Ultrastructural aspects of spermiogenesis and synspermia in the brown spider Loxosceles intermedia (Araneae: Sicariidae)

This study reports ultrastructural and cytochemical aspects of spermiogenesis and synspermia in the brown spider Loxosceles intermedia. The roundish early spermatids are initially interconnected by cytoplasmic bridges, forming groups of four cells. During spermiogenesis, these cells pass through a series of modifications: (1) progressive nuclear condensation brings chromatin into a fibrillar arrangement; (2) the nucleus becomes long and asymmetric, with a short post-centriolar elongation; (3) formation of the long, cone-shaped acrosome and the F-actin acrosomal filament; (4) establishment of the implantation fossa and the 9x2+3 pattern flagellum, which extends away from the sperm cell body. Eventually, the entire cell undergoes twisting and folding resulting in a synspermium, containing four sperm cells in which the flagellum and nucleus are delimitated by the plasma membrane, as individualized structures, but remain involved by the fused remaining cytoplasm and plasma membrane. Reaching the vas deferens, the synspermia are surrounded by a basic glycoproteic secretion. Synspermia are considered a derivative character, probably developed in this Sicariidae species, as well as in other Haplogynae, as an adaptation to improve the reproductive strategy.     

Loxosceles niedeguidonae (Araneae, Sicariidae) a new species of brown spider from Brazilian semi-arid region

A new species of recluse spider, Loxosceles niedeguidonaesp. n., is described from the Parque Nacional Serra da Capivara, State of Piauí, Brazil. This is the first endemic species described from Brazilian semi-arid environment. The species is included in gaucho group of Gertsch (1967) due to its spermathecal shape and is considered close to Loxosceles chapadensis Bertani, Fukushima & Nagahama, 2010 by the unusual long male palpal tibia, a character not common for species of this group. An updated key for Loxosceles species of gaucho group is presented.     

Experimental evidence for a direct cytotoxicity of Loxosceles intermedia (brown spider) venom in renal tissue.

Brown spider (Loxosceles genus) venom causes necrotic lesions often accompanied by fever, hemolysis, thrombocytopenia, and acute renal failure. Using mice exposed to Loxosceles intermedia venom, we aimed to show whether the venom directly induces renal damage. The experimental groups were composed of 50 mice as controls and 50 mice that received the venom. Light microscopic analysis of renal biopsy specimens showed alterations including hyalinization of proximal and distal tubules, erythrocytes in Bowman's space, glomerular collapse, tubule epithelial cell blebs and vacuoles, interstitial edema, and deposition of eosinophilic material in the tubule lumen. Electron microscopic findings indicated changes including glomerular epithelial and endothelial cell cytotoxicity as well as disorders of the basement membrane. Tubule alterations include epithelial cell cytotoxicity with cytoplasmic membrane blebs, mitochondrial changes, increase in smooth endoplasmic reticulum, presence of autophagosomes, and deposits of amorphous material in the tubules. We also found that the venom caused azotemia with elevation of blood urea levels but did not decrease C3 complement concentration or cause hemolysis in vivo. Confocal microscopy with antibodies against venom proteins showed direct binding of toxins to renal structures, confirmed by competition assays. Double-staining immunofluorescence reactions with antibodies against type IV collagen or laminin, antibodies to venom toxins, and fluorescent cytochemistry with DAPI revealed deposition of toxins in glomerular and tubule epithelial cells and in renal basement membranes. Two-dimensional electrophoresis showed venom rich in low molecular mass and cationic toxins. By immunoblotting with antibodies to venom toxins on renal extracts from venom-treated mice, we detected a renal binding toxin at 30 kD. The data provide experimental evidence that L. intermedia venom is directly involved in nephrotoxicity.     

Biotechnological applications of brown spider (Loxosceles genus) venom toxins

Loxoscelism (the term used to define accidents by the bite of brown spiders) has been reported worldwide. Clinical manifestations following brown spider bites are frequently associated with skin degeneration, a massive inflammatory response at the injured region, intravascular hemolysis, platelet aggregation causing thrombocytopenia and renal disturbances. The mechanisms by which the venom exerts its noxious effects are currently under investigation. The whole venom is a complex mixture of toxins enriched with low molecular mass proteins in the range of 5–40 kDa. Toxins including alkaline phosphatase, hyaluronidase, metalloproteases (astacin-like proteases), low molecular mass (5.6–7.9 kDa) insecticidal peptides and phospholipases-D (dermonecrotic toxins) have been identified in the venom. The purpose of the present review is to describe biotechnological applications of whole venom or some toxins, with especial emphasis upon molecular biology findings obtained in the last years.     

