ABSTRACT. Venom systems can be utilised to provide novel insights into the selective drivers underlying evolutionary change as their direct genetic basis and clearly defined functional phenotypes are amenable for exploring the evolutionary history of these adaptations. Spitting cobras are a particularly suitable vertebrate model for such research as three lineages of these elapid snakes have convergently evolved the ability to spit venom at adversaries, while retaining the predatory use of their venom via biting events. These snakes thus allow us to test hypotheses investigating whether the repeated origins of a novel defensive use of venom corresponds with the evolution of defence-specific toxin components to complement the morphological adaptations that underpin spitting behaviours. Further, they also enable exploration of whether spitting cobras can modulate the composition of their expelled venom when exposed to different ecological stimuli (i.e. prey vs aggressors), in a manner analogous to certain venomous invertebrates. In this talk I will highlight how a variety of productive and generous toxinological collaborations enabled the generation and integration of diverse data types required to robustly investigate these topics, and reveal how the repeated origin of venom spitting provides compelling evidence of convergent venom evolution driven by selection for defensive use.

ABSTRACT. Aim The insect suborder Heteroptera represents a highly diverse group of phytophagous, zoophagous and hematophagous species, which have exploited a variety of different habitats all over the world. The piercing-sucking mouthparts allow them to inject a complex cocktail of salivary proteins that facilitates the extra-oral digestion of various tissues. Predaceous Heteroptera have evolved venomous saliva, which additionally aids in prey capture and defense. The African assassin bug Psytalla horrida is capable of deploying venoms from different salivary gland parts in a context-dependent manner. Venom from the posterior main gland (PMG) is used to overwhelm, kill and predigest prey. Although the general composition of P. horrida venom is known, the function and mode of action of many components are unclear and need to be analyzed. Methods We fractionated PMG venom from P. horrida using cation-exchange chromatography and screened the fractions for cytotoxic, antimicrobial and hemolytic activity. The protein composition of the active fractions was analyzed using LC-MS/MS. Results The observed toxic effects were mainly caused by two fractions, which strongly reduced insect cell viability and bacterial growth and caused up to 15% hemolysis of erythrocytes. Proteomic analysis of the respective fractions revealed that they mainly contained gelsolin, S1 proteases, uncharacterized venom proteins and redulysins. Main Conclusions The selection and analysis of active venom fractions is a crucial step in the identification and characterization of toxic components in P. horrida venom. In order to find out which proteins are responsible for the observed effects, we will heterologously express candidate proteins and screen them for activity. The results of our study contribute to a better understanding of the ecology of venomous Heteroptera and provide new insights into venom functions and novel compounds.

ABSTRACT. Aim Venoms have evolved independently in many animal lineages and show a huge diversity on different scales—ranging from structures to inject venom, to venom producing tissues, to venom function and venom composition¬—which makes venoms a great source to study evolutionary interactions between traits across different levels of biological complexity. However, venom research often focusses on pharmacological activity of isolated toxins, while behavioral and morphological studies are scarce. We use centipede venoms as a model to show how different aspects of venom use are interlinked, and demonstrate why behavioral and morphological studies need to be included in venom research. Methods We used a combination of behavioral experiments, mass spectrometry imaging, proteomics, and immunohistochemistry to investigate venom modulation in the red-headed centipede Scolopendra morsitans. Results Our results show that S. morsitans is able to regulate the composition of its venom and uses specific peptides for predation and defense. The ability to modulate venom composition is probably due to the presence of a complex neuronal network that innervates the venom gland and enables a fine-tuned adjustment of venom secretion. Main Conclusions This multimethodological study on centipede venoms demonstrates how behavioral, morphological and molecular aspects of venom use are interlinked and that venom research would benefit from more holistic approaches.

ABSTRACT. Aim The venoms of prey specialists are often taxon-specific and less complex than the venoms of generalists, but venom composition was not studied in detail in these predators. In our study, we investigated the composition and efficacy of venom of the white-tailed spider (Lamponidae: Lampona sp.). Lampona spiders utilise specialised morphological and behavioural adaptations to capture spider prey, but their venom has not been investigated. Methods We combined RNA-Seq transcriptomics of dissected venom glands with LC-MS/MS proteomics of the crude milked venom to analyse the composition of Lampona venom. To test the relative efficacy of Lampona venom on different prey types, we injected venom into a preferred prey type (spiders) and non-preferred prey (crickets), with the venom of a related generalist spider (Gnaphosidae: Gnaphosa sp.) for comparison. We also tested the efficacy of two venom fractions in Lampona (smaller and larger than 10 kDa). Results The proteo-transcriptomic analysis identified 208 toxin sequences, comprising 103 peptides and 105 proteins. Peptides were more abundant and bigger compared to typical araneomorph or mygalomorph toxins. Most of the peptides were Cysteine-rich and contained eight or ten Cysteine residues. Their scaffolds likely represent variations on the inhibitor-cystine-knot (ICK) motif. Proteins included various toxins similar to galectins, leucine-rich repeat proteins, trypsins and neprilysins. The venom of Lampona was spider-specific, as it was more potent against the preferred spider prey than against the alternative prey represented by a cricket. Only the high mass fraction (> 10 kDa) was effective on both spider and cricket prey. On the other hand, the crude venom of the related generalist was similarly potent against both prey types. Main Conclusions We demonstrated venoms of prey-specialised spiders, such as the white-tailed spider, represent natural libraries of novel bioactive molecules with an expected specific mode of action.

ABSTRACT. Aim The modulation of distinct venom components has been well documented in various venomous organisms, including cnidarians (jellyfish, hydroids, sea anemones, corals) that possess a decentralized venom system through the continuous production of venom-containing stinging cells (nematocysts) across the body. Variation in venom expression has been shown across morphologically distinct structures in multiple types of sea anemones, which can be used to infer the ecological role of different toxin components. The colonial hydrozoan Hydractinia symbiolongicarpus is a useful model to explore venom partitioning through a division of labor via different polyp types, including gastrozooids (prey-capture and digestion) and gonozooids (reproduction, no feeding). Methods We created a novel reporter line for minicollagen 1 (Ncol-1), an early marker for nematocysts. In combination with fluorescence-activated cell sorting (FACS), we sequenced and assembled a nematocyst-enriched transcriptome. Using this as a reference for polyp type-specific RNA-seq data, we evaluated differences in venom-like gene expression of two functionally distinct polyp types. Results Using our FACS approach, we detected venom-like sequences within our nematocyst-enriched transcriptome that were not expressed in the nematocyst-depleted sample, and found differential expression of putative toxins between gastrozooids and gonozooids that correlated with their respective ecological roles. We additionally observed a difference in the nematocyst types across these two polyp types, suggesting some putative toxins may be nematocyst-type specific. Main Conclusions The venom repertoire of H. symbiolongicarpus and nematocyst composition differs significantly between functionally distinct polyp types. This suggests that cnidarians modulate venoms according to ecological roles across functionally specialized tissues, likely through the positioning of different nematocyst types that possess distinct venom compositions. Characterization of venom composition in different nematocysts in combination with functional genomic approaches will help further clarify mechanisms for modulation of venoms in cnidarians.

