Morphological changes during the post-embryonic ontogeny of mesothelan spiders and aspects of character evolution in early spiders.

Most morphological studies focus on adult specimens, or if developmental studies are pursued, especially in Euarthropoda, they focus on embryonic development. Araneae (spiders) is one of these groups, in consequence with understudied post-embryonic development. Here we present aspects of the post-embryonic stages of different species of Mesothelae, sister group to the remaining spiders (when fossil species are not taken into account). We used different imaging methods and measured different external morphological structures to detect possible ontogenetic changes. One structure exhibiting post-embryonic changes is the chelicera. Here the significant change occurs between the last immature stage and the adult, yet only in males. For the spinnerets, we could not detect ontogenetic changes, but instead a high variability in length and width, probably due to their lack of pivot joints between the elements. The strongest morphological change during ontogeny occurred on the sternum, which begins with a rather roundish shape in the first stage and changes to being fairly elongate in shape in the last immature stages and the adult. This specific sternum shape only occurs in adults of mesothelan spiders, while opisthothelan spiders have a broader sternum also in the adult. We discuss our results in an evolutionary context, also taking into account recent finds of fossil spiders.

Nephila edulis-breeding and care under laboratory conditions.

Due to fascinating mechanical and biological characteristics spider silk is of great interest in many research fields. Among the orb-weavers Nephila edulis is one of the species used as source for natural spider silk in laboratories. Under appropriate conditions, animals can be kept and bred easily. This manuscript gives information about the spiders' natural habitat, behavior, and breeding and compares them with the established methods and conditions within a research laboratory. Keeping conditions and methods of rearing are described in detail. Within a keeping facility with reliable supply of food, cannibalism rate is significantly reduced and spiders mate all year long. Cohabitants of the genus Steatoda are routinely found in laboratory keeping. While these small spiders do not pose a threat to Nephila edulis, cellar spiders (family Pholcidae) have to be extracted as they have been observed hunting for Nephila spiders.

Sex differences in spiders: from phenotype to genomics.

Sexual reproduction is pervasive in animals and has led to the evolution of sexual dimorphism. In most animals, males and females show marked differences in primary and secondary sexual traits. The formation of sex-specific organs and eventually sex-specific behaviors is defined during the development of an organism. Sex determination processes have been extensively studied in a few well-established model organisms. While some key molecular regulators are conserved across animals, the initiation of sex determination is highly diverse. To reveal the mechanisms underlying the development of sexual dimorphism and to identify the evolutionary forces driving the evolution of different sexes, sex determination mechanisms must thus be studied in detail in many different animal species beyond the typical model systems. In this perspective article, we argue that spiders represent an excellent group of animals in which to study sex determination mechanisms. We show that spiders are sexually dimorphic in various morphological, behavioral, and life history traits. The availability of an increasing number of genomic and transcriptomic resources and functional tools provides a great starting point to scrutinize the extensive sexual dimorphism present in spiders on a mechanistic level. We provide an overview of the current knowledge of sex determination in spiders and propose approaches to reveal the molecular and genetic underpinnings of sexual dimorphism in these exciting animals.

Distribution and development of the external sense organ pattern on the appendages of postembryonic and adult stages of the spider Parasteatoda tepidariorum.

Spiders are equipped with a large number of innervated cuticular specializations, which respond to various sensory stimuli. The physiological function of mechanosensory organs has been analysed in great detail in some model spider species (e.g. Cupiennius salei); however, much less is known about the distribution and function of chemosensory organs. Furthermore, our knowledge on how the sense organ pattern develops on the spider appendages is limited. Here we analyse the development of the pattern and distribution of six different external mechano- and chemosensory organs in all postembryonic stages and in adult male and female spiders of the species Parasteatoda tepidariorum. We show that except for small mechanosensory setae, external sense organs appear in fixed positions on the pedipalps and first walking legs, arranged in longitudinal rows along the proximal-distal axis or in invariable positions relative to morphological landmarks (joints, distal tarsal tip). A comparison to other Entelegynae spiders shows that these features are conserved. We hope that this study lays the foundation for future molecular analysis to address the question how this conserved pattern is generated.

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.

six3 acts upstream of foxQ2 in labrum and neural development in the spider Parasteatoda tepidariorum.

