Morphological Analysis Reveals a Compartmentalized Duct in the Venom Apparatus of the Wasp Spider (Argiope bruennichi)

Spiders are one of the most successful groups of venomous animals, but surprisingly few species have been examined in sufficient detail to determine the structure of their venom systems. To learn more about the venom system of the family Araneidae (orb-weavers), we selected the wasp spider (Argiope bruennichi) and examined the general structure and morphology of the venom apparatus by light microscopy. This revealed morphological features broadly similar to those reported in the small number of other spiders subject to similar investigations. However, detailed evaluation of the venom duct revealed the presence of four structurally distinct compartments. We propose that these subunits facilitate the expression and secretion of venom components, as previously reported for similar substructures in pit vipers and cone snails.     

Primary culture of venom glands from the Brazilian armed spider, Phoneutria nigriventer (Araneae, Ctenidae).

Phoneutria spider venoms are a rich source of bioactive components. The limited amounts of crude material available, however, can be considered as a major hindrance for a faster development in the field. In the present study, we attempted to establish primary cultures of venom glands of Phoneutria nigriventer as an alternative, in vitro source of venom. Three different developmental stages were tried as starting materials: whole embryo (inside the cocoon), nymph (early after cocoon hatching) and young adult (1 year after cocoon hatching). The embryonic cells remained in suspension in the primary cultures, with no signs of adhesion or differentiation, for about 6 months. Nevertheless, this culture was useful for the first chromosome C-banding of Phoneutria. An average of 29+/-1 acrocentric chromosomes were found. Striated muscle cells were the only kind of cells in the culture of venom glands from Phoneutria nymphs. The most promising results were achieved with 1-year-old specimens. Besides muscle, adherent epithelial cells were also obtained in culture. Although these cells remained in culture for a short time (up to 48 h) immunochemical analysis of the culture supernatant evidenced the presence of Phoneutria venom components. This can be considered as a first step toward the functional cultures of venom glands of Phoneutria spiders.     

Tetracycline and penicillin resistant Clostridium perfringens isolated from the fangs and venom glands of Loxosceles laeta: Its implications in loxoscelism treatment

The venom of Loxosceles spiders produces severe dermonecrotic damage, intravascular hemolysis, systemic alterations and risk of death. Clostridium perfringens is present in the microbial flora of the fangs and venom glands of Loxosceles intermedia. Its inoculation with the venom may infect the wound site and exacerbate the dermonecrotic damage. This anaerobic bacterium is widely distributed in nature and capable of damage with similar characteristics and severity to the spider venom. In this study we isolated and characterized species of Clostridium from the fangs and venom glands of Loxosceles laeta, including C. perfringens. The sensitivity patterns of different isolates of C. perfringens were evaluated by minimum inhibitory concentration against penicillin, ampicillin, erythromycin, gentamicin, chloramphenicol, clindamycin and tetracycline, under anaerobic conditions, using the method of microdilution in broth. Strain C. perfringens H28 showed resistance to penicillin, ampicillin, tetracycline and chloramphenicol. Resistance to penicillin and ampicillin was mediated by β-lactamase. In vivo evaluation of dermonecrosis in rabbits using L. laeta venom co-inoculated with isolate C. perfringens H28 produced an increase in the area of dermonecrotic lesions in the presence of penicillin and tetracycline, but not with gentamicin. Antibiotic therapy Loxosceles poisoning should be re-evaluated, considering the existence of multi-resistant strains of C. perfringens.     

Venom and Social Behavior: The Potential of Using Spiders to Evaluate the Evolution of Sociality under High Risk

Risks of sociality, including competition and conspecific aggression, are particularly pronounced in venomous invertebrates such as arachnids. Spiders show a wide range of sociality, with differing levels of cannibalism and other types of social aggression. To have the greatest chance of surviving interactions with conspecifics, spiders must learn to assess and respond to risk. One of the major ways risk assessment is studied in spiders is via venom metering, in which spiders choose how much venom to use based on prey and predator characteristics. While venom metering in response to prey acquisition and predator defense is well-studied, less is known about its use in conspecific interactions. Here we argue that due to the wide range of both sociality and venom found in spiders, they are poised to be an excellent system for testing questions regarding whether and how venom use relates to the evolution of social behavior and, in return, whether social behavior influences venom use and evolution. We focus primarily on the widow spiders, Latrodectus, as a strong model for testing these hypotheses. Given that successful responses to risk are vital for maintaining sociality, comparative analysis of spider taxa in which venom metering and sociality vary can provide valuable insights into the evolution and maintenance of social behavior under risk.     