ABSTRACT. Aim Venom is one of the most widespread convergent adaptive traits in the animal kingdom. This prompts the question - have animals repeatedly adopted the same genetic toolkit for venom production? Methods Here, we performed the first comparative analysis of venom gland transcriptomes from 20 venomous species spanning the main Metazoan lineages, to test whether different animals have independently adopted similar molecular mechanisms to perform the same function. Results We found a strong convergence in gene expression profiles, with venom glands being more similar to each other than to any other tissue from the same species, and their differences closely mirroring the species phylogeny. Although venom glands secrete some of the fastest evolving molecules (toxins), their gene expression does not evolve faster than evolutionarily older tissues. We found 15 venom gland specific gene modules enriched in endoplasmic reticulum stress and unfolded protein response pathways, indicating that animals have independently adopted stress response mechanisms to cope with mass production of toxins. This, in turns, activates regulatory networks for epithelial development, cell turnover and maintenance which seem composed of both convergent and lineage-specific factors, possibly reflecting the different developmental origins of venom glands. Main Conclusions Our findings provide a first evidence that venom gland regulatory networks have evolved, to some extent, from the co-option of pre-existing genetic regulatory circuits from the tissue most closely related to each venom glands. This study represents the first step towards an understanding of the molecular mechanisms underlying the repeated evolution of one of the most successful adaptive traits in the animal kingdom.

ABSTRACT. Scorpion envenoming is a serious public health problem in numerous subtropical parts of the world. In North African countries, about 100,000 accidents are reported annually. The species Androctonus australis (Aa), Buthus occitanus (Bo) and A. mauritanicus (Am), are regarded as the most dangerous. The envenomations are more severe in children in whom mortality is dramatically higher than in adults. A significant percentage of these patients require intensive care treatment, i.e. thousands suffer severe effects and hundred patients die annually as a direct result of the scorpion sting.

The specific treatment of scorpion envenomation is complex and controversial for many reasons. The general objective of this work is to improve specific treatment in the Maghreb, especially in Morocco where only symptomatic treatments are used.

Our presentation will report on the improvement of immunotherapy in North Africa by the conception of an efficient antivenom against the venoms of the most dangerous scorpions mentioned above. In this context, we will describe data obtained on I) proteomic, biochemical, pharmacological and toxicological characterization of the most dangerous scorpions in Morocco; II) production of two types of horse-derived antivenoms, the first being a polyclonal antivenom derived from a mixture of Aa and Bo venoms, while the second is monovalent and produced against Am venom; and III) assessment of protective activity and cross-reactivity of the produced antivenoms.

ABSTRACT. Aim This work aims to isolate and identify a protease from Palythoa caribaeorum venom.

Methods To isolate and characterize the protease, a cation exchange chromatographic separation was performed as the first step, using a previously packed CM Sepharose matrix. Then, the active fraction was separated using centricons with 10 KDa cut membranes at 2791.2 RCF and 4ºC. Finally, the supernatant was subjected to a SHODEX KW-802 size exclusion column chromatography. Finally, the resulting fraction was analyzed by MALDI-TOF mass spectrometry. During each purification step, an electrophoretic analysis (SDS-PAGE) was performed and to follow the proteolytic activity we employed zymograms and EnzChekProtease Assay Kit. To identify whether the enzyme was a serine or metalloprotease type, the effect of metal ions such as ZnCl2, MgCl2, FeCl2, CaCl2 and a metalloprotease activator (APMA) was evaluated. Likewise, the effect of specific inhibitors EDTA, EGTA, 1,10-phenanthroline, TPCK and PMSF were evaluated too.

Results A fraction with serine protease activity was identified from the venom of P. caribaeorum. The electrophoretic analysis of the proteolytic fraction showed a single band. The MALDI-TOF analysis of this fraction identified six proteins ranging from 12 to 64 KDa which correspond to the molecular masses reported for proteases from other venomous animals. PMSF inhibitor reduced the activity of the fraction, suggesting the existence of serine proteases. Additionally, the metal ions did not increase the hydrolysis activity, which suggests that this fraction does not require activators to improve its activity.

Main Conclusion The venom of P. caribaeorum contains proteins that can be associated with those registered for serine proteases in other venomous animals. Likewise, the effect that the inhibitors had on the fraction suggests the presence of at least one serine protease.

ABSTRACT. Aim The cone snail Varioconus guanche (Lauer, 1993) was described from the Canary Islands, but its range also includes southern Morocco, Western Sahara and Mauritania. This species is phylogenetically very close to other species of cone snails from mainland Africa (Senegal and Angola). Following the recent publication of comparative transcriptomic studies of the venoms of continental and insular radiations of West African cones, we aimed to analyze the proteome of the venom duct of V. guanche in order to verify the presence of peptides previously identified in the transcriptome. Methods The venom ducts of five specimens of V. guanche from Fuerteventura were dissected, pooled, lyophilized and extracted with 0.1% formic acid. The extracts were subjected to LC/MS. A PTM database of annotated sequences based upon the published transcriptomes was built using the tool ConoMass in the portal Conoserver. Monoisotopic masses were identified by means of this tool. A fraction of the venom extracts was reduced with dithiotreitol, alkylated with iodoacetamide, and digested with trypsin. The resulting digested solution was used for LC-MS/MS on a Bruker timsTOF-Pro. Data analysis was performed with PEAKS-XPro software. Results A total of 139 monoisotopic masses were obtained by means of LC/MS analysis. Up to 42 of these masses were identified with sequences present on the transcriptome of V. guanche. Additionally, 18 masses were ascribed to sequences present on the transcriptome of other Varioconus from Senegal. Shotgun proteomics (LC-MS/MS) identified a total of 244 sequences in 86 protein groups, including venom-related proteins, hormones and metavenom network proteins in addition to conotoxins. Main Conclusions A total of 146 sequences in the proteome of V. guanche were identified, of which 76 were present on the transcriptome of this species. The rest corresponds to sequences present in other species of cones plus widespread venom proteins and hormones.

