Snake Venomics at the Crossroads Between Ecological and Clinical

Home , Philodryas olfersii, Rhabdophis tigrinus, Tachymenis

Venoms and Toxins Snake venomics at the crossroads between ecological

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Juan J. Calvete Venoms are complex secretions used for predatory and defensive purposes by a wide range of (Instituto de Biomedicina organisms. Venoms and venom production represent fascinating systems to study fundamental de Valencia, Spain) evolutionary processes. Understanding the evolution of venom generation demands the integration of the selective interactions and mechanisms, which transformed ordinary genes into deadly toxins, in the context of the natural history of the producing organism. Humans are not prey for any extant venomous creature on Earth, and thus human envenomings result from unexpected encounters with venomous animals, e.g., snakes. Research on snake venoms conducted on mammalian prey from an ecologically informed perspective is conceptually transferable to the clinic, highlighting the mutually enlightening relationship between evolutionary and translational venomics.

Brief introduction to the interactions generate coevolutionary dynamics through evolutionary context of snake venom an escalating arms race, characterized by asymmetrical selection between the predator’s toxic arsenal and the Venoms, and their associated venom-delivery systems, prey’s evolved counter-adaptative resistance mechanisms. represent innovations that have evolved independently Adaptations to ecosystems require evolutionary in a broad phylogenetic range of animal lineages across changes of both morphological and molecular all major phyla of the animal kingdom. phenotypic traits that maximize the organism’s Venom is an intrinsically ecological trait used by fitness in local environments, e.g., the success of a more than 250,000 species, for the purpose of subjugating snake foraging on preferred prey. Thus, unveiling the prey, deterring competitors or defending themselves evolutionary potential and history of those characters from predators. The reason for possessing toxic weaponry requires a deep knowledge of the patterns of functional is simple. Ancestral snakes, such as Titanoboa variation generated by spatially discrete selection cerrejonensis and its living relatives such as the boas, among individuals, populations and species underlying anacondas and pythons, use constriction to crush and adaptive changes. The genome of an organism contains eat animals by wrapping their mouths around their information of its full repertoire of genes, those that are prey and swallowing them whole. In contrast, venom actively transcribed, but also of the many additional represents a more sibylline means by which one can genetic features, such as introns, intergenic regions subdue potentially dangerous prey whilst minimizing and regulatory elements, that play pivotal roles in the the risk to self in any struggle. control of gene expression and in the physiology of Venom emerged as a key evolutionary innovation the organism. that underpinned the explosive radiation of caenophidian In addition, the genome also encodes traces of snakes, in the wake of the Cretaceous–Paleogene (around events from its evolutionary history, both from 66 million years ago). The evolutionary success of venom functionally failed recombinations and from those that is highlighted by the fact that venomous animals have passed the natural selection filter and contributed to the arisen in every ecosystem of our planet where organisms functional genome of the species. Hence, the sequencing compete for resources. Venom is also a useful defensive of snake genomes, but especially inter- and intra-specific strategy. Painful venoms are used to deter predators, comparative genomics will uncover a treasure trove of and there are many examples of Batesian mimicry by biological information to reconstruct the molecular which a harmless species protects itself from predators bases of the evolution of venom genes. Genomic data by deceitfully imitating the genuine aposematic warning is tremendously informative for phylogenies. It can be signal of a noxious species. Venomous predator–prey used to estimate rates of speciation and extinction, to

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reconstruct historical diversification scenarios, and to have been reported to be involved. This scenario implies Figure 1: A) Consensus link these to ecological and evolutionary factors, such the need to analyse individual venom proteomes, rather phylogeny of New World as climate or organismal traits, as well as population than pooled venoms in different contexts, in order to coral snakes highlighting genetics (phylogeography) and species delimitation. understand the spatio-temporal variability landscape the scattered distribution

