Ecdysteroids are key hormones that regulate molting and developmental processes in arthropods, including insect metamorphosis. Understanding the molecular roles of the genes involved in ecdysteroid biosynthesis could provide crucial insights into the evolutionary conservation of regulation and regulatory modifications in arthropods. While extensively studied in insects, the functions of the so-called Halloween genes, encoding enzymes responsible for the conversion of cholesterol to ecdysone and 20-hydroxyecdysone, remain unclear in arachnids. Therefore, this study aims to elucidate the functions of the Halloween gene shadow (Pt-sad) in the spider Parasteatoda tepidariorum in both embryonic and postembryonic stages and to assess its potential role in molting. In situ hybridization was employed to characterize the expression patterns of Pt-sad, and RNAi-mediated knockdown (RNA interference) was conducted to explore its effects on development and molting. Knockdown of Pt-sad resulted in increased embryonic mortality and developmental delays, while it disrupted molt cycles in postembryonic stages, supporting a role in ecdysteroidogenesis. Expression analysis revealed Pt-sad activity in specific regions of embryos and juveniles, including the central nervous system, appendages, and circulatory system. These findings highlight the multifunctionality of shadow in the spider. While the involvement of this gene in the ecdysteroid pathway is most likely conserved in arthropods, this role may have diversified in spiders to include additional, unique functions that differ from their insect counterparts.

Hox genes are known for their role in the specification of typical body plan features in animals. Evolutionary changes in Hox gene function are believed to be involved in the emergence of the diverse body plans we observe in animals today. Spiders share many body plan features with other arthropods, but also have numerous unique traits of their own. Studies of spider Hox genes have already provided insights into evolutionarily conserved and derived features of the spider body plan and their genetic basis. However, many aspects of Hox gene biology have been insufficiently studied in spiders so far. In this review, we highlight previous comparative studies of Hox genes in spiders and their significance for our understanding of the evolution of the spider body plan. We also identify aspects of Hox gene biology that need to be studied in greater detail. Many spider Hox genes have not been investigated beyond their mRNA expression patterns, and the role of Hox genes with apparently plesiomorphic or dual functions, like ftz and Hox3 is still unclear. Spiders have a duplicated Hox gene cluster, but possible sub- or neofunctionalisation of duplicates have not yet been studied systematically. Future research should therefore focus on these issues, in addition to the role of Polycomb and trithorax-mediated regulation, the identification of regulatory regions, cofactors or spider-specific target genes, and the significance of non-coding RNAs transcribed from within the Hox cluster and even from the antisense strand of particular Hox genes.

Leg axis formation in Drosophila is organized by Wingless (Wg) and Decapentaplegic (Dpp) that control a number of downstream factors to pattern the dorsoventral (DV) and proximodistal (PD) axis. The T-box genes are important downstream factors mainly involved in dorsoventral leg axis formation. The ventral side is specified by H15 and midline, whereas optomotor-blind (omb) and Dorsocross (Doc1) are factors to specify dorsal cell fates. We show here that omb also organizes PD leg axis patterning in the beetle Tribolium castaneum. In the legs, Tc-omb is expressed along the dorsal side and represses ventral factors like wg and H15. Intriguingly, removing Tc-omb function leads to the activation of the Dpp pathway along the dorsal side of the legs, thus mimicking normal dpp expression in Drosophila. Dpp activity along the dorsal side leads to altered expression of proximal-distal patterning genes such as Distal-less (Dll) and dachshund (dac). Our results indicate a cell-autonomous activation of Dll and repression of dac by dpp. These findings are compatible with the cross-regulatory "cascade model" of proximal-distal leg imaginal disc patterning of Drosophila.

Fox genes encode transcription factors that contain a DNA binding domain, the forkhead domain, and are known from diverse animal species. The exact homology of the Fox genes of different species is debated and this makes inferences about the evolution of the Fox genes, and their duplications and losses difficult. We have performed phylogenetic analyses of the Fox gene complements of 32 panarthropod species. Our results confirm an ancestral complement of FoxA, FoxB, FoxC, FoxD, FoxF, FoxG, FoxJ1, FoxJ2/3, FoxK, FoxL1, FoxL2, FoxN1/4, FoxN2/3, FoxO, FoxP, and FoxQ2 in the Arthropoda, and additionally FoxH and FoxQ1 in the Panarthropoda (including tardigrades and onychophorans). We identify a novel Fox gene sub-family, that we designate as FoxT that includes two genes in Drosophila melanogaster, Circadianly Regulated Gene (Crg-1) and forkhead domain 3F (fd3F). In a very recent paper, the same new Fox gene sub-family was identified in insects (Lin et al. 2021). Our analysis confirms the presence of FoxT and shows that its members are present throughout Panarthropoda. We show that the hitherto unclassified gene CG32006 from the fly Drosophila melanogaster belongs to FoxJ1. We also detect gene losses: FoxE and FoxM were lost already in the panarthropod ancestor, whereas the loss of FoxH occurred in the arthropod ancestor. Finally, we find an ortholog of FoxQ1 in the bark scorpion Centruroides sculpturatus, confirmed not only by phylogenetic analysis, but also by forming an evolutionarily conserved gene cluster with FoxF, FoxC, and FoxL1. This suggests that FoxQ1 belongs to the ancestral Fox gene complement in panarthropods and also in chelicerates, but has been lost at the base of the mandibulate arthropods.