ABSTRACT. The saucer bug Ilyocoris cimicoides and the common backswimmer Notonecta glauca are heteropteran predators of a wide variety of arthropods. They secrete venomous saliva, which they inject into their prey to perform extra-oral digestion. Although several studies have characterized the venom composition of true bugs, most of them focused on species belonging to the Reduviidae or Belastomatidae family. In this study, we analyzed transcriptomic and proteomic data of the aquatic bugs I. cimicoides and N. glauca and performed bioassays to characterize their venom activity. In both species, the salivary glands secreted protein mixtures containing, among others, proteases, venom protein family 2 proteins, carbohydrases, lipases and many uncharacterized proteins. While I. cimicoides secreted hemolysin-like proteins and lyases N. glauca venom also contained serine protease inhibitors. There were considerable differences in enzyme activity and bioactivity between the two species. While venom extracts from both insects exhibited protease and lipase activity, only venom from I. cimicoides showed amylase and hemolytic activity. The amylase present in I. cimicoides was able to cleave both starch and glycogen, an unexpected result due to its strict predatory behavior. Although I. cimicoides and N. glauca are present in similar habitats, we show that their digestive enzymes are distinctly different. The bioactivity and composition of N. glauca and I. cimicoides venom assist further efforts to characterize the function and pharmacological activity of insect venom and to understand the complex biomolecular process carried on by true water bugs. Here we demonstrate the importance of combining molecular and enzymatic profiling experiments to characterize the complexity of the venom systems in novel taxa. Our results provide a solid foundation for understanding the role of insect venom composition in prey capture and the extra-oral digestion process.

ABSTRACT. Aim In venomous predatory groups, venom potency is expected to reflect ecological and evolutionary drivers including diet. Previous studies have determined that the venoms of numerous predatory groups exhibit prey specific effects, driven by their diet breadth. The aim of our study is to determine if spider venoms have evolved prey specific effects. Methods We performed a preliminary study using the venoms of six species of tarantula in lethal dose 50% (LD50) and incapacitation dose 50% (ED50) experiments. We tested the potency of each tarantula venom on two prey types: crickets, which are orthopteran insects commonly eaten by tarantulas in the wild and woodlice, which are terrestrial crustaceans, a rather uncommon prey for tarantulas. For each tarantula species, we tested four different raw venom dilutions: 4%, 2%, 1%, and 0.5%, on both prey species, in cohorts of 10, using 5 µL injections. Number of deaths/incapacitations per cohort were recorded at the 1 minute, 5 minute, 10 minute, 15 minute, 30 minute, 1 hour, 2 hour and 24 hour timepoints after each initial venom injection. Results Crickets were disabled more rapidly and more consistently compared to woodlice, across all four venom dilutions, and for all six venoms tested. While crickets were disabled within five minutes of venom injection, most of them recovered 2 to 24 hours after venom injection, most woodlice gradually died off over the 24 hour observation period. Main Conclusions LD50 values recorded at a single timepoint (in this case the 24hr timepoint) do not capture the complex nature of venom, making ED50 a more appropriate tool to investigate the functional efficacy of a venom in situ. Our results suggest tarantulas may have evolved prey specific toxins to disable common prey types found in their diet. This matches the observed pattern found in other venomous predators.

ABSTRACT. Ribbon worms (Nemertea) are active predators that inject toxins to subdue prey through a muscular eversible proboscis and defend themselves with toxic epidermal secretions. These toxins are not produced by distinct multicellular glands, but instead, by secretory cells lining the body wall and proboscis epithelia, making the task of investigating nemertean venom especially challenging. Here we aim to identify putative novel toxins in the heteronemertean Lineus longissimus. We have performed an RNA-Seq differential gene expression (DGE) analysis comparing the proboscis and the posterior end of the body of Lineus longissimus, to identify genes up-regulated in the proboscis which might represent putative predatory toxins. We identified 375 genes upregulated in the proboscis, of which 74 encode putative toxin sequences. Among these, we have found some previously known nemertean toxins, including cytotoxin A-III and the recently discovered α-nemertide. We have also annotated 24 putative novel predatory toxins with sequence homology to toxins previously described in other animal venoms, including cytotoxins, neurotoxins and CRVP (Cysteine Rich Venom Proteins), which are characterized by paralytic, cytotoxic and inflammatory effects that could facilitate the capture and digestion of nemertean prey. Moreover, within the sequences found up-regulated in the proboscis, we also identified 50 that did not show homology with previously known genes but had a signal peptide and cysteine patterns similar to those of conotoxins and could thus represent putative novel toxins exclusive of L. longissimus. This study contributes to a better understanding of the hidden diversity of venoms and toxins of nemertean worms, a group that has been poorly studied up until now but seems to be an untapped source of novel bioactive compounds with biomedical potential.

ABSTRACT. Aim All medically important venomous snakes across Turkey are vipers except for one species of cobra restricted to the southeast of the country. A comprehensive study comparing the enzymatic activities of the Anatolian viper venoms is missing. Herein, we aim to determine and compare the enzymatic activities and protein profiles of the venoms of 11 venomous snake taxa distributed in Turkey. Methods Venoms from 10 viperid taxa (Macrovipera lebetinus, Montivipera xanthina, M. raddei, M. wagneri, M. b. bulgardaghica, M. b. albizona, Vipera ammodytes transcaucasiana, V. kaznakovi, V. berus barani, V. darevskii) and one elapid species (W. morgani) were included in the study. Phospholipase A2, L-amino acid oxidase, protease, hyaluronidase, phosphodiesterase and nucleotidase activities were determined spectrophotometrically. Protein profiles were compared by SDS-PAGE and reversed phase HPLC methods using C-18 column. Results Venom proteins mostly observed between 10 and 75 kDa on SDS-PAGE gels and major band numbers varied from 7 to 11. Chromatograms obtained by HPLC provided more detailed overview of venom proteins and peptides with higher resolution for comparisons and separated more proteoforms. The venoms included in the study showed enzymatic activities at varying levels. Main Conclusions The study includes all medically important venomous snakes in Turkey and provides an overview of their enzyme activities and protein profiles. Similarities of the protein band pattern on SDS-PAGE gel and HPLC chromatograms showed correlation with the taxonomic levels. Enzymatic activities of most of the included taxa were determined for the first time. The results of the study will guide future studies aiming to purify the enzymes and will be useful to assess the pathology of snakebites. Determining the dominant enzyme activities for each species is also important for assessing the antivenom efficacy.