Merging morphological and molecular datasets for fossil underlying the adaptations that drive intra-specific of PLA2-rich (blue) and the and extant taxa would also give a more complete view venom evolution. 3FTx-predominant (red) of the natural history of snakes. Genomic resources venom phenotypes. are currently only available for specific snake species; Snake venomics B) Geographic distribution however, the steady growth in annotated snake genomes of currently available represents the tip of an ‘-omic’ revolution. These will Venom proteomics, ‘venomics’, began to be applied micrurine venom play a fundamental role in addressing the connection modestly in the field of toxinology at the turn of the proteomes. The pie between genotype and phenotype for fitness-related century. To date, the venom proteomes of more than charts display the relative traits and more explicitly—the relative importance of 200 species and subspecies of snakes, particularly abundances of 3FTxs (res), structural changes in proteins, versus gene regulatory from the families Viperidae (340 species of true vipers PLA2s (red) and other toxins changes as the basis for adaptive variation in the and pit vipers) and Elapidae (360 species of elapids, (grey). Green triangles venom phenotype. for example, cobras, kraits, mambas and sea snakes) highlight the outlier venom However, despite being traits of moderate genetic (www.reptiledatabase.org) have been provided. The proteomes of Caatinga complexity in terms of the number of genes that field continues to expand at an accelerated expansion coral snakes; the red disc encode toxin families, within- and between-species pace, which is mainly due to analytical advances over identifies the streamlined venom variability in-space (geographic) and in-time the last decade. In particular, the combination of next- venom proteome of the (ontogenetic) seems to be a common feature at all generation transcriptomics and well-established bottom- aquatic coral snake, taxonomic levels. The mechanisms that generated such up mass spectrometry-based proteomics platforms, has M. surinamensis. biodiversity remain largely elusive, although genomic demonstrated capabilities for the rapid identification reorganizations and post-transcriptional regulation of and relative quantification of toxins in snake venoms in the expression patterns of messengers encoding toxins unprecedented detail.

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However, these figures still represent only approx- with bottom-up approaches, top-down venomics is imately 12–27% of the venom proteomes of front-fanged gaining momentum, particularly for the analysis of (solenoglypha and proteroglypha) snakes, and the venoms from species for which a homologous situation is much more extreme regarding the venoms of transcriptomic reference database is available. This rear-fanged (opistoglypha) snakes, where only a handful point is illustrated by the recent transcriptomics- of venoms have been characterized out of the approx- guided bottom-up and top-down study of the changes imately 700 species that produce venom. This bias undergone by the venom of the arboreal rear-fanged may be ascribed to the high incidence of human brown treesnake, Boiga irregularis, which correlate with morbidity and mortality from envenomation by front- a pronounced ontogenetic shift in prey preference, from fanged snakes, which inject venom into at least 1.8–2.7 a diet consisting almost exclusively of poikilothermic million people worldwide per year, with combined upper ectotherms (small lizards) in neonates, to both

estimates of mortality ranging from 80,000 to 138,000 ectotherms and homeoendothermic vertebrates (birds Downloaded from http://portlandpress.com/biochemist/article-pdf/41/6/28/862003/bio041060028.pdf by guest on 30 September 2021 deaths. On the other hand, although the large majority and mammals) being consumed by adults. of rear-fanged snakes (>2200 species in the family Colubridae) are unable to deliver sufficient quantities Identification of evolutionary of toxin to be lethal, at least three species (Dispholidus trends through clade venomics typus, Thelotornis capensis and Rhabdophis tigrinus) have caused human fatalities, and bites by two additional The identification of evolutionary trends across whole species (Philodryas olfersii and Tachymenis peruviana) genera, taxonomic clades and phylogenetic families is of have resulted in serious human envenomations, clearly increasing interest in venom analysis. indicating that the study of their venoms should not Unveiling the origin and phylogenetic distribution be further neglected. In addition, rear-fanged snakes of venom traits is key to understanding the underlying are a phylogenetically diverse collection of species that evolutionary processes (local adaptation, balancing feed on a variety of prey and show varying prey capture selection) and reconstructing the historical ecological strategies, from constriction to envenomation, and prey- constraints that moulded snake venoms to their present- specific toxins have been identified. It is hypothesized day variability. These traits include: that venoms of rear-fanged snakes, particularly those of • Paedomorphic traits, for example the retention of

species with highly specialized diets, may contain novel juvenile β-neurotoxic heterodimeric PLA2 crotoxin- prey-specific toxin genes, moulded by adaptive trophic rich venom phenotypes in adult South American evolution and exhibit unique pharmacological activities. Crotalus durissus species. Clearly, understanding the nature of the venoms of a • Ontogenetic traits, such as the age-dependent group of organisms (i.e., organisms that share a common transition from crotoxin-rich (type A) to ancestor), which includes the vast majority of ecological haemorrhagic PIII-metalloproteases-rich (type B) variants amongst extant snakes, is fundamental to venom in Costa Rican Crotalus simus. understanding venom evolution in advanced snakes. • Dichotomic traits, such as type A versus type B

