Venom extraction method influences venom composition and potency in the giant house spider Eratigena atrica (C. L. Koch, 1843).

Extraction is the first step when investigating venom composition and function. In small invertebrates, widely used extraction methods include electrostimulation and venom gland extraction, however, the influence of these methods on composition and toxicology is poorly understood. Using the Giant House Spider Eratigena atrica as a model, we show that electrostimulation and venom gland removal extraction methods produce different protein profiles as assessed by Coomassie-stained SDS-PAGE and significantly different potencies in the cricket Acheta domesticus.

Inhibition of bacterial biofilms by the snake venom proteome.

Snake venoms possess a range of pharmacological and toxicological activities. Here we evaluated the antibacterial and anti-biofilm activity against methicillin-susceptible and methicillin-resistant Staphylococcus aureus (MSSA and MRSA) of venoms from the Samar spitting cobra Naja samarensis and the Puff adder Bitis arietans. Both venoms prevented biofilm production by pathogenic S. aureus in a growth-independent manner, with the B. arietans venom being most potent. Fractionation showed the active molecule to be heat-labile and >10 kDa in size. Proteomic profiles of N. samarensis venom revealed neurotoxins and cytotoxins, as well as an abundance of serine proteases and three-finger toxins, while serine proteases, metalloproteinases and C-lectin types were abundant in B. arietans venom. These enzymes may have evolved to prevent bacteria colonising the snake venom gland. From a biomedical biotechnology perspective, they have valuable potential for anti-virulence therapy to fight antibiotic resistant microbes.

Worldwide Web: High Venom Potency and Ability to Optimize Venom Usage Make the Globally Invasive Noble False Widow Spider Steatoda nobilis (Thorell, 1875) (Theridiidae) Highly Competitive against Native European Spiders Sharing the Same Habitats.

Venom compositions include complex mixtures of toxic proteins that evolved to immobilize/dissuade organisms by disrupting biological functions. Venom production is metabolically expensive, and parsimonious use is expected, as suggested by the venom optimisation hypothesis. The decision-making capacity to regulate venom usage has never been demonstrated for the globally invasive Noble false widow Steatoda nobilis (Thorell, 1875) (Theridiidae). Here, we investigated variations of venom quantities available in a wild population of S. nobilis and prey choice depending on venom availability. To partially determine their competitiveness, we compared their attack rate success, median effective dose (ED50) and lethal dose (LD50), with four sympatric synanthropic species: the lace webbed spider Amaurobius similis, the giant house spider Eratigena atrica, the missing sector orb-weaver Zygiella x-notata, and the cellar spider Pholcus phalangioides. We show that S. nobilis regulates its venom usage based on availability, and its venom is up to 230-fold (0.56 mg/kg) more potent than native spiders. The high potency of S. nobilis venom and its ability to optimize its usage make this species highly competitive against native European spiders sharing the same habitats.

Scorpion Species with Smaller Body Sizes and Narrower Chelae Have the Highest Venom Potency.

Scorpionism is a global health concern, with an estimation of over one million annual envenomation cases. Despite this, little is known regarding the drivers of scorpion venom potency. One widely held view is that smaller scorpions with less-developed chelae possess the most potent venoms. While this perception is often used as a guide for medical intervention, it has yet to be tested in a formal comparative framework. Here, we use a phylogenetic comparative analysis of 36 scorpion species to test whether scorpion venom potency, as measured using LD50, is related to scorpion body size and morphology. We found a positive relationship between LD50 and scorpion total length, supporting the perception that smaller scorpions possess more potent venoms. We also found that, independent of body size, scorpion species with long narrow chelae have higher venom potencies compared to species with more robust chelae. These results not only support the general perception of scorpion morphology and potency, but also the presence of an ecology trade-off with scorpions either selected for well-developed chelae or more potent venoms. Testing the patterns of venom variations in scorpions aids both our ecological understanding and our ability to address the global health burden of scorpionism.

Bites by the noble false widow spider Steatoda nobilis can induce Latrodectus-like symptoms and vector-borne bacterial infections with implications for public health: a case series.

