrica and exposure of their embryos to temperature fluctuations. By exposing spider embryos to alternating temperatures of 14 and 32°C for the first 10 days of embryonic development, we obtained eight two-headed individuals, subsequently divided into three groups according to morphological differences. We described in detail morphological abnormalities of the prosoma identified in members of each group. Histological examination confirmed a close relationship between morphological deformities and the brain structure of teratogenically changed spiders. The fusion of appendages (pedipalps and chalicerae) was accompanied by the fusion of corresponding ganglia. The absence of appendages (pedipalps) was accompanied by the absence of corresponding ganglia. This correlation may have resulted from previously impaired neuromere development which led to changes in the morphological structure of the prosoma. Since no deformities were identified in control animals, it can be concluded that bicephaly was caused by exposing embryos to alternating temperatures.