Daily thermal preference variation of the sand recluse spider Sicarius thomisoides (Araneae: Sicariidae)

Preferential temperature as a physiological feature is crucial for spiders, since it determines the selection of key habitats for their survival and reproduction. In this work, we study the daily and geographical variation of the preferential temperature of the spider Sicarius thomisoides subjected to different degrees of daily thermal oscillation in their habitats. Preferred temperatures differ between coastal and inland populations, but in both cases, there is a marked bimodality in the daily pattern of temperature preference, with two peaks per day that would be given by the changes in the hours of activity. These nocturnal spiders select higher temperatures in the evening (active period) and select lower temperatures during late morning (resting period). In laboratory, spiders have preferred temperatures that differ from those found in their habitats, so they must tolerate or compensate non-preferred temperatures by active thermoregulation in natural conditions.     

Molecular cloning and functional characterization of two isoforms of dermonecrotic toxin from Loxosceles intermedia (brown spider) venom gland

Brown spider (Genus Loxosceles) bites are normally associated with necrotic skin degeneration, gravitational spreading, massive inflammatory response at injured region, platelet aggregation causing thrombocytopenia and renal disturbances. Brown spider venom has a complex composition containing many different toxins, of which a well-studied component is the dermonecrotic toxin. This toxin alone may produce necrotic lesions, inflammatory response and platelet aggregation. Biochemically, dermonecrotic toxin belongs to a family of toxins with 30-35 kDa characterized as sphingomyelinase-D. Here, employing a cDNA library of Loxosceles intermedia venom gland, we cloned and expressed two recombinant isoforms of the dermonecrotic toxin LiRecDT2 (1062 bp cDNA) and LiRecDT3 (1007 bp cDNA) that encode for signal peptides and complete mature proteins. Phylogenetic tree analysis revealed a structural relationship for these toxins compared to other members of family. Recombinant molecules were expressed as N-terminal His-tag fusion proteins in Escherichia coli and were purified to homogeneity from cell lysates by Ni(2+) chelating chromatography, resulting in proteins of 33.8 kDa for LiRecDT2 and 34.0 kDa for LiRecDT3. Additional evidence for related toxins containing sequence/epitopes identity comes from antigenic cross-reactivity using antibodies against crude venom toxins and antibodies raised with a purified dermonecrotic toxin. Recombinant toxins showed differential functionality in rabbits: LiRecDT2 caused a macroscopic lesion with gravitational spreading upon intradermal injection, while LiRecDT3 evoked transient swelling and erythema upon injection site. Light microscopic analysis of skin biopsies revealed edema, a collection of inflammatory cells in and around blood vessels and a proteinaceous network at the dermis. Moreover, differential functionality for recombinant toxins was also demonstrated by a high sphingomyelinase activity for LiRecDT2 and low activity for LiRecDT3 as well as greater in vitro platelet aggregation and blood vessel permeability induced by LiRecDT2 and residual activity for LiRecDT3. Cloning and expression of two recombinant dermonecrotic toxins demonstrate an intraspecific family of homologous toxins that act in synergism for deleterious activities of the venom and open possibilities for biotechnological applications for recombinant toxins as research tools for understanding the inflammatory response, vascular integrity and platelet aggregation modulators.     

Identification, cloning and functional characterization of a novel dermonecrotic toxin (phospholipase D) from brown spider (Loxosceles intermedia) venom

Brown spider bites are associated with lesions including dermonecrosis, gravitational spreading and a massive inflammatory response, along with systemic problems that may include hematological disturbances and renal failure. The mechanisms by which the venom exerts its noxious effects are currently under investigation. It is known that the venom contains a major toxin (dermonecrotic toxin, biochemically a phospholipase D) that can experimentally induce dermonecrosis, inflammatory response, animal mortality and platelet aggregation. Herein, we describe cloning, heterologous expression, purification and functionality of a novel isoform of the 33 kDa dermonecrotic toxin. Circular dichroism analysis evidenced correct folding for the toxin. The recombinant toxin was recognized by whole venom serum antibodies and by a specific antibody to a previously described dermonecrotic toxin. The identified toxin was found to display phospholipase activity and dermonecrotic properties. Additionally, the toxin caused a massive inflammatory response in rabbit skin dermis, evoked platelet aggregation, increased vascular permeability, caused edema and death in mice. These characteristics in combination with functional studies for other dermonecrotic toxins illustrate that a family of dermonecrotic toxins exists, and includes a novel member with high activity that may be useful for future structural and functional studies.     

