Embryonic expression patterns of Wnt genes in the RTA-clade spider Cupiennius salei

Spiders represent widely used model organisms for chelicerate and even arthropod development and evolution. Wnt genes are important and evolutionary conserved factors that control and regulate numerous developmental processes. Recent studies comprehensively investigated the complement and expression of spider Wnt genes revealing conserved as well as diverged aspects of their expression and thus (likely) function among different groups of spiders representing Mygalomorphae (tarantulas), and both main groups of Araneae (true spiders) (Haplogynae/Synspermiata and Entelegynae). The allegedly most modern/derived group of entelegyne spiders is represented by the RTA-clade of which no comprehensive data on Wnt expression were available prior to this study. Here, we investigated the embryonic expression of all Wnt genes of the RTA-clade spider Cupiennius salei. We found that most of the Wnt expression patterns are conserved between Cupiennius and other spiders, especially more basally branching species. Surprisingly, most differences in Wnt gene expression are seen in the common model spider Parasteatoda tepidariorum (a non-RTA clade entelegyne species). These results show that data and conclusions drawn from research on one member of a group of animals (or any other organism) cannot necessarily be extrapolated to the group as a whole, and instead highlight the need for comprehensive taxon sampling.     

Stick insects (Phasmida) as prey of spiders: size,palatability and defence mechanisms in feeding tests

Summary The maximum body length of the phasmid Carausius morosus which the ctenid spider Cupiennius salei can overwhelm is 2–3x larger than the length of crickets. Eight phasmid species were offered to Cupiennius salei in feeding tests. Among them, Carausius morosus was raised on 4 different food plants. All species of prey items were accepted by the spider predator very readily (>80%). This indicates that phasmids are a highly palatable prey group for spiders. The possible importance of chemical defence and its obvious lack under laboratory conditions are discussed.     

The biocontrol potential of Philodromus (Araneae,Philodromidae) spiders for the suppression of pome fruit orchard pests

A number of different generalist (polyphagous) predators occur in agroecosystems. Yet their biocontrol potential has been little investigated in detail. Philodromus species (Philodromidae) belong to the dominant spider species occurring in commercial orchards. We studied in detail the trophic functional traits of Philodromus albidus, Philodromus aureolus, and Philodromus cespitum (Philodromidae) by means of (1) the analysis of natural prey; and (2) experiments on acceptance of a variety of prey taxa. We found that the three philodromids are euryphagous. We classified prey species into three categories according to their function in the orchard: beneficial species, indifferent species, and pests. Philodromid spiders captured mostly other spiders in the field because spiders were most available. As concerns pests, the philodromids preyed mostly on Brachycera and Sternorrhyncha. They selected Acari and Brachycera. Indifferent species, such as Collembola and Nematocera, were also highly selected. In the laboratory, philodromids accepted mostly pests, such as lepidopterans, brachycerans, and aphids, while other spiders were accepted the least. The three philodromids have differentiated trophic niches with respect to prey size not only in the adult stage but throughout their ontogenetic development: P. albidus utilized smaller prey than the other two species. We conclude that the philodromids have a potential as biocontrol agents because they prey mostly on pests but their predation pressure is reduced due to higher selectivity for the indifferent fauna.     

Purification, cDNA structure and biological significance of a single insulin-like growth factor-binding domain protein (SIBD-1) identified in the hemocytes of the spider Cupiennius salei

Cupiennius salei single insulin-like growth factor-binding domain protein (SIBD-1), which exhibits an IGFBP N-terminal domain-like profile, was identified in the hemocytes of the spider C. salei. SIBD-1 was purified by RP-HPLC and the sequence determined by a combination of Edman degradation and 5′–3′- RACE PCR. The peptide (8676.08 Da) is composed of 78 amino acids, contains six intrachain disulphide bridges and carries a modified Thr residue at position 2. SIBD-1 mRNA expression was detected by quantitative real-time PCR mainly in hemocytes, but also in the subesophageal nerve mass and muscle. After infection, the SIBD-1 content in the hemocytes decreases and, simultaneously, the temporal SIBD-1 expression seems to be down-regulated. Two further peptides, SIBD-2 and IGFBP-rP1, also exhibiting IGFBP N-terminal domain variants with unknown functions, were identified on cDNA level in spider hemocytes and venom glands. We conclude that SIBD-1 may play an important role in the immune system of spiders.     