Anterior patterning in animals is based on a gene regulatory network, which comprises highly conserved transcription factors like six3, pax6 and otx. More recently, foxQ2 was found to be an ancestral component of this network but its regulatory interactions showed evolutionary differences. In most animals, foxQ2 is a downstream target of six3 and knockdown leads to mild or no epidermal phenotypes. In contrast, in the red flour beetle Tribolium castaneum, foxQ2 gained a more prominent role in patterning leading to strong epidermal and brain phenotypes and being required for six3 expression. However, it has remained unclear which of these novel aspects were insect or arthropod specific. Here, we study expression and RNAi phenotype of the single foxQ2 ortholog of the spider Parasteatoda tepidariorum. We find early anterior expression similar to the one of insects. Further, we show an epidermal phenotype in the labrum similar to the insect phenotype. However, our data indicate that foxQ2 is positioned downstream of six3 like in other animals but unlike insects. Hence, the epidermal and neural pattering function of foxQ2 is ancestral for arthropods while the upstream role of foxQ2 may have evolved in the lineage leading to the insects.

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.

Transformational Mimicry in a Myrmecomorphic Spider.

Species that are Batesian mimics during postembryonic development shift between mimetic models as they grow in size. However, it has not yet been tested whether these successive mimetic phenotypes are similarly protected from predators. Early-instar phenotypes could represent an inaccurate phenotype or an accurate phenotype because of selection from different predators. Here, we tested the hypotheses of transformational Batesian mimicry in the ant-mimicking jumping spider Leptochestes berolinensis. We quantified the mimetic accuracy of different ontogenetic stages to potential ant models by using a multitrait approach. We measured movement, body profile, body size, and coloration. Analysis revealed adults to be more accurate mimics than juveniles. Adults were similar to smaller morphs of Camponotus or Lasius ants, whereas juveniles were more similar to Lasius and Colobopsis ants. We tested whether predators, mantises, and Pisaura spiders were deceived by mimics after having experience with ant models. These predators never captured any ant or a mimic but always captured the nonmyrmecomorphic spider. We conclude that L. berolinensis is a Batesian mimic of ants undergoing transformational mimicry, with all stages being accurate mimics.

Tachinobia repanda (Hymenoptera: Eulophidae) From Egg Sacs of a Colonial Spider, Cyrtophora moluccensis (Araneae: Araneidae) in Papua New Guinea.

We report the discovery of the wasp Tachinobia repanda Boucek collected from egg sacs of the colonial spider Cyrtophora moluccensis (Doleschall) in Morobe Province, Papua New Guinea (PNG) by Lubin, Y.D. in 1980. This is the first record of T. repanda from egg sacs of a colonial spider. The likely host of this eulophid wasp was the larvae of a sarcophagid fly that parasitizes the egg sacs of these spiders. The 67 T. repanda collected were all females and varied little in body size. We suggest that this species is a gregarious hyperparasitoid.

Allometry of reaching and contact structures in Kukulcania hibernalis (Araneae: Filistatidae).

Large part of the morphological diversity observed across taxa is attributed to the effect of sexual selection; and the static allometry of these structures vary largely from highly positive to negative, depending on their function, and position on the animal's body. In arthropods, information of how sexually selected contact and reaching male structures use during courtship scale on body size is scarce. We tested two complementary hypotheses: the reaching structure hypothesis and the contact-function hypothesis, in the spider Kukulcania hibernalis. We used the length of the proximal segments of the male pedipalp to test the reaching structure hypothesis, and claw features to test the contact-function hypothesis. Our results support both hypotheses. Small males have disproportionally longer pedipalps (highly negative allometry) than large males, increasing the probability of small-bodied males to inseminate even large females. We also found that both distal contact and noncontact homologous structures scaled shallow (slope < 1) on body size, but allometry was significantly shallower for contact than for noncontact distal structures, providing support to the contact-function hypothesis, and allowing teasing apart the effect of sexual selection on distal contact structures with dual functions.

Prevalence of a vertically transmitted single-stranded DNA virus in spinybacked orbweavers (Gasteracantha cancriformis) from Florida, USA.