Surgical technique for isolation of the main venom gland of viperid, crotalid and elapid snakes

The main venom glands of venomous snakes were isolated in situ by a harmless surgical technique. The surgery had no apparent effect on the snakes' appearance, disposition or feeding habits more than 2 years after surgery. The snake, after surgery, could serve as an excellent preparation for studying the venom secretory process in the main venom gland as well as studying the secretions of the accessory glands. Surgery also eliminates the risk of lethal bites occurring to zoo personnel and contributes to the study and treatment of diseases of venomous snakes.     

Recent Advances in Research on Widow Spider Venoms and Toxins

Widow spiders have received much attention due to the frequently reported human and animal injures caused by them. Elucidation of the molecular composition and action mechanism of the venoms and toxins has vast implications in the treatment of latrodectism and in the neurobiology and pharmaceutical research. In recent years, the studies of the widow spider venoms and the venom toxins, particularly the α-latrotoxin, have achieved many new advances; however, the mechanism of action of the venom toxins has not been completely clear. The widow spider is different from many other venomous animals in that it has toxic components not only in the venom glands but also in other parts of the adult spider body, newborn spiderlings, and even the eggs. More recently, the molecular basis for the toxicity outside the venom glands has been systematically investigated, with four proteinaceous toxic components being purified and preliminarily characterized, which has expanded our understanding of the widow spider toxins. This review presents a glance at the recent advances in the study on the venoms and toxins from the Latrodectus species.     

Biochemical and molecular characterization of the venom from the Cuban scorpion Rhopalurus junceus

This communication describes the first general biochemical, molecular and functional characterization of the venom from the Cuban blue scorpion Rhopalurus junceus, which is often used as a natural product for anti-cancer therapy in Cuba. The soluble venom of this arachnid is not toxic to mice, injected intraperitoneally at doses up to 200 μg/20 g body weight, but it is deadly to insects at doses of 10 μg per animal. The venom causes typical alpha and beta-effects on Na⁺ channels, when assayed using patch-clamp techniques in neuroblastoma cells in vitro. It also affects K⁺ currents conducted by ERG (ether-a-go-go related gene) channels. The soluble venom was shown to display phospholipase, hyaluronidase and anti-microbial activities. High performance liquid chromatography of the soluble venom can separate at least 50 components, among which are peptides lethal to crickets. Four such peptides were isolated to homogeneity and their molecular masses and N-terminal amino acid sequence were determined. The major component (RjAa12f) was fully sequenced by Edman degradation. It contains 64 amino acid residues and four disulfide bridges, similar to other known scorpion toxins. A cDNA library prepared from the venomous glands of one scorpion allowed cloning 18 genes that code for peptides of the venom, including RjA12f and eleven other closely related genes. Sequence analyses and phylogenetic reconstruction of the amino acid sequences deduced from the cloned genes showed that this scorpion contains sodium channel like toxin sequences clearly segregated into two monophyletic clusters. Considering the complex set of effects on Na⁺ currents verified here, this venom certainly warrant further investigation.     

Inflammatory mediators generated at the site of inoculation of Loxosceles gaucho spider venom

Patients bitten by Loxosceles spiders generally manifest marked local inflammatory reaction and dermonecrosis. This report evaluated edema formation, leukocyte infiltration and release of inflammatory mediators at the injection site of Loxosceles gaucho venom. BALB/c mice were i.d. injected with venom and thereafter paws were disrupted and homogenized to obtain differential counts of migrated cells, as well to assay the levels of cytokines, chemokines and lipid mediators. Increased footpad thickness was detected as soon as 30 min after venom injection, and 24 h later was similar to that of the control group. Loxosceles venom mildly augmented the recruitment of leukocytes to the footpad in comparison with PBS-injected mice. Moreover, it stimulated the release of IL-6, MCP-1 and KC at 2 and 24 h after venom injection. In addition, higher levels of PGE₂ were detected 30 min after venom injection in comparison with control group. However, the venom failed to increase levels of IL-1β, TNF-α, TXB₂ and LTB₄. Our results demonstrate that L. gaucho venom evokes an early complex inflammatory reaction, stimulating the secretion of pro-inflammatory cytokines and lipid mediators (PGE₂), and recruiting leukocytes to the footpad which contribute to the local reaction induced by L. gaucho venom.     

Isolation and identification of Clostridium perfringens in the venom and fangs of Loxosceles intermedia (brown spider): enhancement of the dermonecrotic lesion in loxoscelism.

Loxoscelism or the envenoming by the brown spiders (Loxosceles genus spiders), may produce extensive dermonecrosis and hemorrhage at the bite site and, eventually, systemic reactions that may be lethal. Isolation and identification of many different bacteria, among them Clostridium perfringens, of great medical importance due to its involvement in dermonecrotizing and systemic conditions, was carried out from the venomous apparatus (fangs and venom) of spiders obtained directly from nature, through microbiological cultures in aerobic and anaerobic conditions. Working with Loxosceles intermedia venom (alone) and with the venom conjugated with Clostridium perfringens using rabbits as experimental models for dermonecrosis, allowed for the observation that venom and anaerobic bacteria conjugated resulted in a striking increase of the dermonecrotic picture when compared to venom alone, suggesting a role for Clostridium perfringens in the severe dermonecrotic picture of these patients and opening the possibility for the association of antibiotic therapy in treating loxoscelism.     