Fox genes represent an evolutionary old class of transcription factor encoding genes that evolved in the last common ancestor of fungi and animals. They represent key-components of multiple gene regulatory networks (GRNs) that are essential for embryonic development. Most of our knowledge about the function of Fox genes comes from vertebrate research, and for arthropods the only comprehensive gene expression analysis is that of the fly Drosophila melanogaster. For other arthropods, only selected Fox genes have been investigated. In this study, we provide the first comprehensive gene expression analysis of arthropod Fox genes including representative species of all main groups of arthropods, Pancrustacea, Myriapoda and Chelicerata. We also provide the first comprehensive analysis of Fox gene expression in an onychophoran species. Our data show that many of the Fox genes likely retained their function during panarthropod evolution highlighting their importance in development. Comparison with published data from other groups of animals shows that this high degree of evolutionary conservation often dates back beyond the last common ancestor of Panarthropoda.

The formation of extraembryonic membranes (EEMs) contributes to the proper development of many animals. In arthropods, the formation and function of EEMs have been studied best in insects. Regarding the development of extraembryonic tissue in chelicerates (spiders and relatives), most information is available for spiders (Araneae). Especially two populations of cells have been considered to represent EEMs in spiders. The first of these potential EEMs develops shortly after egg deposition, opposite to a radially symmetrical germ disc that forms in one hemisphere of the egg and encloses the yolk. The second tissue, which has been described as being extraembryonic is the so-called dorsal field, which is required to cover the dorsal part of the developing spider germ rudiment before proper dorsal closure. In this review, we summarize the current knowledge regarding the formation of potential extraembryonic structures in the Chelicerata. We describe the early embryogenesis of spiders and other chelicerates, with a special focus on the formation of the potential extraembryonic tissues. This article is part of the theme issue ‘Extraembryonic tissues: exploring concepts, definitions and functions across the animal kingdom’.

Background Arthropods comprise the largest and most diverse phylum on Earth and play vital roles in nearly every ecosystem. Their diversity stems in part from variations on a conserved body plan, resulting from and recorded in adaptive changes in the genome. Dissection of the genomic record of sequence change enables broad questions regarding genome evolution to be addressed, even across hyper-diverse taxa within arthropods. Results Using 76 whole genome sequences representing 21 orders spanning more than 500 million years of arthropod evolution, we document changes in gene and protein domain content and provide temporal and phylogenetic context for interpreting these innovations. We identify many novel gene families that arose early in the evolution of arthropods and during the diversification of insects into modern orders. We reveal unexpected variation in patterns of DNA methylation across arthropods and examples of gene family and protein domain evolution coincident with the appearance of notable phenotypic and physiological adaptations such as flight, metamorphosis, sociality, and chemoperception. Conclusions These analyses demonstrate how large-scale comparative genomics can provide broad new insights into the genotype to phenotype map and generate testable hypotheses about the evolution of animal diversity.

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.

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.

The same genes and signaling pathways control the formation of limbs in vertebrates, arthropods and cuttlefish.

Forkhead box (Fox) transcription factors evolved early in animal evolution and represent important components of conserved gene regulatory networks (GRNs) during animal development. Most of the researches concerning Fox genes, however, are on vertebrates and only a relatively low number of studies investigate Fox gene function in invertebrates. In addition to this shortcoming, the focus of attention is often restricted to a few well-characterized Fox genes such as FoxA (forkhead), FoxC (crocodile) and FoxQ2. Although arthropods represent the largest and most diverse animal group, most other Fox genes have not been investigated in detail, not even in the arthropod model species Drosophila melanogaster. In a general gene expression pattern screen for panarthropod Fox genes including the red flour beetle Tribolium castaneum, the pill millipede Glomeris marginata, the common house spider Parasteatoda tepidariorum, and the velvet worm Euperipatoides kanangrensis, we identified a Fox gene with a highly conserved expression pattern along the ventral ectoderm of arthropod and onychophoran limbs. Functional investigation of FoxB in Parasteatoda reveals a hitherto unrecognized important function of FoxB upstream of wingless (wg) and decapentaplegic (dpp) in the GRN orchestrating dorsal-ventral limb patterning.

The same genes and signaling pathways control the formation of limbs in vertebrates, arthropods and cuttlefish.