Functional and Biochemical Characterization of Alvinella pompejana Cys-Loop Receptor Homologues

Cys-loop receptors are membrane spanning ligand-gated ion channels involved in fast excitatory and inhibitory neurotransmission. Three-dimensional structures of these ion channels, determined by X-ray crystallography or electron microscopy, have revealed valuable information regarding the molecular mechanisms underlying ligand recognition, channel gating and ion conductance. To extend and validate the current insights, we here present promising candidates for further structural studies. We report the biochemical and functional characterization of Cys-loop receptor homologues identified in the proteome of Alvinella pompejana, an extremophilic, polychaete annelid found in hydrothermal vents at the bottom of the Pacific Ocean. Seven homologues were selected, named Alpo1-7. Five of them, Alpo2-6, were unidentified prior to this study. Two-electrode voltage clamp experiments revealed that wild type Alpo5 and Alpo6, both sharing remarkably high sequence identity with human glycine receptor α subunits, are anion-selective channels that can be activated by glycine, GABA and taurine. Furthermore, upon expression in insect cells fluorescence size-exclusion chromatography experiments indicated that four homologues, Alpo1, Alpo4, Alpo6 and Alpo7, can be extracted out of the membrane by a wide variety of detergents while maintaining their oligomeric state. Finally, large-scale purification efforts of Alpo1, Alpo4 and Alpo6 resulted in milligram amounts of biochemically stable and monodisperse protein. Overall, our results establish the evolutionary conservation of glycine receptors in annelids and pave the way for future structural studies.     

Evidence for a Diverse Cys-Loop Ligand-Gated Ion Channel Superfamily in Early Bilateria

The genome sequences of Caenorhabditis elegans and Drosophila melanogaster reveal a diversity of cysteine-loop ligand-gated ion channels (Cys-loop LGICs) not found in vertebrates. To better understand the evolution of this gene superfamily, I compared all Cys-loop LGICs from rat, the primitive chordate Ciona intestinalis, Drosophila, and C. elegans. There are two clades of GABA receptor subunits that include both verterbate and invertebrate orthologues. In addition, I identified nine clades of anion channel subunits found only in invertebrates, including three that are specific to C. elegans and two found only in Drosophila. One well-defined clade of vertebrate cation channel subunits, the α7 nicotinic acetylcholine receptor subunits (nAChR), includes invertebrate orthologues. There are two clades of invertebrate nAChRs, one of α-type subunits and one of non-α subunits, that are most similar to the two clades of vertebrate neuronal and muscle α and non-α subunits. There is a large group of divergent C. elegans nAChR-like subunits partially resolved into clades but no orthologues of 5HT3-type serotonin receptors in the invertebrates. The topology of the trees suggests that most of the invertebrate-specific Cys-loop LGIC clades were present in the common ancestor of chordates and ecdysozoa. Many of these disappeared from the chordates. Subsequently, selected subunit genes expanded to form large subfamilies. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Rafael Zardoya]     

Active sensing in a freely walking spider: look where to go

The Central American hunting spider Cupiennius salei, like most other spiders, has eight eyes, one pair of principal eyes and three pairs of secondary eyes. The principal eyes and one pair of the secondary eyes have almost completely overlapping visual fields, and presumably differ in function. The retinae of the principal eyes can be moved independently by two pairs of eye muscles each, whereas the secondary eyes do not have such eye muscles. The behavioural relevance of retinal movements of freely moving spiders was investigated by a novel dual-channel telemetric registration of the eye muscle activities. Walking spiders shifted the ipsilateral retina with respect to the walking direction before, during and after a turning movement. The change in the direction of vision in the ipsilateral anterior median eye was highly correlated with the walking direction, regardless of the actual light conditions. The contralateral retina remained in its resting position. This indicates that Cupiennius salei shifts it visual field in the walking direction not only during but sometimes previous to an intended turn, and therefore “peers” actively into the direction it wants to turn.     

Mutagenic Analysis of the Intracellular Portals of the Human 5-HT3A Receptor

Structural models of Cys-loop receptors based on homology with the Torpedo marmorata nicotinic acetylcholine receptor infer the existence of cytoplasmic portals within the conduction pathway framed by helical amphipathic regions (termed membrane-associated (MA) helices) of adjacent intracellular M3-M4 loops. Consistent with these models, two arginine residues (Arg⁴³⁶ and Arg⁴⁴⁰) within the MA helix of 5-hydroxytryptamine type 3A (5-HT₃A) receptors act singularly as rate-limiting determinants of single-channel conductance (γ). However, there is little conservation in primary amino acid sequences across the cytoplasmic loops of Cys-loop receptors, limiting confidence in the fidelity of this particular aspect of the 5-HT₃A receptor model. We probed the majority of residues within the MA helix of the human 5-HT₃A subunit using alanine- and arginine-scanning mutagenesis and the substituted cysteine accessibility method to determine their relative influences upon γ. Numerous residues, prominently those at the 435, 436, 439, and 440 positions, were found to markedly influence γ. This approach yielded a functional map of the 5-HT₃A receptor portals, which agrees well with the homology model.     