Identification, cloning, expression and functional characterization of an astacin-like metalloprotease toxin from Loxosceles intermedia (brown spider) venom

Injuries caused by brown spiders (Loxosceles genus) are associated with dermonecrotic lesions with gravitational spreading and systemic manifestations. The venom has a complex composition containing many different toxins, of which metalloproteases have been described in many different species of this genus. These toxins may degrade extracellular matrix constituents acting as a spreading factor. By using a cDNA library from an Loxosceles intermedia venom gland, we cloned and expressed a 900 bp cDNA, which encoded a signal peptide and a propeptide, which corresponded to a 30 kDa metalloprotease, now named LALP (Loxosceles astacin-like protease). Recombinant LALP was refolded and used to produce a polyclonal antiserum, which showed cross-reactivity with a 29 kDa native venom protein. CD analysis provided evidence that the recombinant LALP toxin was folded correctly, was still in a native conformation and had not aggregated. LALP addition to endothelial cell cultures resulted in de-adhesion of the cells, and also in the degradation of fibronectin and fibrinogen (this could be inhibited by the presence of the bivalent chelator 1,10-phenanthroline) and of gelatin in vitro. Sequence comparison (nucleotide and deduced amino acid), phylogenetic analysis and analysis of the functional recombinant toxin revealed that LALP is related in both structure and function to the astacin family of metalloproteases. This suggests that an astacin-like toxin is present in a animal venom secretion and indicates that recombinant LALP will be a useful tool for future structural and functional studies on venom and the astacin family.     

Predation by cosmopolitan spiders upon the medically significant pest species Loxosceles reclusa (Araneae: Sicariidae): limited possibilities for biological control

Interspecific predation of three cosmopolitan house spiders, Achearanea tepidariorum (Kock 1841) (Theridiidae), Steotoda triangulosa (Walckenaer 1802) (Theridiidae), and Pholcus phalangioides (Doleschall 1859) (Pholcidae), and the medically significant brown recluse spider, Loxosceles reclusa (Sicariidae) were examined to evaluate transitive predatory relationships and to explore the potential use of cosmopolitan spiders as effective biological control agents on L. reclusa. Fifty houses from northeastern Kansas were visually inspected from May to December 2002 for cosmopolitan spiders and L. reclusa. In 25 houses, insect monitoring traps were used to sample spider diversity and abundance. The remaining 25 houses were monitored to examine intraguild predation and spider behavior. If cosmopolitan spiders have the ability to regulate or decrease L. reclusa populations, houses with large cosmopolitan spider populations are expected to have significantly fewer L. reclusa than houses without cosmopolitan spiders. Predation and/or evidence of predation by all three cosmopolitan spiders on L. reclusa was detected in 68% of houses. Spearman's rank correlation analysis showed overall positive relationships between population densities of cosmopolitan spiders and L. reclusa. When evaluated independently, the presence of both A. tepidariorum and S. triangulosa showed negative, yet nonsignificant, relationships with L. reclusa densities, whereas P. phalangioides showed a positive nonsignificant relationship. Although statistical tests showed a decrease in L. reclusa population densities with increased population densities of two cosmopolitan species, alluding to a potential beneficial interaction for biological control, observations of spider behavior, web positioning (niche partitioning), and predation showed little possibility of biological control capabilities.     

Two novel dermonecrotic toxins LiRecDT4 and LiRecDT5 from brown spider (Loxosceles intermedia) venom: from cloning to functional characterization

Loxoscelism (the condition produced by the bite of brown spiders) has been reported worldwide, but especially in warmer regions. Clinical manifestations include skin necrosis with gravitational spreading while systemic loxoscelism may include renal failure, hemolysis and thrombocytopenia. The venom contains several toxins, of which the best biochemically and biologically studied is the dermonecrotic toxin, a phospholipase-D. Purified toxin induces cutaneous and systemic loxoscelism, especially necrotic lesions, hematological disturbances and renal failure. Herein, we describe cloning, heterologous expression and purification of two novel dermonecrotic toxins: LiRecDT4 and LiRecDT5. The recombinant proteins stably expressed in Escherichia coli cells were purified from culture supernatants in a single step using Ni(2+)-chelating chromatography producing soluble proteins of 34 kDa (LiRecDT4) and 37 kDa (LiRecDT5). Circular dichroism analysis evidenced correctly folding for toxins but differences in secondary structures. Both proteins were recognized by whole venom serum antibodies and by a specific antibody to dermonecrotic toxin. Also, recombinant toxins with phospholipase activity induced experimental skin lesions and caused a massive inflammatory response in rabbit skin dermis. Nevertheless, toxins displayed different effects upon platelet aggregation, increase in vascular permeability and not caused death in mice. These characteristics in combination with functional studies illustrates that a family of dermonecrotic toxins exists, and includes two novel members that are useful for future structural and functional studies. They will also be useful in biotechnological ends, for example, as inflammatory and platelet aggregating studies, as antigens for serum therapy source and for lipids biochemical research.     

