Marine biotoxins constitute one of the major hazards associated with seafood consumption. Risk assessments are essential for the effective management of problems arising from marine biotoxins occurrence, as they are a prerequisite for the establishment or periodic re-evaluation of marine biotoxins regulatory limits and for the adoption of appropriate risk management plans. Risk assessments are science-based data-intensive processes, and their successful outcomes are largely dependent on the quality of data used when they are carried out. In fact, data-related challenges are the most frequently reported issues rendering most marine biotoxins’ risk assessments conducted to date as inconclusive. Notably, data quality perceptions among the stakeholders involved in risk assessments may vary significantly, which may be a human factor influencing data quality. As such, the problem addressed in this thesis is the shortage of empirical information on how data quality is perceived by the different stakeholder roles involved in risk assessments relevant to marine biotoxins hazards. The focus of this thesis is thus to investigate the perceptions of diverse stakeholders within the information chain, namely data producers, collectors and consumers/users, regarding the quality of data used in risk assessments of marine biotoxins hazards, to provide a contribution directed towards data quality improvement. This was done by means of a survey, gathering data through interviewing a number of recognized marine biotoxins experts with documented experience in risk assessments. The research question of this study is: “What are the perceptions of data quality among diverse stakeholders along the information chain relevant to marine biotoxins’ risk assessments?” To answer the research question, the concept of data quality for marine biotoxins data destined for risk assessments was dissected into seven individual subtopics on which the perceptions of expert participants of all three roles were captured. The subtopics explored included: data quality challenges; changes in marine biotoxins data quality during the last decade; awareness on data quality legislation and standardization; importance of data quality dimensions, objectives and key performance indicators; importance of data quality-related feedback exchange between stakeholders of the relevant information chain; factors for successful adoption of harmonized standardized formats for marine biotoxins data collection; and (vii) suggestions for data quality improvement. The perceptions gathered per subtopic were analyzed using inductive thematic analysis, yielding a total of twelve themes, namely communication, compound, data/quality control, Information Technology or Data Collection Framework, legislation, method, organization, people, policy, risk assessment procedure, society/environment and toxicological aspects, with each subtopic containing items categorized within several of these themes. Certain differences were observed in the perceptions between participants of diverse data roles, in the sense that data producers and to a lesser extent data users mostly focused on themes relevant to analytical methodology, compound particularities, data and quality control, toxicological aspects and policies. On the other hand, data collectors’ views were more concentrated on items relevant to Information Technology or Data Collection Framework and organization. It is noted, however, that interpretation of these trends needs to consider that in many of the study participants different roles overlapped in the same person. This indicates that results should be cautiously generalized. Nevertheless, they could constitute a basis for further research to generate deeper knowledge in the field of data quality in risk assessments relevant to marine biotoxins and gain further insights on the differences in perceptions among data roles.

Major variations in venom composition can occur between juvenile and adult venomous snakes. However, due to logistical constraints, antivenoms are produced using adult venoms in immunising mixtures, possibly resulting in limited neutralisation of juvenile snake venoms. Daboia russelii is one of the leading causes of snakebite death across South Asia. Its venom is potently procoagulant, causing stroke in prey animals but causing in humans consumptive coagulopathy—a net anticoagulant state—and sometimes death resulting from hemorrhage. In this in vitro study, we compared the venom activity of—and antivenom efficacy against—six 2-week-old D. russelii relative to that of their parents. Using a coagulation analyser, we quantified the relative coagulotoxicity of these venoms in human, avian, and amphibian plasma. The overall potency on human plasma was similar across all adult and neonate venoms, and SII (Serum Institute of India) antivenom was equipotent in neutralising these coagulotoxic effects. In addition, all venoms were also similar in their action upon avian plasma. In contrast, the neonate venoms were more potent on amphibian plasma, suggesting amphibians make up a larger proportion of neonate diet than adult diet. A similar venom potency in human and avian plasmas but varying selectivity for amphibian plasma suggests ontogenetic differences in toxin isoforms within the factor X or factor V activating classes, thereby providing a testable hypothesis for future transcriptomics work. By providing insights into the functional venom differences between adult and neonate D. russelii venoms, we hope to inform clinical treatment of patients envenomated by this deadly species and to shed new light on the natural history of these extremely medically important snakes.