The diversity,recombination and horizontal transmission of Wolbachia in spiders in China

Wolbachia are maternally inherited endosymbiotic bacteria. These intracellular bacteria are common in arthropods and could manipulate host reproduction in diverse ways, such as feminization, parthenogenesis, male killing and cytoplasmic incompatibility. In spiders, infection by Wolbachia has been found in a total of 99 species belonging to 62 genera and 17 families. Furthermore, recent studies analyzed the phylogeny of Wolbachia in Hylyphantes graminicola, 2 cave spiders and Agelenopsis species using multilocus sequence typing (MLST) approach. However, the diversity of Wolbachia strains determined by MLST in spiders from China is still largely unknown.In this study, we collected 1153 spider individuals from Mangshan in China and screened for Wolbachia in 975 individuals representing 68 spider species belonging to 45 genera of 16 families. We analyzed the phylogenetic relationship between Wolbachia and their host spiders by MLST approach. We found novel infections of Wolbachia in 1 family, 9 genera and 20 species of spiders. We found 13 new Wolbachia strains and suggest that group A is more common than group B in Wolbachia that infect spiders. Our results revealed three recombination events of the concatenated multilocus sequences in Wolbachia that infect spiders. Furthermore, our results demonstrated the phylogenetic incongruence between Wolbachia and spiders, suggesting the horizontal transmission of Wolbachia in spiders.We suggest that recombination and horizontal transmission may play an important role in the diversity and evolution of Wolbachia in spiders.     

Divergence in gut bacterial community between females and males in the wolf spider Pardosa astrigera

Sex is one of the important factors affecting gut microbiota. As key predators in agroforestry ecosystem, many spider species show dramatically different activity habits and nutritional requirements between females and males. However, how sex affects gut microbiota of spiders remains unclear. Here, we compared the composition and diversity of gut bacteria between female and male Pardosa astrigera based on bacterial 16S rRNA gene sequencing. Results showed that the richness of bacterial microbiota in female spiders was significantly lower than in male spiders (p < .05). Besides, β‐diversity showed a significant difference between female and male spiders (p = .0270). The relative abundance of Actinobacteriota and Rhodococcus (belongs to Actinobacteriota) was significantly higher in female than in male spiders (p < .05), whereas the relative abundance of Firmicutes and Acinetobacter (belongs to Proteobacteria) and Ruminococcus and Fusicatenibacter (all belong to Firmicutes) was significantly higher in male than in female spiders (p < .05). The results also showed that amino acid and lipid metabolisms were significantly higher in female than in male spiders (p < .05), whereas glycan biosynthesis and metabolism were significantly higher in male than in female spiders (p < .05). Our results imply that sexual variation is a crucial factor in shaping gut bacterial community in P. astrigera spiders, while the distinct differences of bacterial composition are mainly due to their different nutritional and energy requirements.     

Ultrastructure of spider thread anchorages

Spiders attach silken threads to substrates by means of glue-coated nanofibers (piriform silk), spun into disc-like structures. The organization and ultrastructure of this nano-composite silk are largely unknown, despite their implications for the biomechanical function and material properties of thread anchorages. In this work, the ultrastructure of silken attachment discs was studied in representatives of four spider families with Transmission Electron Microscopy to facilitate a mechanistic understanding of piriform silk function across spiders. Based on previous findings from comparative studies of piriform silk gland morphology, we hypothesized that the fibre-glue proportion of piriform silk differs in different spiders, while the composition of fibre and glue fractions is consistent. Results confirmed large differences in the relative proportion of glue with low amounts in the orb weaver Nephila senegalensis (Araneidae) and the hunting spider Cupiennius salei (Ctenidae), larger amounts in the cobweb spider Parasteatoda tepidariorum (Theridiidae) and a complete reduction of the fibrous component in the haplogyne spider Pholcus phalangioides (Pholcidae). We rejected our hypothesis that glue ultrastructure is consistent. The glue is a colloid with polymeric and fluid fractions that strongly differ in proportions and assembly. We further confirmed that in all species studied both dragline and piriform silk fibres do not make contact with the environmental substrate. Instead, adhesion is established by a thin dense skin layer of the piriform glue. These results advance our understanding of piriform silk function and the interspecific variation of its properties, which is significant for spider biology, web function and the bioengineering of silk.     