Proteome analysis of brown spider venom: identification of loxnecrogin isoforms in Loxosceles gaucho venom

Brown spiders of the Loxosceles genus are distributed worldwide. In Brazil, eight species are found in Southern states, where the envenomation by Loxosceles venom (loxoscelism) is a health problem. The mechanism of the dermonecrotic action of Loxosceles venom is not totally understood. Two isoforms of dermonecrotic toxins (loxnecrogins) from L. gaucho venom have been previously purified, and showed sequence similarities to sphingomyelinase. Herein we employed a proteomic approach to obtain a global view of the venom proteome, with a particular interest in the loxnecrogin isoforms' pattern. Proteomic two-dimensional gel electrophoresis maps for L. gaucho, L. intermedia, and L. laeta venoms showed a major protein region (30-35 kDa, pI 3-10), where at least eight loxnecrogin isoforms could be separated and identified. Their characterization used a combined approach composed of Edman chemical sequencing, matrix-assisted laser desorption/ionization-time of flight mass spectrometry, and electrospray ionization-quadropole-time of flight tandem mass spectrometry leading to the identification of sphingomyelinases D. The venom was also pre-fractionated by gel filtration on a Superose 12 fast protein liqiud chromatography column, followed by capillary liquid chromatography-mass spectrometry. Eleven possible loxnecrogin isoforms around 30-32 kDa were detected. The identification of dermonecrotic toxin isoforms in L. gaucho venom is an important step towards understanding the physiopathology of the envenomation, leading to improvements in the immunotherapy of loxoscelism.     

Molecular cloning, heterologous expression and functional characterization of a novel translationally-controlled tumor protein (TCTP) family member from Loxosceles intermedia (brown spider) venom

Envenoming with brown spiders (Loxosceles genus) is common throughout the world. Cutaneous symptoms following spider bite accidents include dermonecrosis, erythema, itching and pain. In some cases, accidents can cause hypersensibility or even allergic reactions. These responses could be associated with histaminergic events, such as an increase in vascular permeability and vasodilatation. A protein that may be related to the effects of spider venom was identified from a previously obtained cDNA library of the L. intermedia venom gland. The amino acid sequence of this protein is homologous to proteins from the TCTP (translationally-controlled tumor protein) family, which are extracellular histamine-releasing factors (HRF) that are associated with the allergic reactions to parasites. Herein, we described the cloning, heterologous expression, purification and functional characterization of a novel member of the TCTP family from the Loxosceles intermedia venom gland. This recombinant protein, named LiRecTCTP, causes edema, enhances vascular permeability and is likely related to the inflammatory activity of the venom. Moreover, LiRecTCTP presents an immunological relationship with mammalian TCTPs.     

Activities against hemostatic proteins and adrenal gland ultrastructural changes caused by the brown widow spider Latrodectus geometricus (Araneae: Theridiidae) venom

Brown widow spider (BrWS) (Latrodectus geometricus) venom produces intense systemic reactions such as cramps, harsh muscle nociceptive, nauseas, vomiting and hypertension. The proposed pathogenic mechanisms resulting in these accidents have principally been damages occurring at the nervous system. However, it is suspected that there is also damage of the adrenal glands, as a result of the experimental animal's clinical manifestations, which developed symptoms compatible with acute adrenal insufficiency. We have currently found that the adrenal gland is damaged by this venom gland homogenates (VGH) producing severe alterations on cortex cells resulting in death by acute adrenal insufficiency. In general, the ultrastructural study on the glands of mice under transmission electronic microscopy observations showed alterations in the majority of the intracellular membranes within 3 to 24 h. BrWSVGH also showed specific actions on extracellular matrix proteins such as fibronectin, laminin and fibrinogen. In addition, zymogram experiments using gelatin as substrates detected gelatinolytic activity. The molecular exclusion fractionation of crude BrWSVGH resulted in 15 fractions, of which F1 and F2 presented α/β-fibrinogenase and fibronectinolytic activities. Fractions F6, F14 and F15 showed only α-fibrinogenase activity; in contrast, the gelatinolytic action was only observed in fraction F11. Only metalloproteinase inhibitors abolished all these proteolytic activities. Our results suggest that adrenal cortex lesions may be relevant in the etiopathogenesis of severe brown widow spider envenoming. To our knowledge, this is the first report on adrenal gland damages, fibrinogenolytic activity and interrelations with cell-matrix adhesion proteins caused by L.geometricus VGH. The venom of this spider could be inducing hemostatic system damages on envenomed patients.     