CONTEXT: In recent years, the Noble false widow spider Steatoda nobilis (Thorell, 1875) has expanded its range globally and may represent a potential threat to native ecosystems and public health. Increasing numbers in synanthropic habitats have led to more human encounters and envenomations. Steatoda nobilis bites were previously classed as medically significant with similarities to bites from true black widows of the genus Latrodectus but deemed milder in onset, with symptoms generally ranging from mild to moderate. CASE DETAILS: In this manuscript we present 16 new cases of S. nobilis envenomations bringing the total number of confirmed cases reported in the literature to 24. We report new symptoms and provide discussion on the contributing factors to pathology following bites by S. nobilis. DISCUSSION: We report a range of pathologies including necrosis, Latrodectus-like envenomation symptoms that include debilitating pain, tremors, fatigue, nausea, hypotension, and vectored bacterial infections including cellulitis and dermatitis. Symptoms ranged from mild to severe, requiring hospitalisation in some cases.

Synanthropic spiders, including the global invasive noble false widow Steatoda nobilis, are reservoirs for medically important and antibiotic resistant bacteria.

The false widow spider Steatoda nobilis is associated with bites which develop bacterial infections that are sometimes unresponsive to antibiotics. These could be secondary infections derived from opportunistic bacteria on the skin or infections directly vectored by the spider. In this study, we investigated whether it is plausible for S. nobilis and other synanthropic European spiders to vector bacteria during a bite, by seeking to identify bacteria with pathogenic potential on the spiders. 11 genera of bacteria were identified through 16S rRNA sequencing from the body surfaces and chelicerae of S. nobilis, and two native spiders: Amaurobius similis and Eratigena atrica. Out of 22 bacterial species isolated from S. nobilis, 12 were related to human pathogenicity among which Staphylococcus epidermidis, Kluyvera intermedia, Rothia mucilaginosa and Pseudomonas putida are recognized as class 2 pathogens. The isolates varied in their antibiotic susceptibility: Pseudomonas putida, Staphylococcus capitis and Staphylococcus edaphicus showed the highest extent of resistance, to three antibiotics in total. On the other hand, all bacteria recovered from S. nobilis were susceptible to ciprofloxacin. Our study demonstrates that S. nobilis does carry opportunistic pathogenic bacteria on its body surfaces and chelicerae. Therefore, some post-bite infections could be the result of vector-borne bacterial zoonoses that may be antibiotic resistant.

Venomics Approach Reveals a High Proportion of Lactrodectus-Like Toxins in the Venom of the Noble False Widow Spider Steatoda nobilis.

The noble false widow spider Steatoda nobilis originates from the Macaronesian archipelago and has expanded its range globally. Outside of its natural range, it may have a negative impact on native wildlife, and in temperate regions it lives in synanthropic environments where it frequently encounters humans, subsequently leading to envenomations. S. nobilis is the only medically significant spider in Ireland and the UK, and envenomations have resulted in local and systemic neurotoxic symptoms similar to true black widows (genus Latrodectus). S. nobilis is a sister group to Latrodectus which possesses the highly potent neurotoxins called α-latrotoxins that can induce neuromuscular paralysis and is responsible for human fatalities. However, and despite this close relationship, the venom composition of S. nobilis has never been investigated. In this context, a combination of transcriptomic and proteomic cutting-edge approaches has been used to deeply characterise S. nobilis venom. Mining of transcriptome data for the peptides identified by proteomics revealed 240 annotated sequences, of which 118 are related to toxins, 37 as enzymes, 43 as proteins involved in various biological functions, and 42 proteins without any identified function to date. Among the toxins, the most represented in numbers are α-latrotoxins (61), δ-latroinsectotoxins (44) and latrodectins (6), all of which were first characterised from black widow venoms. Transcriptomics alone provided a similar representation to proteomics, thus demonstrating that our approach is highly sensitive and accurate. More precisely, a relative quantification approach revealed that latrodectins are the most concentrated toxin (28%), followed by α-latrotoxins (11%), δ-latroinsectotoxins (11%) and α-latrocrustotoxins (11%). Approximately two-thirds of the venom is composed of Latrodectus-like toxins. Such toxins are highly potent towards the nervous system of vertebrates and likely responsible for the array of symptoms occurring after envenomation by black widows and false widows. Thus, caution should be taken in dismissing S. nobilis as harmless. This work paves the way towards a better understanding of the competitiveness of S. nobilis and its potential medical importance.