Several snake toxins are widely studied due to their broad therapeutic potential. The venom produced by snakes of the genus Crotalus sp presents a wide variety of pharmacological uses of its isolated fractions, such as antifungal, antiviral, antibacterial, and antitumor properties, among other therapeutic potentialities. Regarding its antitumor action, this venom has a direct effect on tumor cells, and the isolation of its compounds is important for the characterization of the anticarcinogenic actions. Toxins derived from snake venoms have proven to be excellent biological tools to evaluate the inhibition of metastasis in vitro and in vivo, as well as the direct action on tumor cells. Crotalus durissus terrificus venom and its toxins have been widely evaluated as potential candidates for the development of new antineoplastic therapies, efficient against different tumor lines and cellular targets. This work highlights this snake’s venom toxins, focusing on the antineoplastic properties.

Insects mostly use their venom to subdue prey, or for both subduing prey and defense from predators, but some rare species such as larval lepidopterans (caterpillars) use venom solely for deterring predators. Of the 133 lepidopteran families, nine (Erebidae, Eucliedae, Lasiocampidae, Limacodidae, Megalopygidae, Notodontidae, Nymphalidae, Saturniidae and Zygaenidae) can cause serious pathophysiological conditions in humans and animals. Although they present a health hazard, caterpillars are under-represented in venom research and little is known about the structure and function of their toxins.

Limacodidae is a family of more than 1,500 species, over half of which, known as stinging nettles, have venomous larvae (1). Envenomation by limacodids mainly causes intense pain, itch, and erythema, and occasionally numbness, weakness, nausea, and dizziness. Using a combination of imaging technologies, transcriptomics, proteomics and functional assays, we recently provided a holistic portrait of the venom system of one species, Doratifera vulnerans, which produces a complex peptide-rich venom, contrary to the common belief that defensive venoms have simple composition (2). Three of the most abundant families of venom peptides are: (i) disulfide-rich knottins similar to the inhibitor cystine knot (ICK) peptides that dominate spider venoms; (ii) linear, cationic cecropin-like peptides that cause pain when injected into mammals, and also kill bacteria, insects and parasites; and (iii) homologues of adipokinetic hormone/corazonin-related neuropeptide (ACPs) (2).

Using proteomics and transcriptomics, I am currently examining the venom of additional limacodid species, including the saddleback caterpillar Acharia stimulea native to eastern North America, and an undescribed species collected in Townsville, Australia. Surprisingly, I found that the North American species possesses venom that is very similar to that of the Australian D. vulnerans whereas venom of the undescribed Australian species is radically different, hinting at complex evolution of venom composition within Limacodidae. I am also expanding knowledge of the evolutionary trajectory of individual toxins by synthesising and characterising peptides intermediate in structure between the ancestral cecropin of non-venomous Lepidoptera and D. vulnerans cecropin-like venom toxins. Finally, I am producing a library of limacodid venom peptides using solid-phase peptide synthesis and recombinant expression that I will test on mammalian ion channels including those involved in pain. This research will provide novel insights into multiple aspects of limacodid venoms.

References:

1. Epstein M, ed. (1996) Revision and phylogeny of the Limacodid-group families, with evolutionary studies on slug caterpillars (Lepidoptera:Zygaenoidea), Smithsonian Institution Press, Washington DC.
2. Walker AA, Robinson SD, Paluzzi JV, Merritt DJ, Nixon SA, Schroeder CI, et al. (2021) Production, composition, and mode of action of the painful defensive venom produced by a limacodid caterpillar, Doratifera vulnerans. Proc Natl Acad Sci USA. 118, e2023815118.

Ciguatoxins (CTXs), produced by microalgae within the Gambierdiscus and Fukuyoa genera, and tetrodotoxins (TTXs), produced by bacteria, are one of the most potent emerging natural marine toxins in Europe, which act by selectively blocking the action potentials of voltage-gated sodium channels (VGSCs) in nerves, skeletal and cardiac muscle membranes, with opposite mechanisms of action. CTXs block them in an open position, whilst TTXs do it in a closed state.