ABSTRACT. Aim Voltage-gated calcium channel CaV3.2 and sodium channel NaV1.7 have been implicated in pain-related pathologies. Given that animal venoms are a rich source of selective ligands for many ion channels, the aim of this study was to discover novel spider toxins with dual inhibitory activity at both CaV3.2 and NaV1.7 channels. Methods Using electrophysiology, HPLC and MS techniques, the venom of several spiders was screened on the voltage-gated calcium CaV3.2 and sodium NaV1.7 channels. Results The crude venom of Pterinochilus murinus showed potent inhibitory activity at both targets. Bioassay-guided HPLC fractionation of the crude venom revealed a 36-amino acid peptide toxin (PmuTx1) containing six cysteine residues. Sequence alignment and structural modelling of PmuTx1 (Fig. 1) showed high similarity with Pn3a, a known NaV1.7 sodium channel inhibitor1, including conservation of K22 and K24 identified as crucial for Pn3a activity2. Main Conclusions PmuTx1 shows potent inhibitory activity at both CaV3.2 and NaV1.7 channels. Altogether, our results suggest the potential of PmuTx1 as an antinociceptive agent with dual activity on two therapeutic targets known to be implicated in pain pathways.

ABSTRACT. The karst viper (Vipera ursinii ssp.) favors high-mountain dry grasslands in southern and south-eastern Croatia. It is medically less important than other Vipera species, because of its remote habitat and the very small amount of venom that it injects by its relatively short fangs. The scientific literature on Vipera ursinii deals mostly with the morphology, ecology and distribution range of this snake, due to the species’ conservation issues, while the toxinological aspects of its venom have not so far been investigated. Here we report on the composition and biological activity of the Vipera ursinii ssp. venom. Using a proteomics approach, we have identified 25 proteins in the venom that belong to seven protein families: snake venom metalloproteinase, serine protease, secreted phospholipase A2, cysteine-rich secretory protein, snake C-type lectin-like protein, serine protease inhibitor and nerve growth factor. The Vipera ursinii ssp. venom was found to be distinctively insecticidal. Its lethal toxicity towards crickets was more than five times greater than that of Vipera ammodytes ammodytes venom, while the opposite held in mice. Interestingly, the mode of dying after injecting a mouse with Vipera ursinii ssp. venom may suggest the presence of a neurotoxic component. Neurotoxic effects of European vipers have so far been ascribed exclusively to ammodytoxins and ammodytoxin-like basic secreted phospholipases A2. Structural and immunological analyses of the Vipera ursinii ssp. venom, however, confirmed that ammodytoxin-like proteins are not present in this venom.

ABSTRACT. Aim Snake venom proteins can be grouped into 15-20 major protein families. However, these proteins show variation based on the taxonomic level, age, diet, sex and ecological factors. In this study, we aimed to investigate the individual venom variation of Macrovipera lebetinus obtusa, a medically important and the biggest viper species of Turkey. Methods The venomic protein profiles of 9 different M. lebetinus individuals from Sanliurfa province (southeastern Turkey) were compared by SDS-PAGE and reversed phase HPLC methods using C-18 column. Additionally, phospholipase A2 (PLA2) activity was determined by a spectrophotometry-based method and proteolytic activity was compared by zymography using gelatin and fibrinogen as substrates. Results Thirteen protein bands were observed on SDS-PAGE jel, most of the proteins were separated between 75 and 10 kDa. PLA2 activity varied between 4.66 and 8.27 nmol/dk/ml/mg. Zymogram gel results showed the activity of proteases with different molecular weights. A certain level of individual variation was observed in SDS-PAGE protein bands and enzyme activities. Moreover, more significant qualitative and quantitative differences were observed in the HPLC chromatograms. HPLC method provided higher resolution compared to SDS-PAGE and separated more proteoforms. Main Conclusions Data on the individual venom variation of M. lebetinus venom was obtained. Results of the study showed that M. l. obtusa venom shows individual variation in the protein content and PLA2 and protease activities even within the narrow range of distribution. Determination of the intraspecific venom variation is important for assessing the pathology of snakebite and production of antivenoms, i.e.to prepare more potent venom pools for the hyper-immunization.

ABSTRACT. Phospholipases A2 are one of the most abundant protein fraction in cobra’s venom. They specifically hydrolyze the ester bond at the sn-2-glycerophospholipid position, releasing fatty acid and lysophospholipid. Their presence in the venom can cause such effects as edema, haemolysis, necrosis, as well as cardio- and neurotoxicity. However, currently, phospholipases play an important role in the food industry in the production of bread, dairy products or in the refining of vegetable oils. Therefore, the aim of these studies was to compare the enzymatic activity of PLA2 from Naja ashei venom with phospholipases currently used in food industry. PLA2 from cobra venom were purified in two step scheme including size-exclusion and ion-exchange chromatography. Then proteins in the fractions were identified and quantitated using shotgun LC-MS. Fractions containing more than 95% of PLA2 were analyzed for enzymatic activity using the sPLA2 Assay Kit (Cayman Chemical) We obtained four fractions (labelled as E2,E3, F2 and F3) containing such a high percentage of PLA2 that they can be considered homogeneous. We compared their catalytic activity with phospholipases used in food industry: Lecitase Ultra and Lecitase 10L as well as PLA2 from bee venom. Two of the analyzed fractions (E2 and F2) have significantly higher enzymatic activity than the tested standards. With the use of Teorell-Stenhagen buffer we also observed that phospholipases A2 from Naja ashei venom work over a wide pH range. The purified phospholipase A2 fractions from Naja ashei venom have a very high catalytic activity, exceeding the enzymes currently used in the food industry. Moreover, the fact that they are active over a wide pH range makes them interesting from an industrial point of view. The conducted research indicates that the components of snake venoms can be an interesting research object not only due to their pharmacological but also industrial potential.