Diet Breadth Mediates the Prey Specificity of Venom Potency in Snakes.

Venoms are best known for their ability to incapacitate prey. In predatory groups, venom potency is predicted to reflect ecological and evolutionary drivers relating to diet. While venoms have been found to have preyspecific potencies, the role of diet breadth on venom potencies has yet to be tested at large macroecological scales. Here, using a comparative analysis of 100 snake species, we show that the evolution of prey-specific venom potencies is contingent on the breadth of a species' diet. We find that while snake venom is more potent when tested on species closely related to natural prey items, we only find this prey-specific pattern in species with taxonomically narrow diets. While we find that the taxonomic diversity of a snakes' diet mediates the prey specificity of its venom, the species richness of its diet was not found to affect these prey-specific potency patterns. This indicates that the physiological diversity of a species' diet is an important driver of the evolution of generalist venom potencies. These findings suggest that the venoms of species with taxonomically diverse diets may be better suited to incapacitating novel prey species and hence play an important role for species within changing environments.

The kiss of (cell) death: can venom-induced immune response contribute to dermal necrosis following arthropod envenomations?

Introduction: Snakes, insects, arachnids and myriapods have been linked to necrosis following envenomation. However, the pathways involved in arthropod venom-induced necrosis remain a highly controversial topic among toxinologists, clinicians and the public. On the one hand, clinicians report on alleged envenomations based on symptoms and the victims' information. On the other hand, toxinologists and zoologists argue that symptoms are incompatible with the known venom activity of target species. This review draws from the literature on arthropod envenomations, snakebite, and inflammatory processes to suggest that envenomation by a range of organisms might trigger an intense inflammatory cascade that ultimately lead to necrosis. If confirmed, these processes would have important implications for the treatment of venom-induced necrosis. Objectives: To describe two inflammatory pathways of regulated necrosis, tumour necrosis factor (necroptosis) and Neutrophil Extracellular Traps (NETosis); to discuss existing knowledge about snake venom and arachnid-induced necrosis demonstrating the involvement of tumour necrosis factor and neutrophils in the development of tissue necrosis following envenomation and to contribute to the understanding of venom-induced necrosis by arthropods and provide clinicians with an insight into little known inflammatory processes which may occur post envenomation. Methods: ISI Web of Science databases were searched using the terms "spider bite necrosis", "arthropod envenomation necrosis", "venom necrosis", "venom immune response", "loxoscelism", "arachnidism", "necroptosis venom", "necroptosis dermatitis", "tumour necrosis factor TNF venom", "scorpionism", "scolopendrism", "centipede necrosis", "NETosis venom", "NETosis necrosis". Searches produced 1737 non-duplicate citations of which 74 were considered relevant to this manuscript. Non-peer-reviewed sources or absence of voucher material identifying the organism were excluded. What is necrosis? Necrosis is the breakdown of cell membrane integrity followed by inflowing extracellular fluid, organelle swelling and the release of proteolytic enzymes into the cytosol. Necrosis was historically considered an unregulated process; however, recent studies demonstrate that necrosis can also be a programmed event resulting from a controlled immune response (necroptosis). Tumour necrosis factor and the necroptosis pathway: Tumour necrosis factor is a pro-inflammatory cytokine involved in regulating immune response, inflammation and cell death/survival. The pro-inflammatory cytokine TNF-α participates in the development of necrosis after envenomation by vipers. Treatment with TNF-α-antibodies may significantly reduce the manifestation of necrosis. Neutrophil Extracellular Traps and the NETosis pathway: The process by which neutrophils discharge a mesh of DNA strands in the extracellular matrix to entangle ("trap") pathogens, preventing them from disseminating. Neutrophil Extracellular Traps have been recently described as important in venom-induced necrosis. Trapped venom accumulates at the bite site, resulting in significant localized necrosis. Arthropod venom driving necrosis: Insects, myriapods and arachnids can induce necrosis following envenomation. So far, the processes involved have only been investigated in two arachnids: Loxosceles spp. (recluse spiders) and Hemiscorpius lepturus (scorpion). Loxosceles venom contains phospholipases D which hydrolyse sphingomyelin, resulting in lysis of muscle fibers. Subsequently liberated ceramides act as intermediaries that regulate TNF-α and recruit neutrophils. Experiments show that immune-deficient mice injected with Loxosceles venom experience less venom-induced inflammatory response and survive longer than control mice. Necrosis following Hemiscorpius lepturus stings correlates with elevated concentrations of TNF-α. These observations suggest that necrosis may be indirectly triggered or worsened by pathways of regulated necrosis in addition to necrotic venom compounds. Conclusions: Envenomation often induce an intense inflammatory cascade, which under certain circumstances may produce necrotic lesions independently from direct venom activity. This could explain the inconsistent and circumstantial occurrence of necrosis following envenomation by a range of organisms. Future research should focus on identifying pathways to regulated necrosis following envenomation and determining more efficient ways to manage inflammation. We suggest that clinicians should consider the victim's immune response as an integral part of the envenomation syndrome.