However, differences in gland structure and size, as well venoms in North American Crotalus or PLA2-rich as fang morphology, have resulted in high-pressure and versus 3FTx-rich venoms across the genus Micrurus; low-pressure venom delivery systems in front-fanged dendrotoxin-rich versus 3FTx-predominant venoms and rear-fanged snakes. A derived consequence is that, in African mambas. in comparison to front-fanged snakes, venom extraction The overall picture, rather than the individual venom from rear-fanged snakes is more challenging, time- proteomes, provides hints for reconstructing the origin of consuming and generally results in significantly lower evolutionary trends. Moving from reference proteomes venom yields. to genus-wide and macroevolutionary venom pattern Research on venoms has been continuously enhanced recognition adds the required extra dimensionality for by advances in technology. Challenges remain to be solved comparative venomics to achieve this goal. in order to achieve a compact and automated platform At this point, it must be emphasized that the with which to routinely carry out comprehensive functional evolution of venoms is intimately linked quantitative analysis of all toxins present in a venom. to, and can only be understood in the context of, the However, the introduction in 2015 of highly sensitive and organismal ecology and dietary habits. To this end, resolutive ion-trap spectrometers for top-down venomics research questions should be formulated from an represents a turning point in snake venom analysis. ecologically informed perspective. For instance, the This technology allows the in-depth characterization of physiological state of the prey at the time of the encounter, proteins by sequentially confining, manipulating and which is in turn modulated by the ecosystem’s climate, fragmenting whole toxin ions inside the mass spectrometer. may impose restrictions on the selection of the type of Whether as a stand-alone workflow or in combination venom dominant in the snake population with which

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Ecological venomics Clinical toxinology (Prey subjugation) (Defensive bites in icted to non-prey animals) Natural Selection Downloaded from http://portlandpress.com/biochemist/article-pdf/41/6/28/862003/bio041060028.pdf by guest on 30 September 2021

Figure 2. Cartoon of the exclusive and overlapping domains of ecological venomics and clinical toxinology, with an emphasis on the mutually enlightening relationship between evolutionary and translational venomics. The recognition of adaptive variations that have evolved via natural selection and enhance the snake foraging success on preferred prey, belongs exclusively to the field of ecological venomics, whereas the identification of the relevant toxins that should be neutralized to reverse the symptoms of snakebites inflicted to non-prey animals, such as pets, farm animals or humans, falls into the exclusive scope of clinical toxinology.

it has a shared ecological and evolutionary history. the heterodimeric PLA2 Mojave toxin, acts non- Mojave rattlesnakes (Crotalus scutulatus) exhibit two enzymatically and is thus less affected by temperature. distinct venom phenotypes, type A (neurotoxic) and type Each venom type possesses a slight competitive edge B (haemotoxic), which are geographically segregated over the other in its distribution range. In areas with in populations that exhibit no discernible difference milder winters, type B venom may be a higher effective in diet. However, strong association has been found strategy for rattlesnakes, whereas in habitats with more between venom type and climate, in which the type A extreme temperature variations, snakes with a rate- neurotoxic venom was found in regions with cooler limited toxin arsenal may be outcompeted by those winters and higher rainfall. The effectiveness of type B using a temperature invariant, neurotoxic strategy. venom appeared to be strongly dependent on metallo- The venoms from coral snakes of theMicrurus genus proteinases, whose enzymatic activity is rate limited that have been proteomically characterized to date by temperature; on the other hand, the most lethal also exhibit a puzzling phenotypic dichotomy. This is component of the type A β-neurotoxic venom, namely characterized by the toxin arsenal being dominated,

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either by pre-synaptically acting PLA2s or post-synaptic toxins and identify venoms whose components act 3FTxs, and a general, but imperfect, distributional additively (e.g., Aspidelaps spp.) or synergistically pattern of these venom phenotypes along the North- (Micropechis ikaheka). In most studies, however, venom South axis of the American continent. Due to their toxicity assessments are based upon non-native ‘model’ cryptozoic and fossorial habits, natural history data for laboratory surrogate prey species, which are not most micrurine species, such as diet or feeding behaviour, consumed in the wild by the venomous predator. are scarce. Nevertheless, evidence for strong positive Nonetheless, toxicovenomic analyses performed on selection for the pre-synaptic 3FTx and post synaptic mammalian prey has something to offer clinical

PLA2 toxin families has been reported in M. fulvius toxinology. Toxins bearing the highest mammalian venom, suggesting that dichotomic micrurine venoms prey-incapacitation activity are often also the most may have been shaped through balancing selection. medically important molecules in the context of a

In this evolutionary scenario, fitness is dependent human envenoming, i.e., those toxins that need to be Downloaded from http://portlandpress.com/biochemist/article-pdf/41/6/28/862003/bio041060028.pdf by guest on 30 September 2021 on the frequencies of the two lethal venom toxin classes neutralized to reverse the effects of the venom. In this

involved, with the hetero- (i.e., 3FTx + PLA2) phenotype sense, ecological venomics and clinical toxinology can having an advantage over the homo-phenotypes. M. be mutually enlightening, providing that predictions ibiboboca and micrurine taxa of uncertain taxonomy inferred from toxicovenomics evidence gathered from found within the Brazilian Caatinga biomass express laboratory animals are testable and falsifiable.