ABSTRACT. Aim The aim of the study was to determine antimicrobial and Anti-quorum sensing activities in honey bee venom collected from Mugla in Turkey. Methods Antimicrobial and Quorum Sensing (QS) inhibitory effects were determined in fourteen bee venom samples collected from Mugla region in Turkey. Anti-quorum sensing activity determination of bee venom samples was detected against Chromobacterium violaceum CV026. In addition, the violacein pigment inhibition activities were determined by using C. violaceum CV12472. Detection of antimicrobial effects of bee venom samples was determined by broth microdilution method against S.aureus ATCC 25923, L. monocytogenes ATCC 7644, E.coli ATCC 25922, S.typhimurium ATCC 14028, and C.albicans ATCC 10239. Results It was determined that the bee venom samples used in the study did not show quorum sensing and violacein pigment inhibition activities. According to the antimicrobial activity results, the highest inhibition effect of bee venom samples was against determined against S.aureus. The highest inhibition effect among bee venom samples was detected in the AZ-8 against S.aureus at a concentration of 0.625 mg/ml. Bee venom samples were found to be effective in the range of 1.25-5 mg/ml against S. typhimurium. Also It was determined that bee venom samples were moderately effective against L.monocytogenes strains. In samples, weak antimicrobial effect was detected against E.coli and C.albicans strains. Main Conclusions As a result, while no anti-quorum sensing activity was observed in the bee venom samples used in the study, they were found to have a high antimicrobial effect against S.aureus and S.typhimirium strains. Acknowledgement This study was supported by Mugla Sıtkı Kocman University Scientific Research Projects Coordination Unit (Project Number: 19/087/03/1/2)

ABSTRACT. Aim The cone snail Lautoconus ventricosus (Gmelin, 1791) is known from the Mediterranean Sea and neighboring Atlantic Ocean. Following the recent publication of the genome organization and structure of venom genes of this species, we aimed to analyze the proteome of the venom duct in order to verify the presence of peptides identified in the transcriptomes of the venom gland and foot and in the genome. Methods The venom ducts of five specimens of L. ventricosus from Algarve, Portugal, were dissected, pooled, lyophilized and extracted with 0.1% formic acid. The extracts were subjected to LC/MS. A PTM database of annotated sequences based upon published transcriptomes and genome conotoxin genes was built using the tool ConoMass in the portal Conoserver. Monoisotopic masses were identified by means of this tool. A fraction of the venom extracts was reduced with dithiotreitol, alkylated with iodoacetamide, and digested with trypsin. The resulting digested solution was used for LC-MS/MS on a Bruker timsTOF-Pro. Data analysis was performed with PEAKS-XPro software. Results A total of 270 monoisotopic masses were obtained by means of LC/MS analysis. 83 of these masses were identified with sequences present on the transcriptome of the venom gland of L. ventricosus. Shotgun proteomics (LC-MS/MS) identified a total of 246 sequences in 126 protein groups, including venom-related proteins, hormones and metavenom network proteins in addition to conotoxins. 27 propeptide sequences were also identified. Main Conclusions A total of 199 conotoxin sequences were identified in the proteome of L. ventricosus. 134 sequences were present on the transcriptome of the venom gland of this species, whereas 4 additional sequences were present in the transcriptome of the foot, and one from the genome conotoxin genes. Not only mature conotoxins, but also partially processed conopeptides were detected. Other sequences identified corresponded to widespread venom proteins and hormones.

ABSTRACT. In the Amazon rainforest, ants have colonized a vast array of different habitats from the underground to the tree canopy. The diversity of ecological constraints in these habitats, especially in arboreal species, suggests strong selection pressure that would translate into specific adaptations for displacement, defense, and prey capture, among others. In this context, we hypothesized that venoms, which are biochemical cocktails of insecticidal and pain-triggering peptides, have been subjected to different selection pressures in arboreal and ground-dwelling species, resulting in the production of fast-acting and pain-producing toxins in arboreal species. Indeed, predatory arboreal species need to subdue a prey rapidly to prevent it from escaping by dropping to the ground or flying away. In addition, ant predators in the arboreal environment mainly belong to vertebrate, which deterrence is essentially induced by pain. We selected a suite of 15 Neotropical ant species from 5 genera with contrasted ecological traits. The venom reservoirs, sting length and several other morphological traits were measured in all species. Using RNA-seq sequencing of venom glands and LC-MS/MS of venoms, we also explored the diversity of venom peptidomes. Besides, venoms were injected into blowfly Lucilia caesar and cellular bioassays were conducted on both Drosophila S2 and Mouse F11 cells to evaluate the venom performance to affect insect prey and to trigger pain to vertebrates. The combination of transcriptomic and proteomic analyses revealed markedly different venom peptide compositions, particularly among ant genera. Morphological data highlighted that species using venom for only predatory purposes had lower venom amounts while non-predatory ants had larger venom reservoirs. Insecticidal and pain-triggering activity were not associated with habitat use but rather with phylogeny and venom peptide composition. In conclusion, our study reveals that venoms and venom-associated traits have adapted to specific ecological constraints, suggesting a vast resource of largely unknown, but valuable, molecules.

ABSTRACT. Aim Venom is highly diverse in its composition and the range of species that employ it. However, across this diversity, venoms have evolved to carry our similar functions and hence, venom function and composition is expected to follow macroecological and evolutionary patterns relating to ecological selection pressures and physiological trade-offs. Identifying these general patterns at the largest taxonomic scales has been challenging due to issues relating to confounding factors. To overcome this we use phylogenetic comparative methods that can account for potential confounding factors and test a series of such macroecological patterns in venom across snakes, spiders and scorpions including; whether venom generally evolves to be prey specific and the role of body size and habitat in driving venom potency and yield.

Methods To test these macroecological hypotheses we collated data on LD50, venom yield, body mass and habitat types for over 200 snake, spider and scorpion species. We used Bayesian phylogenetic comparative methods to test (1) whether LD50’s where higher when tested on species evolutionarily similar to natural prey (2) whether habitat type drives venom yields and (3) whether body size drives LD50’s and venom yields as predicted from metabolic theory.

Results We confirm that, in general, venom is prey specific, with significantly lower LD50 values when tested on species more closely related to natural prey. However, we find that this relationship was only found in species with specialist diets. We also find support that species in more complex habitats, such as arboreal environments, have lower venom yields. Finally, we find that venom yield increases with body size as expected from metabolic theory and also find support for lower potency in larger animals.

Main Conclusions We demonstrate that phylogenetic comparative methods can be useful tools to identify and test macroecology patterns at the highest scales of venom diversity.