For the European Food Safety Authority (EFSA), these neurotoxins have been the primary concerns due to the important impact on human health, and their important effects on food safety. The evaluation and characterisation of their risk with adequate detection methods for monitoring procedures are crucial to protect human health. EFSA also encourages the search for occurrence data and the development of reliable and efficient analytical methods. However, despite technological advances, there are still methodological uncertainties and high demand for universal marine toxins detectors. Electrochemical cell-based biosensors seem a promising strategy, which are based on cell-based assays (CBAs) and therefore measure the toxicological effect of toxins on cells and provide an estimation of the composite toxicity of a sample and have the added advantage of the high sensitivity provided by electrochemical detection techniques.

Bearing that in mind, Neuro-2a cells were immobilised on electrodes of different materials, and their viability was assessed using a tetrazolium salt. After optimisation of the assay conditions, the effect of CTX and TTX standard solutions on the immobilised cells was evaluated. Finally, the system was applied to the analysis of fish extracts containing CTX (Variola louti) and TTX (Lagocephalus sceleratus), as a proof of concept. The strategy has been proved to be successful and therefore paves the way towards the development of electrochemical cell-based biosensors for the detection of marine neurotoxins.

Nanomedicine has led to the development of new biocompatible and biodegradable materials able to improve the pharmaceutical effect of bioactive components, broadening the options of treatment for several diseases, including cancer. Additionally, some snake venom toxins have been reported to present cytotoxic activity in different tumor cell lines, making them an auspicious option to be used as cancer drugs. Our research team has been working with the synthesis process and characterization of different polymeric nanoparticles to evaluate their antiproliferative activity in different cancerous cell lines. Chitosan nanoparticles (~400 nm, zeta potential: ~25 mV) had en encapsulation efficency (EE%) of ~50% of the black-tailed rattlesnake, and an IC₅₀ of ~15 µg/mL in the T-47D breast carcinoma cell line. Additionally, we have synthetized and characterized PLGA nanoparticles (~250 nm, zeta potential: ~-25 mV) with an EE% of ~90%. Rattlesnake venoms from the Sonoran Desert in Mexico have the potential to be developed into cancer pharmaceuticals, and, the addition of said venoms into nanoparticles has an interesting potential as a safe drug delivery method.

Naja kaouthia is a medically important Asian snake from the Elapidae family. In Brazil, the Elapidae family is represented by the Micrurus genus, and the antielapidic serum is produced using M. corallinus and M. frontalis venoms. There are still barriers in the antielapidic serum production, such as breeding snakes from the Micrurus genus and the small amount of venom extracted from individuals. Due to evolutionary and venom composition resemblances between N. kaouthia and Micrurus genus, like the large amount of three-finger toxins (3FTx) and phospholipases A₂ (PLA₂), it is interesting to search for similar proteins in N. kaouthia’s venom as a possible alternative to enrich the pool of Micrurus’ venoms used in the antielapid serum production. The venom composition and function of 29 N. kaouthia individuals and three different Micrurus species, M. altirostris, M. lemniscatus and M. spixii, were evaluated by measurement of enzymatic activities of L-amino acid oxidase (LAAO), PLA₂ and proteolytic activity over azocasein, SDS-PAGE, RP-HPLC, ELISA and Western Blotting (using antielapidic and antinaja serums). Analyzing the results obtained, the N. kaouthia venom enzymatic activities were most like to the M. altirostris venom activities, which suggested similarities in venom composition. SDS-PAGE profile of the four species had differences in the high molecular weight bands and only quantitative variations in the region correspondent to PLA₂ and 3FTx, between 10 and 15 kDa. RP-HPLC profiles of the four species were severely different, the Micrurus species presenting more abundance of peaks. Finally, the Western Blotting showed cross reactivity between a band of approximately 10 kDa from M. lemniscatus venom and the antinaja serum and between two bands among 10 and 15 kDa in N. kaouthia venom and the antielapidic serum. So, the recognized bands in N. kaouthia, probably 3FTx and PLA2, have the potential to enrich the antielapid venom pool. Acknowledgements: This work was supported by FAPESP processes 2018/25786-0 and 2021/05405-5.