Envenomation by the noble false widow spider Steatoda nobilis (Thorell, 1875) - five new cases of steatodism from Ireland and Great Britain.

OBJECTIVE: The noble false widow Steatoda nobilis is the only medically significant spider known to occur in the British Isles and Ireland, with a single case of steatodism ever reported from Great Britain. We present here five new cases of envenomations by S. nobilis, three from Ireland and two from Great Britain and describe symptoms not previously reported for the genus Steatoda. CASE PRESENTATION: Four adult males and one adult female with confirmed S. nobilis bites reported their symptoms to the authors. General practitioner chart was obtained for case #3. In all five cases, envenomations were immediately followed by a sharp and prolonged onset of pain, mild to extensive erythema and localised to extensive swelling around the bite site. Additional symptoms include moderate to intense pruritus, vasodilation of the capillaries around the bite site and a possible minor necrotic wound. CONCLUSION: In all cases, symptoms subsided within 48-72 h and no further complications were reported. Envenomations by S. nobilis seem to produce symptoms similar (but not identical) to those previously reported from other Steatoda sp. Considering their benign outcome, envenomations by S. nobilis should still be regarded as of moderate medical importance, requiring monitoring and pain management strategies.

'Degraded' RNA profiles in Arthropoda and beyond.

The requirement for high quality/non-degraded RNA is essential for an array of molecular biology analyses. When analysing the integrity of rRNA from the barnacle Lepas anatifera (Phylum Arthropoda, Subphylum Crustacea), atypical or sub-optimal rRNA profiles that were apparently degraded were observed on a bioanalyser electropherogram. It was subsequently discovered that the rRNA was not degraded, but arose due to a 'gap deletion' (also referred to as 'hidden break') in the 28S rRNA. An apparent excision at this site caused the 28S rRNA to fragment under heat-denaturing conditions and migrate along with the 18S rRNA, superficially presenting a 'degraded' appearance. Examination of the literature showed similar observations in a small number of older studies in insects; however, reading across multiple disciplines suggests that this is a wider issue that occurs across the Animalia and beyond. The current study shows that the 28S rRNA anomaly goes far beyond insects within the Arthropoda and is widespread within this phylum. We confirm that the anomaly is associated with thermal conversion because gap-deletion patterns were observed in heat-denatured samples but not in gels with formaldehyde-denaturing.

Development of the venom ducts in the centipede Scolopendra: an example of recapitulation.

In contrast to previous claims that (a) there is a law of recapitulation and, conversely, (b) recapitulation never happens, the evolutionary repatterning of development can take many forms, of which recapitulation is one. Here, we add another example to the list of case studies of recapitulation. This example involves the development of the venom claws (forcipules) in the centipede Scolopendra subspinipes mutilans, and in particular the development of the duct through which venom flows from the gland that produces it (proximal) to the opening called the meatus (distal) through which it is injected into prey. Most of the information we present is from early postembryonic stages--these have been neglected in previous work on centipede development. We show that the venom ducts arise from sutures that are invaginations of the cuticle. In S. s. mutilans, the invagination in each forcipule forms into a tubular structure that detaches itself from the exoskeleton and moves toward the center of the forcipule. This is in contrast to extant Scutigera, and also, probably, Scolopendra's extinct Scutigera-like ancestors, where the duct remains attached to the cuticle of throughout development. Thus, S. s. mutilans exhibits a recapitulatory repatterning of development.

