Female mating rates and their fitness consequences in the common house spider Parasteatoda tepidariorum

Mating systems, with varying female mating rates occurring with the same partner (monandry) or with multiple mates (polyandry), can have far reaching consequences for population viability and the rate of gene flow. Here, we investigate the mating rates of the common house spider Parasteatoda tepidariorum (Theridiidae), an emerging model for genetic studies, with yet undescribed reproductive behavior. It is hypothesized that spiders belonging to this family have low re-mating rates. We paired females twice with the same male (monandry) or with different males (polyandry), and recorded behaviors, mating success and fitness resulting from single- and double-matings, either monandrous or polyandrous. Despite the study being explorative in nature, we predict successful matings to be more frequent during first encounters, to reduce female risk of remaining unmated. For re-mating to be adaptive, we expect higher fitness of double-mated females, and polyandrous females to experience highest mating success and fitness if reproductive gains are achieved by mating with multiple partners. We show that the majority of the females did not mate, and those that did mated only once, not necessarily on their first encounter. The likelihood of re-mating did not differ between monandrous and polyandrous encounters and female mating experience (mated once, twice monandrous, twice polyandrous) did not affect fitness, indicated by similar offspring production. Female twanging of the web leads to successful matings suggesting female behavioral receptivity. Cannibalism rates were low and mostly occurred pre-copulatory. We discuss how the species ecology, with potentially high mating costs for males and limited female receptivity, may shape a mating system with low mating rates

An atlas of spider development at single-cell resolution provides new insights into arthropod embryogenesis

Spiders are a diverse order of chelicerates that diverged from other arthropods over 500 million years ago. Research on spider embryogenesis, particularly studies using the common house spider Parasteatoda tepidariorum, has made important contributions to understanding the evolution of animal development, including axis formation, segmentation, and patterning. However, we lack knowledge about the cells that build spider embryos, their gene expression profiles and fate. Single-cell transcriptomic analyses have been revolutionary in describing these complex landscapes of cellular genetics in a range of animals. Therefore, we carried out single-cell RNA sequencing of P. tepidariorum embryos at stages 7, 8 and 9, which encompass the establishment and patterning of the body plan, and initial differentiation of many tissues and organs. We identified 20 cell clusters, from 18.5 k cells, which were marked by many developmental toolkit genes, as well as a plethora of genes not previously investigated. We found differences in the cell cycle transcriptional signatures, suggestive of different proliferation dynamics, which related to distinctions between endodermal and some mesodermal clusters, compared with ectodermal clusters. We identified many Hox genes as markers of cell clusters, and Hox gene ohnologs were often present in different clusters. This provided additional evidence of sub- and/or neo-functionalisation of these important developmental genes after the whole genome duplication in an arachnopulmonate ancestor (spiders, scorpions, and related orders). We also examined the spatial expression of marker genes for each cluster to generate a comprehensive cell atlas of these embryonic stages. This revealed new insights into the cellular basis and genetic regulation of head patterning, hematopoiesis, limb development, gut development, and posterior segmentation. This atlas will serve as a platform for future analysis of spider cell specification and fate, and studying the evolution of these processes among animals at cellular resolution

A chelicerate Wnt gene expression atlas: novel insights into the complexity of arthropod Wnt-patterning

The Wnt genes represent a large family of secreted glycoprotein ligands that date back to early animal evolution. Multiple duplication events generated a set of 13 Wnt families of which 12 are preserved in protostomes. Embryonic Wnt expression patterns (Wnt-patterning) are complex, representing the plentitude of functions these genes play during development. Here, we comprehensively investigated the embryonic expression patterns of Wnt genes from three species of spiders covering both main groups of true spiders, Haplogynae and Entelegynae, a mygalomorph species (tarantula), as well as a distantly related chelicerate outgroup species, the harvestman Phalangium opilio. All spiders possess the same ten classes of Wnt genes, but retained partially different sets of duplicated Wnt genes after whole genome duplication, some of which representing impressive examples of sub- and neo-functionalization. The harvestman, however, possesses a more complete set of 11 Wnt genes but with no duplicates. Our comprehensive data-analysis suggests a high degree of complexity and evolutionary flexibility of Wnt-patterning likely providing a firm network of mutational protection. We discuss the new data on Wnt gene expression in terms of their potential function in segmentation, posterior elongation, and appendage development and critically review previous research on these topics. We conclude that earlier research may have suffered from the absence of comprehensive gene expression data leading to partial misconceptions about the roles of Wnt genes in development and evolution