Venoms of snake species inhabiting distinct geographies exhibit significant intraspecific variation. For instance, remarkable biogeographic venom variation has been documented in the venoms of the common cobra (Naja naja) and Russell’s viper (Daboia russelii), two of India’s widely distributed snake species. The distinct ecologies and habitats across their range distribution may determine venom phenotypes and toxin abundances. Moreover, venom variation may also be contributed by inter-and intra-specific competition, distinct sexual ecologies, dietary shifts, varying predator pressures, and ontogenetic shifts in ecology. Here, we assess the ontogenetic variability in the venoms of Naja naja and Daboia russelii. Venoms collected from snakes of these two species across their developmental stages were analysed for their proteomic composition, biochemical activities, and lethality against a variety of prey species. The relative abundances of toxin families in D. russelii venoms significantly varied between the young and adult individuals. While the adult venoms were enriched with higher molecular weight proteases, increased abundances of lower molecular weight toxins, such as phospholipases, were recorded in the venoms of younger individuals. These compositional variations also correlated with the biochemical and pharmacological properties of these venoms. In addition, a light interference-based toxin-receptor binding experiment was also performed to understand the prey-specific neurotoxicity of the adult and juvenile N. naja venoms. The study, for the first time, comprehensively documents the similarities and differences in venoms of two medically important Indian snakes across their developmental stages. These results further augment our knowledge of the toxin repertoire evolution in these lineages.

Variation within species can result from adaptive processes in response to abiotic or biotic variables; however, the importance of neutral processes and population structure when considering intraspecific variation are often ignored. When investigating a trait that is assumed to be under selection, it is essential to test whether variation in the trait is concordant with genetic variation resulting from neutral demographic processes. When discordance is observed between trait variation and neutral variation, selection may be occurring and variables driving variation can be investigated. Here, we compared neutral genomic and venom expression divergence in the Red Diamond Rattlesnake (Crotalus ruber) to understand if and how biotic and/or biotic factors may be driving venom evolution. We assembled a high-quality reference genome of C. ruber to test for neutral variation and estimate venom gland transcriptome expression in multiple individuals. We found discordance between neutral genomic and venom expression variation, suggesting that selection is, at least in part, contributing to trait evolution in this system. We also found significant clinal variation in venom expression for two venom gene families: Snake Venom Metalloproteases and C-type lectins; however, the remaining six venom gene families did not demonstrate significant clinal variation. This suggests that the expression of these venom families is either not variable, or their variation is driven by other factors besides neutral processes. We conclude that abiotic factors are likely not as important in explaining venom variation and that venom variation is most likely affected by biotic variables in C. ruber.

Toxin-antitoxin (TA) systems are modules that are associated with some physiological processes of bacteria, such as the formation of persistent cells, biofilm formation, protection against bacteriophages and responses to stressful environments. In these modules, the toxin is related to the inhibition of physiological processes and the antitoxin provides protection to the cell against the toxin. In the genome of a hybrid pathogenic E. coli strain (atypical enteropathogenic E. coli and extraintestinal E. coli strain - BA1250) were found eight type II TA systems, and some of them with more than one copy. Only TA systems CcdB-CcdA, YhaV-PrlF, MazF-MazE, YoeB-YefM and PasI-PasT were present in one copy. Therefore by transcription analysis under stress conditions such as nutritional, oxidative, osmotic, acid and in antibiotic presence (ciprofloxacin and ampicillin) were analyzed either at log or stationary phase of BA1250 strain. We observed significant changes in the expression levels of the CcdB-CcdA, YhaV-PrlF, YoeB-YefM and PasT-PasI systems in the presence of nutritional stress at stationary phase. It’s worth mentioning that CcdB-CcdA and YoeB-YefM were the most affected systems at the log phase in the presence of antibiotics and nutritional stress. On the contrary, the MazF-MazE system none changes in the expression levels was observed. These data indicate that these four TA systems are related to the bacterial stress response, ongoing experiments will elucidate how these mechanisms may be involved with the pathogenicity of this hybrid strain.