Homeobox genes are key toolkit genes that regulate the development of metazoans and changes in their regulation and copy number have contributed to the evolution of phenotypic diversity. We recently identified a whole genome duplication (WGD) event that occurred in an ancestor of spiders and scorpions (Arachnopulmonata), and that many homeobox genes, including two Hox clusters, appear to have been retained in arachnopulmonates. To better understand the consequences of this ancient WGD and the evolution of arachnid homeobox genes, we have characterized and compared the homeobox repertoires in a range of arachnids. We found that many families and clusters of these genes are duplicated in all studied arachnopulmonates (Parasteatoda tepidariorum, Pholcus phalangioides, Centruroides sculpturatus, and Mesobuthus martensii) compared with nonarachnopulmonate arachnids (Phalangium opilio, Neobisium carcinoides, Hesperochernes sp., and Ixodes scapularis). To assess divergence in the roles of homeobox ohnologs, we analyzed the expression of P. tepidariorum homeobox genes during embryogenesis and found pervasive changes in the level and timing of their expression. Furthermore, we compared the spatial expression of a subset of P. tepidariorum ohnologs with their single copy orthologs in P. opilio embryos. We found evidence for likely subfunctionlization and neofunctionalization of these genes in the spider. Overall our results show a high level of retention of homeobox genes in spiders and scorpions post-WGD, which is likely to have made a major contribution to their developmental evolution and diversification through pervasive subfunctionlization and neofunctionalization, and paralleling the outcomes of WGD in vertebrates.

The bleaching, clearing and handling of tiny specimens with soft tissue and cuticular components for confocal laser scanning microscopy is difficult, because after cuticle bleaching and tissue clearing the specimens are virtually invisible. We have adjusted the design of the specimen container described by Smolla et al. (Arthropod Struct Dev 43:175–81, 2014) to handle tiny specimens.

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.

Background: Gene duplications provide genetic material for the evolution of new morphological and physiological features. One copy can preserve the original gene functions while the second copy may evolve new functions (neofunctionalisation). Gene duplications may thus provide new genes involved in evolutionary novelties.Results: We have studied the duplicated homeobox gene homothorax (hth) in the spider species Parasteatoda tepidariorum and Pholcus phalangioides and have compared these data with previously published data from additional spider species. We show that the expression pattern of hth1 is highly conserved among spiders, consistent with the notion that this gene copy preserves the original hth functions. By contrast, hth2 has a markedly different expression profile especially in the prosomal appendages. The pattern in the pedipalps and legs consists of several segmental rings, suggesting a possible role of hth2 in limb joint development. Intriguingly, however, the hth2 pattern is much less conserved between the species than hth1 and shows a species specific pattern in each species investigated so far.Conclusions: We hypothesise that the hth2 gene has gained a new patterning function after gene duplication, but has then undergone a second phase of diversification of its new role in the spider clade. The evolution of hth2 may thus provide an interesting example for a duplicated gene that has not only contributed to genetic diversity through neofunctionalisation, but beyond that has been able to escape evolutionary conservation after neofunctionalisation thus forming the basis for further genetic diversification.

Zinc finger transcription factors of the Sp6–9 group are evolutionarily conserved in all metazoans and have important functions in, e.g., limb formation and heart development. The function of Sp6–9-related genes has been studied in a number of vertebrates and invertebrates, but data from chelicerates (spiders and allies) was lacking so far. We have isolated the ortholog of Sp6–9 from the common house spider Parasteatoda tepidariorum and the cellar spider Pholcus phalangioides. We show that the Sp6–9 gene in these spider species is expressed in the developing appendages thus suggesting a conserved role in limb formation. Indeed, RNAi with Sp6–9 in P. tepidariorum leads not only to strong limb defects, but also to the loss of body segments and head defects in more strongly affected animals. Together with a new expression domain in the early embryo, these data suggest that Sp6–9 has a dual role P. tepidariorum. The early role in head and body segment formation is not known from other arthropods, but the role in limb formation is evolutionarily highly conserved.

The acquisition of a novel function, or neofunctionalization, protects duplicated genes from redundancy and subsequent loss, and is a major force that drives adaptive evolution. Neofunctionalization has been inferred for many duplicated genes based on differences in regulation between the parental gene and its duplicate. However, only few studies actually link the new function of a duplicated gene to a novel morphological or physiological character of the organism. Here we show that the duplication of dachshund (dac) in arachnids (spiders and allies) is linked with the evolution of a novel leg segment, the patella. We have studied dac genes in two distantly related spider species, the entelegyne spider Parasteatoda tepidariorum and the haplogyne spider Pholcus phalangioides. Both species possess two paralogous dac genes that duplicated before the split between entelegyne and haplogyne spiders. In contrast to the evolutionarily highly conserved dac1, its duplicate dac2 is strongly expressed in the patella leg segment during embryogenesis in both species. Using parental RNA interference in P. tepidariorum we show that dac2 is required for the development of the patella segment. If dac2 function is impaired, then the patella is fused with the tibia into a single leg segment. Thus, removing the function of dac2 experimentally reverts P. tepidariorum leg morphology into a stage before the duplication of dac and the evolution of the patella segment. Our results indicate that the origin of the patella is the result of the duplication and subsequent neofunctionalization of dac in the arachnid lineage.