Comparative studies on the structure and development of the venom-delivery system of centipedes, and a hypothesis on the origin of this evolutionary novelty.

The venom-injecting forcipules of centipedes represent an evolutionary novelty that appeared in the centipede stem lineage more than 400 Ma. No other lineage of arthropods (or indeed of animals) has evolved claws for injecting venom from a pair of walking legs. However, little is known of the development, ultrastructure, or detailed function of centipede forcipules. Here, we provide comparative structural information on the venom duct apparatus that is the main functional system within each forcipule, based on scanning electron microscopy and transmission electron microscopy studies. We also give comparative developmental information, using DAPI staining, on embryonic forcipules from the four main centipede orders, including Scutigeromorpha. The photographs of Scutigera embryos we present are the first to be published for any species belonging to this order. The structure of the venom apparatus within each forcipule represents a discrete element of the novelty, whose origin requires a special explanation. This is in contrast to the novel external shape of the forcipules, which can be arrived at gradually by a series of changes from the starting point of a standard walking leg. Drawing on a proposed structural homology between venom glands and epidermal glands, we present a hypothesis of how the venom gland and duct may have arisen in evolution.

Prey orientation and the role of venom availability in the predatory behaviour of the centipede Scolopendra subspinipes mutilans (Arthropoda: Chilopoda).

Many animal phyla contain clades in which most or all species are venom-injecting predators. An example, in the arthropods, is the class Chilopoda, containing the approximately 3500 species of centipedes. Very little ecological or behavioural work yielding quantitative data has been conducted on centipede predation. Here, we describe a study of this kind. Our experiments employed one centipede species - a large tropical one, Scolopendra subspinipes mutilans - and two species of prey - a cricket, Gryllus assimilis, and a locust, Schistocerca gregaria. We conducted two experiments. The first was aimed at investigating the extent to which the centipedes attacked prey in particular tagmata as opposed to at random over the whole body surface. The results showed that the centipedes were highly selective, preferring to attack the head or thorax rather than the abdomen; indeed, they often reoriented the prey in order to achieve this. A possible explanation of this behaviour is to maximize the speed with which the neurotoxins in the venom reach either the brain or the thoracic ganglia that control limb movement. The second experiment was aimed at investigating the effect of venom-extraction on the attack rate, and specifically at testing if the magnitude of any such effect differed between the two types of prey, which differ considerably in size. The results showed a major effect of venom extraction in relation to both types of prey, but with the time taken to return to a 'normal' attack rate being longer in the case of the larger prey-type, namely the locust. We discuss these results in relation to the 'venom optimization hypothesis' and, more generally, to the principle of minimizing the production/use of venom, which is an energetically expensive resource.

Variation and specialisation of the forcipular apparatus of centipedes (Arthropoda: Chilopoda): a comparative morphometric and microscopic investigation of an evolutionary novelty.

The forcipules of centipedes are the only known example in the animal kingdom of an evolutionary transition from walking legs to venom-injecting appendages. They provide a classic case of an evolutionary novelty under most (but not all) definitions of that concept. Although there is a reasonable literature on forcipules, and on the forcipular segment more generally, it is fragmentary and scattered. Also, many previous studies have been based on a single species and hence have no comparative component. Here, we build on this earlier literature by providing detailed qualitative and quantitative information on the forcipular segments of representatives of the five extant orders of centipedes. Our results reveal notable differences between the orders - as well as considerable variation within some of them. The pattern of inter-group differences can be used to infer, albeit cautiously, a major evolutionary trend from a presumed scutigeromorph-like last common ancestor (LCA), in which the forcipules were probably leg-like (as in present-day scutigeromorphs) to a more specialized claw-like structure with movement restricted to the horizontal plane. This morphological trend may reflect an ecological trend from open-habitat ambush predation to leaf-litter and subterranean predatory opportunism.