Ophiophagus hannah Venom: Proteome,Components Bound by Naja kaouthia Antivenin and Neutralization by N. kaouthia Neurotoxin-Specific Human ScFv

Venomous snakebites are an important health problem in tropical and subtropical countries. King cobra (Ophiophagus hannah) is the largest venomous snake found in South and Southeast Asia. In this study, the O. hannah venom proteome and the venom components cross-reactive to N. kaouthia monospecific antivenin were studied. O. hannah venom consisted of 14 different protein families, including three finger toxins, phospholipases, cysteine-rich secretory proteins, cobra venom factor, muscarinic toxin, L-amino acid oxidase, hypothetical proteins, low cysteine protein, phosphodiesterase, proteases, vespryn toxin, Kunitz, growth factor activators and others (coagulation factor, endonuclease, 5’-nucleotidase). N. kaouthia antivenin recognized several functionally different O. hannah venom proteins and mediated paratherapeutic efficacy by rescuing the O. hannah envenomed mice from lethality. An engineered human ScFv specific to N. kaouthia long neurotoxin (NkLN-HuScFv) cross-neutralized the O. hannah venom and extricated the O. hannah envenomed mice from death in a dose escalation manner. Homology modeling and molecular docking revealed that NkLN-HuScFv interacted with residues in loops 2 and 3 of the neurotoxins of both snake species, which are important for neuronal acetylcholine receptor binding. The data of this study are useful for snakebite treatment when and where the polyspecific antivenin is not available. Because the supply of horse-derived antivenin is limited and the preparation may cause some adverse effects in recipients, a cocktail of recombinant human ScFvs for various toxic venom components shared by different venomous snakes, exemplified by the in vitro produced NkLN-HuScFv in this study, should contribute to a possible future route for an improved alternative to the antivenins.     

Preclinical testing of Peruvian anti-bothropic anti-venom against Bothrops andianus snake venom

Bothrops andianus is a venomous snake found in the area of Machu Picchu (Peru). Its venom is not included in the antigenic pool used for production of the Peruvian anti-bothropic anti-venom. B. andianus venom can elicit many biological effects such as hemorrhage, hemolysis, proteolytic activity and lethality. The Peruvian anti-bothropic anti-venom displays consistent cross-reactivity with B. andianus venom, by ELISA and Western Blotting and is also effective in neutralizing the venom's toxic activities.     

The venom apparatus of Vespa orientalis: morphology and cytology.

The anatomy and histology of the venom apparatus of Vespa orientalis has been investigated. There are two acid glands per hornet each divided into a secretory part made up of two layers of cells containing large amounts of lipids and mucopolysaccharides, and a ‘transporting’ part through which the substances secreted are transported into the venom sac. The alkaline gland contains one layer of cells; muscle fibers are connected to these cells externally and probably play a role in the transport of the secreted substances into the stinging apparatus. The venom sac serves as a reservoir for the secretion of the acid gland and is made of a thick layer of muscle fibers.The toxicity of the different parts of the venom apparatus has been studied both by in vivo and in vitro methods. Extracts of acid and alkaline glands and of venom sac were injected into bees and mice. All the extracts were lethal for the bees, all the extracts were toxic for the mice but only an amount of 4500–7500 μg of venom sac extract was lethal for the mice. The different extracts applied to cultures of liver cells produced an accumulation of fat within the cells, which is considered as a toxic change of the cells.     

Venom from the centipede Scolopendra viridis Say: Purification, gene cloning and phylogenetic analysis of a phospholipase A2

Venom components from the centipede Scolopendra viridis Say were studied, using both the soluble venom and a cDNA library prepared from mRNA of the venomous glands. Separation of the soluble venom by high performance liquid chromatography (HPLC) permitted to obtain at least 54 different fractions. The fraction eluting at 46.24 min showed phospholipase activity. The enzyme was purified to homogeneity and the first 25 amino acid residues were identified by Edman degradation. From the cDNA library several genes were cloned, one of which codes for a protein with identical amino acid sequence as the one experimentally determined. The cloned gene codes for a signal peptide of 28 amino acids and a mature peptide of 119 residues. The molecular weight of the enzyme was estimated by mass spectrometry and shown to be 13,752 Da, which matches exactly with the molecular mass expected from the deduced amino acid sequence of the gene. Phylogenetic analysis of this sequence, in comparison with other known from venomous animals, showed that it is more similar to snake phospholipases than to insect or arachnid sequences, suggesting that it has been submitted to convergent evolution. To the best of our knowledge this is the first time that a phospholipase from this species of animal is fully characterized. We have named it Scol/Pla.     