Lack of evidence for conserved parasegmental grooves in arthropods

In the arthropod model species Drosophila melanogaster, a dipteran fly, segmentation of the anterior-posterior body axis is under control of a hierarchic gene cascade. Segmental boundaries that form morphological grooves are established posteriorly within the segmental expression domain of the segment-polarity gene (SPG) engrailed (en). More important for the development of the fly, however, are the parasegmental boundaries that are established at the interface of en expressing cells and anteriorly adjacent wingless (wg) expressing cells. In Drosophila, both segmental and transient parasegmental grooves form. The latter are positioned anterior to the expression of en. Although the function of the SPGs in establishing and maintaining segmental and parasegmental boundaries is highly conserved among arthropods, parasegmental grooves have only been reported for Drosophila, and a spider (Cupiennius salei). Here, we present new data on en expression, and re-evaluate published data, from four distantly related spiders, including Cupiennius, and a distantly related chelicerate, the harvestman Phalangium opilio. Gene expression analysis of en genes in these animals does not corroborate the presence of parasegmental grooves. Consequently, our data question the general presence of parasegmental grooves in arthropods

Expression and function of the zinc finger transcription factor Sp6-9 in the spider Parasteatoda tepidariorum

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

It takes Two: Discovery of Spider Pax2 Duplicates Indicates Prominent Role in Chelicerate Central Nervous System, Eye, as Well as External Sense Organ Precursor Formation and Diversification After Neo- and Subfunctionalization

Paired box genes are conserved across animals and encode transcription factors playing key roles in development, especially neurogenesis. Pax6 is a chief example for functional conservation required for eye development in most bilaterian lineages except chelicerates. Pax6 is ancestrally linked and was shown to have interchangeable functions with Pax2. Drosophila melanogaster Pax2 plays an important role in the development of sensory hairs across the whole body. In addition, it is required for the differentiation of compound eyes, making it a prime candidate to study the genetic basis of arthropod sense organ development and diversification, as well as the role of Pax genes in eye development. Interestingly, in previous studies identification of chelicerate Pax2 was either neglected or failed. Here we report the expression of two Pax2 orthologs in the common house spider Parasteatoda tepidariorum, a model organism for chelicerate development. The two Pax2 orthologs most likely arose as a consequence of a whole genome duplication in the last common ancestor of spiders and scorpions. Pax2.1 is expressed in the peripheral nervous system, including developing lateral eyes and external sensilla, as well as the ventral neuroectoderm of P. tepidariorum embryos. This not only hints at a conserved dual role of Pax2/5/8 orthologs in arthropod sense organ development but suggests that in chelicerates, Pax2 could have acquired the role usually played by Pax6. For the other paralog, Pt-Pax2.2, expression was detected in the brain, but not in the lateral eyes and the expression pattern associated with sensory hairs differs in timing, pattern, and strength. To achieve a broader phylogenetic sampling, we also studied the expression of both Pax2 genes in the haplogyne cellar spider Pholcus phalangioides. We found that the expression difference between paralogs is even more extreme in this species, since Pp-Pax2.2 shows an interesting expression pattern in the ventral neuroectoderm while the expression in the prosomal appendages is strictly mesodermal. This expression divergence indicates both sub- and neofunctionalization after Pax2 duplication in spiders and thus presents an opportunity to study the evolution of functional divergence after gene duplication and its impact on sense organ diversification

Regressive evolution of the arthropod tritocerebral segment linked to functional divergence of the Hox gene

The intercalary segment is a limbless version of the tritocerebral segment and is present in the head of all insects, whereas other extant arthropods have retained limbs on their tritocerebral segment (e.g. the pedipalp limbs in spiders). The evolutionary origin of limb loss on the intercalary segment has puzzled zoologists for over a century. Here we show that an intercalary segment-like phenotype can be created in spiders by interfering with the function of the Hox gene labial. This links the origin of the intercalary segment to a functional change in labial. We show that in the spider Parasteatoda tepidariorum the labial gene has two functions: one function in head tissue maintenance that is conserved between spiders and insects, and a second function in pedipalp limb promotion and specification, which is only present in spiders. These results imply that labial was originally crucial for limb formation on the tritocerebral segment, but that it has lost this particular subf unction in the insect ancestor, resulting in limb loss on the intercalary segment. Such loss of a sub-function is a way to avoid adverse pleiotropic effects normally associated with mutations in developmental genes, and may thus be a common mechanism to accelerate regressive evolution

FoxB, a new and highly conserved key factor in arthropod dorsal-ventral (DV) limb patterning

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

Neofunctionalization of a Duplicate dachshund Gene Underlies the Evolution of a Novel Leg Segment in Arachnids

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