Oldest true orb-weaving spider (Araneae: Araneidae)

The aerial orb web woven by spiders of the family Araneidae typifies these organisms to laypersons and scientists alike. Here we describe the oldest fossil species of this family, which is preserved in amber from Alava, Spain and represents the first record of Araneidae from the Lower Cretaceous. The fossils provide direct evidence that all three major orb web weaving families: Araneidae, Tetragnathidae and Uloboridae had evolved by this time, confirming the antiquity of the use of this remarkable structure as a prey capture strategy by spiders. Given the complex and stereotyped movements that all orb weavers use to construct their webs, there is little question regarding their common origin, which must have occurred in the Jurassic or earlier. Thus, various forms of this formidable prey capture mechanism were already in place by the time of the explosive Cretaceous co-radiation of angiosperms and their flying insect pollinators. This permitted a similar co-radiation of spider predators with their flying insect prey, presumably without the need for a 'catch-up lag phase' for the spiders.     

Scavenging by spiders (Araneae) and its relationship to pest management of the brown recluse spider

Experiments reported in Sandidge (2003; Nature 426: 30) indicated that the brown recluse spider, Loxosceles reclusa Gertsch & Mulaik, preferred to scavenge dead prey over live prey and that the spiders were not detrimentally affected when fed insecticide-killed crickets. Extrapolations made in subsequent media coverage disseminating the results of this research made counter-intuitive statements that pesticide treatment in houses would increase brown recluse populations in homes. This information was presented as if the scavenging behavior was specialized in the brown recluse; however, it was more likely that this behavior has not been well studied in other species. To provide a comparison, the current laboratory study examined the likelihood of non-Loxosceles spiders to scavenge dead prey. Of 100 non-Loxosceles spiders that were tested (from 11 families, 24 genera, and at least 29 species from a variety of spider hunting guilds), 99 scavenged dead crickets when offered in petri dishes. Some of the spiders were webspinners in which real-world scavenging of dead prey is virtually impossible, yet they scavenge when given the opportunity. Therefore, scavenging is a flexible opportunistic predatory behavior that is spread across a variety of taxa and is not a unique behavior in brown recluses. These findings are discussed in relation to pest management practices.     

Thermal niche overlap of the corner recluse spider Loxosceles laeta (Araneae; Sicariidae) and its possible predator,the spitting spider Scytodes globula (Scytodidae)

Loxoscelism is a health problem caused by the bite of spiders of the genus Loxosceles. In Chile all cases are attributable to Loxosceles laeta. It has been suggested that the spitting spider Scytodes globula may be a predator of L. laeta and control its population, which is only possible if they share the microhabitat. This study compared the thermal preferences and tolerances of the two species. Later, spiders acclimated to 15 °C and 25 °C were exposed to decreasing and increasing temperatures to determine the lower and upper critical temperatures. The preferred temperatures were lower during the morning, but there were no differences between the species. The thermal niche breadths were similar for the species, with a large overlap. Both species showed tolerance to extreme temperatures, but L. laeta showed greater tolerance to low temperatures. Both species showed acclimation of the lower critical temperatures to changes in acclimation temperatures. The similarity of preferred and tolerated temperatures was partly an expected fact, since the species share the same macrohabitat; these spider species are very common in domestic environments of central Chile. However, the results imply that their microhabitat choices are also very similar, indicating a high probability of meeting and predation, which could have important consequences in loxoscelism epidemiology.     

Revision and cladistic analysis of the jumping spider genus Onomastus (Araneae: Salticidae)

The jumping spider genus Onomastus Simon, 1900 is revised. Four new species: Onomastus indra sp. nov. , Onomastus kaharian sp. nov. , Onomastus pethiyagodai sp. nov. , and Onomastus rattotensis sp. nov. are described. Parsimony analysis of 26 morphological characters supported the monophyly of Onomastus. Lyssomanes is sister to Onomastus. Onomastus separates into two clades: the widespread South‐East Asia clade and the South Asia clade. The South Asia clade is restricted to the Sri Lanka–Western Ghats biodiversity hotspot. Species of the South Asia clade appear to be spot endemics, highly in danger of extinction because of habitat loss and climate change. Male palps are complex and species‐specific, suggesting rapid divergent evolution. © 2010 The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 159 , 711–745.