Structural analysis of the venom glands of the armed spider Phoneutria nigriventer (Keyserling, 1891): microanatomy, fine structure and confocal observations

Spiders belonging to the genus Phoneutria (Perty, 1833), most commonly known as 'armed' spiders, are among the most dangerous species in Brazil due to high toxicity of their venom, associated with their habit of invading domestic or specific areas such as banana plantations. The venom of Phoneutria spiders is secreted by a pair of venom glands located inside their cephalothoraxes and connected to the chelicerae by two independent ducts. In the present study, the microanatomy and histological structure of the venom glands of Phoneutria nigriventer (Keyserling, 1891) were examined in detail by histochemical and conventional stains with laser confocal, scanning and transmission electron microscopies. The analysis confirmed the bulbous-shaped organ previously observed by others. The venom glands of P. nigriventer are covered externally by a double layer of striated muscles, which are arranged in a spiral fashion. This disposition of the external muscle fibers might provide the contraction movement of the venom gland to release their contents during a sting aggression. The presence of pore-like openings between the muscle fibers that cover the venom glands of P. nigriventer was considered quite remarkable. The presence of axon-like structures between the muscle fibers seen in the gland surface was also quite remarkable. The secretory epithelium of P. nigriventer invaginates into the gland lumen, contributing to the increase of the secretory surface area and also accommodating a higher number of secretory cells. Our observation of histological sections and SEM showed that the secretory cells in the venom gland of P. nigriventer form complex structures, secretory units, which originate at the base near the muscular layer and that extend into the central area until the gland lumen. Our study also identified a possible holocrine secretory mechanism of P. nigriventer venom gland, at least in the first venom milking, since we were able to see nuclei stained on confocal laser microscopy. However, our observation cannot disregard other possible types of secretory pathways in subsequent milkings, since we found no nuclei in the second and in the third venom secretions.     

Comparative Studies of the Venom of a New Taipan Species,Oxyuranus temporalis,with Other Members of Its Genus

Taipans are highly venomous Australo-Papuan elapids. A new species of taipan, the Western Desert Taipan (Oxyuranus temporalis), has been discovered with two specimens housed in captivity at the Adelaide Zoo. This study is the first investigation of O. temporalis venom and seeks to characterise and compare the neurotoxicity, lethality and biochemical properties of O. temporalis venom with other taipan venoms. Analysis of O. temporalis venom using size-exclusion and reverse-phase HPLC indicated a markedly simplified “profile” compared to other taipan venoms. SDS-PAGE and agarose gel electrophoresis analysis also indicated a relatively simple composition. Murine LD₅₀ studies showed that O. temporalis venom is less lethal than O. microlepidotus venom. Venoms were tested in vitro, using the chick biventer cervicis nerve-muscle preparation. Based on t₉₀ values, O. temporalis venom is highly neurotoxic abolishing indirect twitches far more rapidly than other taipan venoms. O. temporalis venom also abolished responses to exogenous acetylcholine and carbachol, indicating the presence of postsynaptic neurotoxins. Prior administration of CSL Taipan antivenom (CSL Limited) neutralised the inhibitory effects of all taipan venoms. The results of this study suggest that the venom of the O. temporalis is highly neurotoxic in vitro and may contain procoagulant toxins, making this snake potentially dangerous to humans.     

Brown spider venom toxins interact with cell surface and are endocytosed by rabbit endothelial cells

Bites from the Loxosceles genus (brown spiders) cause severe clinical symptoms, including dermonecrotic injury, hemorrhage, hemolysis, platelet aggregation and renal failure. Histological findings of dermonecrotic lesions in animals exposed to Loxosceles intermedia venom show numerous vascular alterations. Study of the hemorrhagic consequences of the venom in endothelial cells has demonstrated that the degeneration of blood vessels results not only from degradation of the extracellular matrix molecule or massive leukocyte infiltration, but also from a direct and primary activity of the venom on endothelial cells. Exposure of an endothelial cell line in vitro to L. intermedia venom induce morphological alterations, such as cell retraction and disadhesion to the extracellular matrix. The aim of the present study was to investigate the interaction between the venom toxins and the endothelial cell surface and their possible internalization, in order to illuminate the information about the deleterious effect triggered by venom. After treating endothelial cells with venom toxins, we observed that the venom interacts with cell surface. Venom treatment also can cause a reduction of cell surface glycoconjugates. When cells were permeabilized, it was possible to verify that some venom toxins were internalized by the endothelial cells. The venom internalization involves endocytic vesicles and the venom was detected in the lysosomes. However, no damage to lysosomal integrity was observed, suggesting that the cytotoxic effect evoked by L. intermedia venom on endothelial cells is not mediated by venom internalization.