PLA2-rich venom phenotypes. The finding of PLA2 To this end, ‘antivenomics’, a proteomics-based outliers within a 3FTx biogeographic spot may affinity chromatography protocol to quantify the extent indicate the occurrence of local adaptive evolution to of cross-reactivity of antivenoms against homologous the unique Caatinga ecosystem, and highlights the often and heterologous venoms, is available. In its current underappreciated consequences of phenotypic plasticity of format, the so-called ‘third-generation antivenomics’ balanced phenotypes in ecological speciation (Figure 1). platform, antivenomics reveals information on the relative Conversely, streamlined phenotypes may be immunogenicity of the resolved venom components, and indicative of strong selection in snakes that have the maximal capacity of the antivenom antibodies to acquired morphological adaptations towards very immunodeplete each of the venom toxins. The combination specific dietary regimes. An example is Micrurus of antivenomics and in vivo neutralization tests (e.g.,

surinamensis, a semi-aquatic coral snake, endowed with the Median Effective Dose, ED50) constitutes a powerful an extreme venom arsenal dominated by 3FTxs (>95%), toolbox for assessing the neutralizing efficacy of an which is highly toxic to banded knifefish, Gymntus antivenom, and hence a convenient and easy way to test

carapo (LD50 of 0.01 µg of total venom/g of fish body toxicovenomics predictions. weight), one of the most preferred prey species of this snake. In comparison to terrestrial congenerics, M. The overlapping domains of ecological surinamensis exhibits morphological modifications that venomics and clinical toxinology reflect adaptations to life in an aquatic environment. In line with this hypothesis, similar adaptations have Snakebite envenoming is a disease of poverty, which convergently evolved in the Amazon water snake Hydrops annually kills more people than any other disease martii (Colubridae, Dipsadinae), known to consume on the neglected tropical diseases (NTDs) list of the elongated fishes of the same taxa asM. surinamensis. World Health Organization (WHO), residing in some of the world’s most disadvantaged subsistence farming Toxicovenomics communities in rural impoverished African, Asian and Latin American regions. These events leave over 300,000 Recent studies have employed a strategy combining surviving victims with permanent physical disabilities, compositional analysis and functional assays, referred to stigmatizing disfigurements and chronic mental as ‘toxicovenomics’ at the 18th World Congress of the morbidity. Antivenoms constitute the only scientifically International Society on Toxinology (IST) held in Oxford validated therapy for snakebite envenomings, provided in 2015. The essence of the toxicovenomics approach they are safe, effective, affordable, accessible and lies in screening the resolved profile of venom fractions administered appropriately. provided by the venomics workflow for specific toxic The wide spectrum of pathological and patho- activities. The combination of a toxin’s incapacitation physiological manifestations of envenomings, due to the potency and abundance into a toxicity score allows a concerted actions of the unpredictable venom variability more realistic view of the relevance of particular toxins across the phylogeny and distribution range of extant in prey incapacitation than toxic potency alone. snakes, represents a great challenge for the development Applying toxicovenomics on natural prey serves and preclinical evaluation of the efficacy of antivenoms. to rank the adaptive potential of individual venom From a biotechnological standpoint, this goal requires

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knowing the phylogeographical patterns of present-day neutralize the deleterious activities of individual venom snake venoms, identifying their most medically important toxins (‘antivenomics’) have conceptually the same molecules in the context of human envenoming, and applicability in clinical toxinology as in the ecological assessing the specific and para-specific efficacy of current context, highlighting the mutually enlightening antivenoms against the different medically relevant relationship between evolutionary and translational snake venoms. venomics. From an evolutionary ecology perspective, human The recognition of adaptive variations that have snake envenomings result from defensive bites inflicted evolved via natural selection and enhance the snake by sympatric venomous snakes when snakes and humans foraging success on preferred prey, belongs exclusively to have a chance encounter in their shared natural the field of ecological venomics, whereas the identification environment (Figure 2). In this context, the same ‘-omics’ of the relevant toxins that should be neutralized to reverse Downloaded from http://portlandpress.com/biochemist/article-pdf/41/6/28/862003/bio041060028.pdf by guest on 30 September 2021 strategies applied for unravelling the composition the symptoms of snakebites inflicted to non-prey animals, of venoms (‘venomics’), the adaptive potential of such as pets, farm animals or humans, falls into the individual venom toxins towards mammalian prey exclusive scope of clinical toxinology. ■ (‘toxicovenomics’) and the efficacy of antivenoms to