Spider acetylcholine binding proteins: An alternative model to study the interaction between insect nAChRs and neonicotinoids

Acetylcholine binding proteins (AChBPs) are homologs of extracellular domains of nicotinic acetylcholine receptors (nAChRs) and serve as models for studies on nAChRs. Particularly, studies on invertebrate nAChRs that are limited due to difficulties in their heterologous expression have benefitted from the discovery of AChBPs. Thus far, AChBPs have been characterized only in aquatic mollusks, which have shown low sensitivity to neonicotinoids, the insecticides targeting insect nAChRs. However, AChBPs were also found in spiders based on the sequence and tissue expression analysis. Here, we report five AChBP subunits in Pardosa pseudoannulata, a predator enemy against rice insect pests. Spider AChBP subunits shared higher sequence similarities with nAChR subunits of both insects and mammals compared with mollusk AChBP subunits. The AChBP1 subunit of P. pseudoannulata (Pp-AChBP) was then expressed in Sf9 cells. The Ls-AChBP from Lymnaea stagnalis was also expressed for comparison. In both AChBPs, one ligand site per subunit was present at each interface between two adjacent subunits. Neonicotinoids had higher affinities (7.9–18.4 times based on K_d or K_i values) for Pp-AChBP than for Ls-AChBP, although epibatidine and α-bungarotoxin showed higher affinities for Ls-AChBP. These results indicate that spider AChBP could be used as an alternative model to study the interaction between insect nAChRs and neonicotinoids.     

The Functioning of Species-Specific Olfactory Pheromones in the Biology of a Mosquito-Eating Jumping Spider from East Africa

Having unique, complex eyes and vision based on exceptional spatial acuity, salticid spiders are known for their vision-based predatory and mating strategies. Yet Evarcha culicivora, the salticid we consider here, is known for predatory and mating strategies based strongly on the interplay of vision and olfaction. This unusual East African species feeds indirectly on vertebrate blood by choosing blood-carrying female mosquitoes as preferred prey, which it can identify by sight and by smell. Moreover, E. culicivora’s mating strategy is unusual because, unlike the prevailing general pattern among spiders, where males are more active in courtship and females are more active in mate choice, both roles are characteristic of both sexes of E. culicivora. There is also an unusual relationship between diet and mating in this species, with blood meals making both sexes more attractive as potential mates. However, findings from the present study demonstrate that, regardless of source-spider diet and even when restricted to using chemoreception in the absence of seeing another spider, both sexes can discriminate between opposite-sex conspecifics and opposite-sex heterospecifics. Yet there is no evidence that diet (blood versus no blood) influences the attractiveness of opposite-sex heterospecific individuals to E. culicivora. Evidence that the odor of opposite-sex conspecifics is salient to E. culicivora comes from three different experimental designs (retention testing, choice testing and courtship-initiation testing). The effective odor source can be either the presence of the spider or the presence of the spider’s draglines alone.     

Die Feinde der Raubmilben als Reduzierender Faktor der Spinnmilben

Summary The relations betweenMetatetranychus ulmi living on apple trees and its different predators are complex. The most important natural enemy of this spider mite is the predacious miteTyphlodromus pyri (= T. tiliae). It destroys more spider mites than the beneficial insects do. In the district of Stuttgart-Hohenheim about 38 species of insects and spiders feed onTyphlodromus mites. For instanceT. pyri is reduced considerably byOrius minutus. This bug is a natural enemy of aphids and spider mites, however it prefers the predatory mites. It attacks the spider mites and aphids only ifTyphlodromus mites are not available.Chrysopa vulgaris andAnthocoris nemorum are similar in their feeding habits. These two destroyTyphlodromus pyri also but they are less important thanO. minutus. The other beneficial insects in our orchards have little effect on spider mites or predacious mites. If we have enough pests on our apple trees to make spraying necessary, we should look forTyphlodromus mites and be careful no to destroy them. We should always examine the composition of the biocoenosis applying chemical agents because the populations of insects and predacious mites may vary from one area to the other.      