ABSTRACT. Pseudoscorpions are tiny arachnids found in crevice habitats. Within this group a unique venom delivery system has evolved enabling venom injections with modified pincers to overcome prey much larger than the individual hunter. Due to their small size and their harmlessness for men, pseudoscorpion venom was not in the focus of venom research. Up to now, only a few studies have been performed on pseudoscorpion venom, which either identified a small fraction of venom compounds, or were based on solely transcriptomic approaches. Only one study addressed the bioactivity of pseudoscorpion venom. Here we present the results of the first comprehensive proteo-transcriptomic analysis of the house pseudoscorpion’s (Chelifer cancroides) venom. Another major achievement of our study are the first activity tests for pseudoscorpion venom, testing its effect on specific ion channels. Regarding the venom composition of the house pseudoscorpion, we identified the first genuine pseudoscorpion venom compounds with similarity to neurotoxins and we discovered the venom to be dominated by novel compounds without matches in the databases. Interestingly, our electrophysiological activity tests revealed not only a modulation for insect potassium channels but also for sodium channels from Varoa mites, which the house pseudoscorpion preys on as a symbiont of the honey bee. The findings of our study provide the first comprehensive insight about the venom composition and activity of one of the smallest venomous animals ever studied. In addition, the large amount of identified novel compounds demonstrates once again the large potential of investigating neglected venomous taxa to open up additional sources of bioactive compounds usable for basic research and industrial application.

ABSTRACT. Aim Research on Cnidaria venom has been mostly focused on sea anemones, Anthozoans belonging to the subclass Hexacorallia. Notably, scarce information is available on the venom produced by the sister subclass of Octocorallia, or soft corals, and it mostly pertains to their small molecules content. Polyps of soft coral colonies are equipped with nematocyst-rich tentacles used to feed on zooplankton and to deter potential predators. The lack of information regarding polypeptide toxins of soft corals is a significant bias towards the understanding of the evolutionary history of Anthozoan venom toxins, and the development of their biotechnological potential. Methods We used a combined transcriptomic-proteomic approach to investigate the venom of the white seafan Eunicella singularis, a Mediterranean soft coral inhabiting rocky bottoms at 20-30 m depth. We isolated venom polypeptides through selective chemical extraction of the nematocysts and we matched MS/MS proteomic data against a whole body assembled transcriptome. Finally, we performed phylogenetic analyses and modeled the structure of relevant proteins, to gain insights on their function and evolution. Results We identified potential cytolytic, hemostasis impairing and neurotoxic molecules in the venom of E. singularis, finding similarities with well characterized families of cnidarian toxins, such as small cysteine rich peptides (SCRPs) and ShK toxins (ShKT), but also detecting uncommon polypeptides with novel cysteine frameworks. In particular, our investigation highlighted the presence of a new family of cytolytic polypeptides, structurally related to saposins, never observed before in Cnidaria. Homology models and surface electrostatic potentials were analyzed to understand their possible mode of action in comparison to well characterized pore forming proteins. Main Conclusions Our results provide the first description of soft coral venom toxins, highlighting their potential activity and shedding light on the evolutionary history of conserved protein families in Anthozoa.

ABSTRACT. Aim The Noble false widow Steatoda nobilis (Thorell, 1875) originates from the Macaronesian archipelago and has recently expanded its range to most of Western Europe and parts of the Americas. In Ireland, Steatoda nobilis is becoming one of the most widespread spiders in synanthropic habitats. The reasons for its success are not understood, but observations suggest that in Ireland, increased S. nobilis density may negatively impact native spider populations. Because of its competitiveness, abundance and resilience in captivity, Steatoda nobilis is an ideal model to investigate questions related to arthropod invasiveness, venom use and predation strategies. Methods Here we set out to 1) Determine if Steatoda nobilis optimises its behaviour based on venom availability and prey size, as suggested by the Venom Optimisation Hypothesis; 2) Investigate the effects of S. nobilis venom on two native Irish species: Amaurobius similis (Blackwall, 1861) and Eratigena atrica (Koch, 1843); 3) Assess if the offensive and defensive strategies of S. nobilis, give it a competitive advantage over native spiders sharing the same habitats. Results Results suggest that 1) Stetoda nobilis optimises its venom use based on venom availability, injecting venom preferably in the head and thorax of prey, and selecting prey of different size depending on how recent venom extraction was performed; 2) Venom of Steatoda nobilis is extremely toxic to Amaurobius similis and Eratigena atrica, while Steatoda nobilis seems resistant to the venom of the other two species; 3) Steatoda nobilis will engage in territorial fights with the native Irish spiders Amaurobius similis, Eratigena atrica, Pholcus phalangioides, and Zygiella x-notata, and kill its opponents. Main Conclusions Long-term experiments should be set to assess the impact of Steatoda nobilis in-situ within and outside its native range. It would also be interesting to assess if Steatoda nobilis is really exhibiting resistance to most spider venoms.

ABSTRACT. 1İscan.A., 1,2 Çalıskan .F 1 Department of Biotechnology and Biosafety, Graduate School of Natural and Applied Sciences, Eşkisehir Osmangazi University, TR-26040 Eşkisehir, Turkey. 2 Department of Biology, Faculty of Science and Letters, Eskisehir Osmangazi University, TR-26040 Eskisehir, Turkey.

Aim The increasing emergence of bacterial and fungal resistance requires the development of new effective antibiotics. In recent studies, it has been reported that venomous animals are potential drug candidates with their active molecules in hemolymph. Therefore, our aim in this study is to investigate antimicrobial peptides in Mesobuthus gibbosus hemolymph.

Methods Scorpions were collected under the UV light at night from Inhisar, a province of Bilecik, Turkey. Hemolymph from the scorpions was collected directly by heart puncture and centrifuged at 5000 rpm. Cell free hemolymph was applied to a reverse-phase high-performance liquid chromatography system. Elution was monitored at 230 nm and individually collected sub-fractions were vacuum dried. Antimicrobial activities of fractionated hemolymph were examined against 6 bacteria and 6 Candida species.

Results At least 70 eluting fractions were obtained by the means of liquid chromatography and examined for their antimicrobial effects. Sub-fractions with retention times 24.8, 25.6 and 47.3 min., were determined with their effective anticandidal properties. Then retention time of 47.3 min was further separated and showed strong inhibitory effects against C. parapsilosis and C. krusei having MIC values 5,6 µg/mL and 2,8 µg/mL, respectively.