Spiders (order Araneae, class Arachnida) are an important group of predatory arthropods in terrestrial ecosystems that have been recently identified as an untapped reservoir of single-stranded (ss)DNA viruses. Specifically, spiders harbour a diversity of ssDNA viruses encoding a replication-associated protein (Rep) within a circular genome. However, little is known about the ecology of novel circular Rep-encoding ssDNA (CRESS DNA) viruses. Here we investigated two CRESS DNA viruses recently identified in spinybacked orbweavers (Gasteracantha cancriformis), namely spinybacked orbweaver circular virus (SpOrbCV) 1 and 2. SpOrbCV-1 was detected in the majority (> 65 %) of spider specimens from all life stages, including eggs, spiderlings and adults, demonstrating that this virus is active within spinybacked orbweavers. In contrast, SpOrbCV-2 was only detected in adults at a lower (36 %) prevalence. Since we also detected SpOrbCV-2 in other spider species and this virus has been reported from a dragonfly, we suggest that SpOrbCV-2 is accumulated in these predators through common insect prey. The prevalence of SpOrbCV-1 in collected specimens allowed us to design assays to characterize this virus, which represents a new group of CRESS DNA viruses, the 'circularisviruses'. To our knowledge, SpOrbCV-1 is the first example of a vertically transmitted virus in spiders, which may explain its high prevalence in spinybacked orbweavers. Since vertically transmitted viruses infecting insects (class Insecta) can manipulate their host's behaviour and physiology, future studies should investigate the ecological role of vertically transmitted viruses in spiders.

Hunting the wolf: A molecular phylogeny of the wolf spiders (Araneae, Lycosidae).

Lycosids are a diverse family of spiders distributed worldwide. Previous studies recovered some of the deeper splits of the family, but with little support. We present a broad phylogenetic analysis of the Lycosidae including a wide geographic sampling of representatives and all the subfamilies described to date. Additionally, we extend the amount of molecular data used in previous studies (28S, 12S and NADH) through the inclusion of two additional markers, the nuclear H3 and the mitochondrial COI. We estimated the divergence times through the inclusion of fossils as calibration points and used the phylogenetic hypothesis obtained to explore the evolution of particular traits associated with dispersal capabilities. We recovered most of the currently recognized subfamilies with high nodal support. Based on these results, we synonymize Piratinae and Wadicosinae with Zoicinae and Pardosinae, respectively, and revalidate the subfamily Hippasinae. We corroborated that lycosids are a family with a relatively young origin that diversified with the reduction of tropical forests and the advance of open habitats. We show that a gradual accumulation of behavioral traits associated with ambulatory dispersal made Lycosidae the most vagrant subfamily of spiders, with an impressive ability to disperse long distances which helps to explain the worldwide distribution of some very young clades, such as the members of the subfamily Lycosinae.

The Role of Sexual Dimorphism and Tissue Selection in Ecotoxicological Studies Using the Riparian Spider Tetragnatha elongata.

Tetragnathid spiders (Tetragnatha spp.) found in riparian habitats have recently been used as bioindicators of sediment contamination and insect-mediated contaminant flux. We investigated whether sexual dimorphism (size and behavior) influenced the female:male ratio in composite samples, stable isotope ratios (carbon [δ13C], nitrogen [δ15N]), and Hg concentrations in the southern United States. Additionally, we explored whether biomass for contaminant analysis could be preserved by using the legs of tetragnathids as a surrogate for whole-body δ13C and δ15N signatures. We found that female tetragnathids were significantly larger than male spiders and represented a larger proportion of spiders collected at all sites. However, despite the difference in size between sexes, no differences in growth dynamics, isotopic signatures (δ13C and δ15N), or mercury concentrations were observed. It was determined that the leg of a tetragnathid can accurately represent the stable isotope signature of an entire spider.

A Probable Case of Incipient Speciation in Schizocosa Wolf Spiders Driven by Allochrony, Habitat Use, and Female Mate Choice.

There is growing evidence that speciation can occur between populations that are not geographically isolated. The emergence of assortative mating is believed to be critical to this process, but how assortative mating arises in diverging populations is poorly understood. The wolf spider genus Schizocosa has become a model system for studying mechanisms of assortative mating. We conducted a series of experiments to identify the factors that control mate pair formation in a Schizocosa population that includes both ornamented and nonornamented males. We show that the population also includes two previously unrecognized female phenotypes. One female phenotype mates mostly or exclusively with ornamented males, and the other mates mostly or exclusively with unornamented males. Assortative mating within these groups is maintained by differences in maturation time, microhabitat use, and female mate preference. We conclude that the population is not a single species, as previously believed, but rather an incipient species pair with multiple overlapping mechanisms of reproductive isolation. The identification of a new incipient species pair in the well-studied and rapidly speciating Schizocosa clade presents new opportunities for the study of speciation without geographic isolation.

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.

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.

Nutrient intake determines post-maturity molting in the golden orb-web spider Nephila pilipes (Araneae: Araneidae).