Impact of flower-dwelling crab spiders on plant-pollinator mutualisms

Indirect effects in interactions occur when a species influences a third species by modifying the behaviour of a second one. It has been suggested that indirect effects of crab spiders (Thomisidae) on pollinator behaviour can cascade down the food web and negatively affect plant fitness. However, it is poorly understood how different pollinator groups react to crab spiders and, thus, when a reduction in plant fitness is likely to occur. Using continuous video surveillance, we recorded the behaviour of pollinators on two flower species and the pollinators’ responses to three crab spider treatments: inflorescences (1) with a pinned dried spider, (2) with a spider model made of paper, and (3) without spiders (control). We found that pollinators avoided inflorescences with dried spiders only on one plant species (Anthemis tinctoria). Pollinators showed no significant avoidance of paper spiders. Honeybees and bumblebees did not react to dried spiders, but solitary bees and syrphid flies showed a strong avoidance. Finally, we found no evidence that inflorescences with dried spiders suffered from a decrease in fitness in terms of a reduced seed set. We hypothesise that top-down effects of predators on plants via pollinators depend on the degree of specialisation of pollinators and their tendency to avoid spiders.     

Appendage patterning in the South American bird spider <Emphasis Type="Italic">Acanthoscurria geniculata</Emphasis> (Araneae: Mygalomorphae)

Pattern formation by the genes dachshund (dac), Distal-less (Dll), extradenticle (exd) and homothorax (hth) in spider appendages has been studied previously only in members of the higher spiders (Araneomorphae). In order to study the diversity and conservation of pattern formation in spiders as a whole, we studied homologs of these genes in embryos of the bird spider Acanthoscurria geniculata, which belongs to the Mygalomorphae, a more primitive spider group. We show that the patterns of dac and Dll are largely conserved in all spiders studied so far. We find a duplication of hth and exd genes as previously identified in the higher spider Cupiennius salei. These data suggest that pattern formation shows little diversity in all spiders, including the duplication of hth and exd that likely occurred before the split of Mygalomorphae and Araneomorphae. We also find that the legs and pedipalps bear endites of which only the pedipalpal endite expresses Dll and is retained in the adult. Similarly, the limb buds of the posterior spinnerets express Dll and become segmented appendages in the adult, whereas the anterior spinnerets lack Dll expression and are absent in postembryonic stages. In both cases, the expression of Dll or the lack of it indicates structures which will be retained as adult traits or rudimentary structures that degenerate, respectively. The presence of embryonic rudiments of leg endites in Acanthoscurria and the leg-like pattern formation in the posterior spinnerets are interpreted as primitive traits that have been lost in the Araneomorphae.     

Bioaccumulation of Cry1Ab Protein from an Herbivore Reduces Anti-Oxidant Enzyme Activities in Two Spider Species

Cry proteins are expressed in rice lines for lepidopteran pest control. These proteins can be transferred from transgenic rice plants to non-target arthropods, including planthoppers and then to a predatory spider. Movement of Cry proteins through food webs may reduce fitness of non-target arthropods, although recent publications indicated no serious changes in non-target populations. Nonetheless, Cry protein intoxication influences gene expression in Cry-sensitive insects. We posed the hypothesis that Cry protein intoxication influences enzyme activities in spiders acting in tri-trophic food webs. Here we report on the outcomes of experiments designed to test our hypothesis with two spider species. We demonstrated that the movement of CryAb protein from Drosophila culture medium into fruit flies maintained on the CryAb containing medium and from the flies to the spiders Ummeliata insecticeps and Pardosa pseudoannulata. We also show that the activities of three key metabolic enzymes, acetylcholine esterase (AchE), glutathione peroxidase (GSH-Px), and superoxide dismutase (SOD) were significantly influenced in the spiders after feeding on Cry1Ab-containing fruit flies. We infer from these data that Cry proteins originating in transgenic crops impacts non-target arthropods at the physiological and biochemical levels, which may be one mechanism of Cry protein-related reductions in fitness of non-target beneficial predators.     

Evidence suggests that modified setae of the crab spiders Stephanopis spp. fasten debris from the background

Crab spiders (Thomisidae) are known by their ability to change their body colouration via change in epithelial pigments. However, the crab spider genus Stephanopis appears to match the colouration of the bark they are sitting on by having debris attached to its dorsal cuticle. The functional morphology, colouration, and evolution of this phenomenon were investigated in Stephanopis cf. scabra and S. cambridgei. Analysis under the microscope revealed that debris originated from the bark they were sitting on. Using scanning electron microscopy, three different types of setae likely related in the retention of debris were found in S. cf. scabra and one in S. cambridgei. These setae are branched and possess barbs, unlike the more filiform setae found in other crab spider species. In addition, the presence of debris improved the brightness background matching of spiders against the bark, but not hue and chroma matching. Ancestral character state reconstruction suggested that presence of debris evolved two to three times within Thomisidae. The evolution of both masking and colour change among crab spiders indicates that they are under a strong selection to avoid detection.