Main Conclusions In this study, we determined new peptide fractions from M. gibbosus hemolymph which could be the source of new therapeutic components. It is anticipated that determining the possible active compounds with antimicrobial activity included in the hemolymph of the venomous organisms, as well as their primary structure, will be useful pharmacologically.

ABSTRACT. Aim We analysed an actinoporin-like protein MiGa found in the Mediterranean mussel (Mytilus galloprovincialis). We expressed recombinant MiGa and performed functional studies to elucidate its biological function. Methods MiGa protein was expressed in E. coli and purified. Hemolytic activity on bovine erythrocytes was determined by absorbance at 630 nm. Permeabilization of cellular membranes was measured as increase in Sytox green fluorescence after binding in the nuclei. Cells were imaged using a fluorescence microscope. Viability after exposure to toxins was measured as conversion of Presto blue substrate. Lipid affinity was determined by sedimentation assay and calcein release assay using vesicles with different lipid composition. Results The lipid affinity of MiGa differs from actinoporins, which preferentially bind to sphingomyelin. MiGa instead showed the highest affinity for 1-palmitoil-2-oleoil-sn-glicero-3-fosfo-(1'-racglicerol) (POPG) as determined by sedimentation assay and calcein release assay. MiGa is capable of forming membrane pores, although its pore-forming activity is weak compared to actinoporins such as Fragacea toxin (FraC). Hemolytic activity on bovine erythrocytes was first demonstrated at a concentration of 50 nM. 10-fold higher MiGa concentrations were required for membrane permeabilization in the fish cell line EPC, whereas hemolytic activity on fish erythrocytes was not detected (tested to 8 µM). MiGa activity against mussel hemocytes and gill epithelium was not detected at the concentrations tested (up to 4 µM). Interestingly, FraC also showed no activity on mussel cells at this concentration, but was highly active on bovine erythrocytes and the EPC cell line. Main Conclusions Low MiGa activities on animal cells and low binding affinity to sphingomyelin-containing membranes do not support a role for MiGa pores in mussel digestion of zooplankton and predation defence.

ABSTRACT. Aim A long-held popular belief suggests that relatively small scorpions with narrow pedipalps are more venomous to vertebrate organisms than comparatively larger species with broader pedipalps. So far however, there has been no large scale comparative test to support this statement. Here, we attempt to assess if, in the case of scorpions and their venom, size does indeed matter.

Methods We performed a systematic literature review and recorded LD50, body size and other morphological features for 49 species across 5 families the Buthidae, Scorpionidae, Chactidae, Iuridae and Ischnuridae families from Asia, Africa, Europe, North and South America. Using general linear models we tested if venom potency decreased with body size.

Results We found a significant positive correlation between LD50 and scorpion body size (slope = 2.16, p < 0.05). This relationship equates to an order magnitude increase in LD50 over the range of body sizes in our dataset. We also found that these results were not affected by the route which the LD50 was tested (eg. Intravenous (IV), subcutaneous (SC) or intraperitoneal (IP))

Main Conclusions We show that large scorpions do indeed have less potent venoms. This correlation is likely linked to an evolutionary trade-off between muscular strength / size and venom toxicity. Scorpions with larger chelae may rely mostly on strength to crush their prey and deter predators, while smaller scorpions with slender chelae rely more heavily on their venom to subdue prey and ward off enemies. Because venom and body mass have a physiological cost, it likely that a single trait (either physical strength or toxicity) is favoured over the other. These results also aid in understanding how different species may contribute to global patterns of medically significant scorpion envenomation.

ABSTRACT. Currently, piscivorous Conus species represent a research hot-spot in the area of drug discovery and venom application. In this study, we are focusing on other groups of the Conus species with different feeding habits, to uncover the potential values hidden in the neglected molluscivorous and vermivorous Conus species.

Conus species from three other genera (Californiconus, Conasprella and Profundiconus) in the Conidae family are assessed. We revisited online databases and constructed a phylogeny tree with available feeding habit information. We assembled a Conus distribution map based on their feeding habits. Pharmaceutical attempts and the relevant omics data have been counted to demonstrate the current research trend, including some stunning correlation between conotoxins and the COVID-19 pandemic.

Noticeably, many species in the warm and shallow waters are adapted to hunt both fishes and worms independently. We have also discussed the Conotoxin-drug discovery potentials and the urgency of protecting the resources of Conus species.

The findings of this work shed light on the Conus evolution, and emphasize the importance of further omics study and more behavior observation studies on the future.

ABSTRACT. Aim Venomous animals are found throughout a wide range of taxa, including fish species, such as stingrays and scorpionfish, which were previously overlooked. Fish venoms are particular difficult to study due to their instability, sampling complications and problems isolating the proteins. Genomic and transcriptomic data offer a gateway into this hard-to-reach information. Methods Recent efforts in fish genome and transcriptome sequencing, in combination with inside generated venom gland transcriptomes, provided the ability to set a much-needed baseline and further explore the evolutionary dynamics of fish venoms. It is currently hypothesized that fish venom evolved independently at least 18 times. Despite this, similarities in venoms across distinct fish taxa are found, with different molecules from the same gene family being part of the venom composition. Results The newly generated datasets allowed the determination of several of these differences, such as different hyaluronidases and various metalloproteases being recruited into the venom network in different fish groups. This proximity of the genes, mostly of the same gene family, suggests very comparable needs in the evolution of many fish species venoms. Such a scenario of convergent evolution is further exacerbated by the fact that fish venoms are commonly used as a defensive tool, instead of a predatory option or else. Main Conclusions This data shows that there is potential in exploring labile fish venoms through genomes and transcriptomes, despite their individual shortcomings. The information presented enhances the understanding of venom composition in fishes, their genetic differences, organization and evolution.