While molting occurs in the development of many animals, especially arthropods, post-maturity molting (PMM, organisms continue to molt after sexual maturity) has received little attention. The mechanism of molting has been studied intensively; however, the mechanism of PMM remains unknown although it is suggested to be crucial for the development of body size. In this study, we investigated factors that potentially induce PMM in the golden orb-web spider Nephila pilipes, which has the greatest degree of sexual dimorphism among terrestrial animals. We manipulated the mating history and the nutrient consumption of the females to examine whether they affect PMM. The results showed that female spiders under low nutrition were more likely to molt as adults, and mating had no significant influence on the occurrence of PMM. Moreover, spiders that underwent PMM lived longer than those that did not and their body sizes were significantly increased. Therefore, we concluded that nutritional condition rather than mating history affect PMM.

Identification and expression of nuclear receptor genes and ecdysteroid titers during nymphal development in the spider Agelena silvatica.

Ecdysteroids play an essential role in the regulation of the molting processes of arthropods. Nuclear receptors of the spider Agelena silvatica that showed high homology with other arthropods especially in the functional domains were identified, two isoforms of ecdysone receptor (AsEcRA, AsEcRB), retinoid X receptor (AsRXR) and two isoforms of E75 (AsE75A, AsE75D). AsEcR and AsRXR mRNA did not show major changes in expression but occurred throughout the third instar nymphal stage. AsE75DBD was low or non-existent at first then showed a sudden increase from D7 to D10. On the other hand, AsE75D was expressed in the first half and decreased from D6 to D10. Ecdysteroid titers showed a peak on D6 in A. silvatica third instar nymphs. LC-MS/MS analysis of the ecdysteroid peak revealed only 20-hydroxyecdysone (20E) was present. The 20E peak on D6 and increase in AsE75DBD from D7 is likely a result of ecdysteroids binding to the heterodimer formed with constant expression of the AsEcR and AsRXR receptors. These findings indicate the mechanisms regulating molting widely conserved in insects and other arthropods also similarly function in spiders.

Socially cued developmental plasticity in web-building spiders.

BACKGROUND: Socially cued anticipatory plasticity (SCAP) has been proposed as a widespread mechanism of adaptive life-history shifts in semelparous species with extreme male mating investment. Such mating systems evolved several times independently in spiders and male reproductive success should critically depend on timely maturation and rapid location of a receptive and, ideally, virgin female. We experimentally investigated socially cued anticipatory plasticity in two sympatric, closely related Nephila species that share many components of their mating systems, but differ in the degree to which male reproductive success depends on mating with virgin females. Juveniles of both species were reared either in the presence or absence of virgin female silk cues. We predicted strong selection on socially cued plasticity in N. fenestrata in which males follow a highly specialized terminal investment strategy, but expected a weaker plastic response in N. senegalensis in which males lost the ability to monopolize females. RESULTS: Contrary to our predictions, N. fenestrata males presented with virgin female silk cues did not mature earlier than siblings reared isolated from such cues. Males in N. senegalensis, however, showed a significant response to female cues and matured several days earlier than control males. Plastic adjustment of maturation had no effect on male size. CONCLUSIONS: Our results indicate that a strong benefit of mating with virgins due to first male sperm priority does not necessarily promote socially cued anticipatory plasticity. We emphasize the bidirectional mode of developmental responses and suggest that this form of plasticity may not only yield benefits through accelerated maturation, but also by avoiding costs of precipitate maturation in the absence of female cues.

Observations on germ band development in the cellar spider Pholcus phalangioides.

Most recent studies of spider embryonic development have focused on representatives of the species-rich group of entelegyne spiders (over 80 % of all extant species). Embryogenesis in the smaller spider groups, however, is less well studied. Here, we describe the development of the germ band in the spider species Pholcus phalangioides, a representative of the haplogyne spiders that are phylogenetically the sister group of the entelegyne spiders. We show that the transition from radially symmetric embryonic anlage to the bilaterally symmetric germ band involves the accumulation of cells in the centre of the embryonic anlage (primary thickening). These cells then disperse all across the embryonic anlage. A secondary thickening of cells then appears in the centre of the embryonic anlage, and this thickening expands and forms the segment addition zone. We also confirm that the major part of the opisthosoma initially develops as a tube shaped structure, and its segments are then sequentially folded down on the yolk during inversion. This special mode of opisthosoma formation has not been reported for entelegyne spiders, but a more comprehensive sampling of this diverse group is necessary to decide whether this peculiarity is indeed lacking in the entelegyne spiders.