ABSTRACT. Aim Cysteine-rich secretory proteins (CRISPs) are found throughout the animal kingdom and exert different functions. CRISPs from snakes have diverse roles in their venoms. Our objective was to biochemically characterize CRISPs from the venom of the nose-horned viper (Vipera a. ammodytes; Vaa) and to uncover their biological roles. Methods To purify Vaa CRISPs from the crude venom, we used size-exclusion chromatography, ion-exchange chromatography, and RP-HPLC. We produced the recombinant VaaCRISP-1 in E. coli. To determine their biophysical, biochemical and structural properties, we used differential scanning fluorimetry (DSF), CD spectroscopy, two-dimensional gel electrophoresis, and mass spectrometry. Pathophysiological effects of VaaCRISPs were studied ex vivo in mice and rats, and in vitro in human blood samples. Results We purified six isoforms of Vaa CRISP to homogeneity. All are slightly acidic proteins with pI values around 5,3 and molecular masses ranging from 24564,6 Da to 24916,6 Da. DSF experiments showed that Vaa CRISPs are stable at pH 6–9 in the presence of mM Ca2+. They also helped us to optimize the isolation conditions to achieve the best possible purification yield. Using CD spectroscopy, we confirmed that the native structure of the Vaa CRISPs indeed remained intact throughout the isolation. The functional significance of Vaa CRISPs in the venom is not yet completely certain. Apart from some effect on blood coagulation reflected by prolonged activated partial thromboplastin and prothrombin times, Vaa CRISPs showed no effect on mouse neuro-muscular preparation (compound action potential and directly or indirectly evoked muscle contraction) and on the contraction of smooth muscles depolarized with increased K+ concentration. Main Conclusions We developed an efficient isolation procedure to purify Vaa CRISPs from crude snake venom and produced the recombinant isoform VaaCRISP-1. Vaa CRISPs inhibited blood coagulation in vitro but did not affect mouse neuromuscular junction or arterial smooth muscle function.

ABSTRACT. Aim Hymenopterans (ants, bees, wasps) are still a valuable and understudied source of venomous species. Their remarkable ecological diversity across most terrestrial environments along with their complex social behaviours and chemical communication have presumably contributed to the diversity of venom types. Here, we analyzed hymenopterans venom diversity, and possible biological approaches to pharmacology and biotechnology.

Methods We gathered 283 revised hymenopteran toxin sequences and meta-data from UniProt knowledgebase (https://www.uniprot.org/). We assembled different datasets based on (1) toxins diversity, (2) ecological toxins approach, (3) frequency of biological activity, and (4) plausible biotechnology and pharmacology approach. Then, we analyzed the relationships between all 4 research foci using different R packages ggplot2, ggallluvial2, ggraph, tidyverse, and igraph (R version 3.6.1 and RStudio 1.2.5001).

Results Our preliminary results indicated that wasp venoms contained higher protein diversity (toxins, proteins, enzymes) whereas ants produced venoms rich in peptides, called aculeatoxins. We found that hymenopteran venoms were primarily specialized to defend the hosts against pathogens (microbes, predators) as well as for hunting. Voltage-gated ion channels are the primary biological targets of the toxins to induce allergy and pain. Hymenopteran venoms are the potential sources for antimicrobial, antifungal, antiviral, autoimmune diseases, and bioinsecticides.

Conclusions Hymenopteran venoms, from ants and wasps, represent a diverse, complex, and valuable source for the discovery of biologically peptides and proteins. With an estimated 100,000 species around the globe, hymenopteran venom research is just starting.

ABSTRACT. Aim

Gene expression dynamics are a major contributor to complex trait variations within and between species. These shifts in gene expression are subject to selective pressures driving mutations to the cis- and trans-regulatory elements, epigenetic modifications, and gene duplication. A complex trait hypothesized to be evolving under strong selective pressure is venom due to its essential ecological roles related to predation and defense.

Methods

Using sea anemones as a model to understand the evolution of venom as a complex trait, we employed a combination of comparative transcriptomics and genomics. The transcriptomes of 29 sea anemone species from three superfamilies were assembled and used to quantify the expression of functionally characterized toxins and used for phylogenetic covariance analysis. We further explored the recent high-quality genome assemblies of three sea anemone species (Nematostella vectensis, Scolanthus callimorphus, and Actinia equina), to gather insights into the biology of their genomes that drives the evolution of venom phenotype in sea anemones.

Results

Phylogenetic covariance analysis revealed that the possible combinations of toxin families are highly promiscuous in sea anemones, yet the venom phylomorphospace has low dimensionality. This suggests that in sea anemones, a single toxin family dominates the venom phenotype of each species. The evolution of the specific dominant toxin family found in each sea anemone can be highly dynamic, even convergently evolving among distantly related species. Our comparative genomic analysis revealed that all three sea anemone species possess more than ten copies of the toxin gene family that is dominant, with members of each family being found in clusters of tandemly duplicated copies.

Main Conclusions

Altogether, these results suggest that gene duplication drives the shift in the dominant toxin family that strongly dictates the venom phenotype and highlights it as a powerful mechanism for the adaptive evolution of a complex trait.

ABSTRACT. Aim Viperid venoms contain proteins interfering with the coagulation cascade. Among those, snake venom thrombin-like (fibrinogenolytic) enzymes draw attention. In this study, it is aimed to compare the fibrinogenolytic activity of Anatolian viper venoms. Methods Venoms of 10 viperids [Macrovipera lebetinus obtusa (MLO), Montivipera xanthina (MX), M. raddei (MR), M. wagneri (MW), M. b. bulgardaghica (MBB), M. b. albizona (MBA), Vipera ammodytes transcaucasiana (VAT), V. kaznakovi (VK), V. (berus) barani (VB), V. darevskii (VD)] were incubated with human fibrinogen for different time intervals at 37 °C. Fibrinogenolytic activity was determined by SDS-PAGE method. In addition, inhibition study was carried out by pre-incubating venoms with different protease inhibitors. Results All venoms showed fibrinogenolytic activity and degraded the fibrinogen Aα-chain, except VB. All venoms also degraded the Bβ-chain at various degrees after 120 minutes incubation, except MR, VAT and VB. Activities of all venoms were inhibited by metalloprotease inhibitors EDTA and 1,10-phenanthroline. On the other hand, serine protease inhibitor PMSF inhibited α-fibrinogenase activity of VAT and β-fibrinogenase activities of MX, MW and MBB whereas aprotinin did not show inhibitory effect. Main Conclusions This is the first study revealing the presence of fibrinogenolytic enzymes in the venoms of MW, MBB, MBA, VK and VD. According to the results of the study, all venoms except VB contain metalloproteases possessing α- and/or β-fibrinogenolytic activity. Moreover, results of the inhibition study also indicated that MX, MW and MBB venoms contain serine proteases degrading Bβ-chain and VAT venom contain serine proteases with α-fibrinogenolytic activity. This study exhibited the potential of undiscovered venoms from Anatolian vipers as a source for novel fibrinogenolytic enzymes that might have diagnostic and therapeutic potential. Our results will guide the following purification and characterization studies.