Molecular Evolution, Functional Variation, and Proposed Nomenclature of the Gene Family That Includes Sphingomyelinase D in Sicariid Spider Venoms

Keywords: Loxosceles, Sicarius, duplication, toxin

Table 1.

Summary of All Known SMaseD DNA and Protein Sequences Published to Date with Summaries of Enzyme Activity, Dermonecrotic Activity, and Studies That Have Been Conducted with Recombinant Forms of the Protein.

Taxonomic Sampling and Collection

The representation of taxa in different analyses is summarized in table 2. Our goal was to capture the breadth of diversity within Sicariidae, guided by previous phylogenetic work (Binford et al. 2008, fig. 1). To do this, we include at least one representative of every described species group in Loxosceles, representatives of each geographic region to which Sicarius is native, and the most appropriate and available outgroup for each particular analysis (table 2). Spiders in the genus Drymusa are putative close relatives of sicariids that have been previously shown to not express SMase D in their venom (Binford and Wells 2003).

All spiders were collected in the field by G.J.B. and colleagues. Details of collecting localities are available from G.J.B. by request. We restricted analyses to mature individuals to allow for proper species-level confirmation using morphology. We also retained legs of spiders for genomic DNA isolation to help with confirmation of species status. If animals were not mature when collected, we reared them to maturity in the laboratory at 35% humidity, 24 °C. Species-level systematics of some taxa we include is unclear, and their taxonomic nomenclature is being revised. This is particularly true of African sicariids. For this work, we follow the same nomenclatural framework used in Binford et al. (2008). Voucher specimens are maintained in the collection of G.J.B. and will be submitted to the California Academy of Sciences, and duplicates from the same populations will be sent to National Museums from the countries of origin upon completion of our work.

SMase D Homolog Sequencing and Analyses

Comparative Protein Composition and SMase D Assays

Amino Acid Sequence Conservation

We analyzed amino acid conservation among a nonredundant set (no two members having higher than 95% amino acid sequence identity) of venom-expressed members of the SicTox gene family by visual inspection of alignments. Conservation levels were also mapped onto the solvent-exposed surface of Loxosceles laeta SMase D (PDB ID 1XX1, chain A) (Murakami et al. 2005) using the Multalign Viewer utility within the program Chimera (Pettersen et al. 2004).

Table 3.

Taxa Screened for Venom-Expressed SMase D Homologs Organized by Species Group

Rationale for Proposed Nomenclature

Our nomenclatural system follows the general guidelines established by the Human Genome Organization (HUGO) gene nomenclatural committee (HGNC) (http://www.genenames.org/). This committee encourages using symbols to distinguish phylogenetically defined stem or root groupings and then using a hierarchical numbering system to distinguish individual members. Rather than create a rigid structure our goal is to establish a general working scaffold within which newly discovered homologs may easily be placed in a hierarchical structure representative of degree of relatedness. This will serve to standardize discussions in the literature and facilitate efficient characterization of structure and function of this gene family by targeting divergent lineages for further analyses. Recent work by Kalapothakis et al. (2007) provides an excellent starting place. The sequences used in their analysis are anchors in our phylogeny that inform hypotheses of the functional evolution within this family. A complete translation of previous published names for toxins in this family to the SicTox nomenclature is in table 1,and a complete list of all known SicTox family members is available in supplemental table 1, Supplementary Material online. We detail the nomenclature and the logic behind it as we describe the structure of diversity of the group.

Isolation and Identification of SMase D Paralogs

We isolated 329 sequences homologous to SMase D, 291 of which were full length. Of those, 190 were nonredundant at the nucleotide level (GenBank Accession numbers FJ17340–FJ17529, detailed in Supplemental table 1, Supplementary Material online), and 168 were nonredundant at the amino acid level. Of the distinct nucleotide sequences, 143 are from 16 species of Loxosceles, and 47 are from 5 species of Sicarius (table 3). Our PCR methods did not amplify any genes from our outgroup taxon, Drymusa serrana. There was a large range in the number of SMase D homologs discovered from within a single mRNA pool. We were particularly successful at amplifying diverse SMase D homologs from Loxosceles hirsuta, Loxosceles apachea, L. arizonica, S sp cf damarensis (Oorlogskloof), and Sicarius peruensis (Olmos) (table 3). The number of distinct paralogs that we isolated from within a single species ranged from 1 to a minimum of 12 (defined by 98% identity) in L. arizonica. Our differential recovery of sequences and paralogs among taxa is not easily explained by numbers of individual spiders whose venom glands were combined in the cDNA pool or by greater success in amplifying cDNAs from species closely related to the source species of the amino acid sequences we used to design degenerate primers (table 3). The primer sphing1f successfully amplified homologs across all taxa, whereas Lpara amplified 12 sequences nonredundant at the nucleotide and amino acid levels (1 in the α clade, L. apachea αIB1b, and 11 in the β clade, all from African Loxosceles in the spinulosa species group).

Phylogenetic Patterns and Delineation of Major Clades

Bayesian analyses resolved two distinct clades that we label α and β, with posterior probabilities of 1.0 and 0.99, respectively (fig. 2). NJ collapses the β clade into two clades, each with low bootstrap support, as an unresolved basal polytomy with the α clade. The average pairwise amino acid distance p-distance between the α and β clades is 0.537. We distinguish major lineages (groups) within the α and β clades by roman numerals (I–VII in α) and (I–III in β) (fig. 2). We delineate groups based on strong posterior probability support of monophyly (fig. 2) and high percent divergences among them (table 4). Some groups either include a large number of sequences structured into well-supported groups (e.g., αI), or include sequences that are highly divergent (e.g., βI & βII). We delineate subgroups within these groups with capital letters (A–C in αI, A–F in βI, and A–B in βII). The number of nonredundant amino acid sequences included in groups and subgroups ranges from 1 (αVI, βIC) to 47 (αIB).

Duplication History in SicTox Gene Family

Although the SicTox gene tree has some clustering of sequences into clades consistent with species groups, duplications complicate this relationship. Recent duplications are most apparent in groups of closely related species for which we have extensive recovery of gene copies. This is particularly true for αIA and αIB members of the spadicea and reclusa groups, respectively. Our sampling includes the only three described species in the spadicea group, and there is solid support of spadicea and hirsuta being each other's closest relatives with intermedia sister to that pair (fig. 1). Within the αIA clade, there have been at least three duplication events since the MRCA of the spadecia group (fig. 2). Estimates of the age of the MRCA of the spadecia group range between 48 and 34 Ma (unpublished, methods as in Binford et al. 2008), meaning the minimally two duplications between the MRCA and hirsuta occurred within this time frame. The αIB clade includes only members of the North American reclusa group. Patterns of relationships among terminal paralog groups within αIB mirror species relationships in the reclusa group (fig. 1) and contain clear duplication events (fig. 2). An example is in the phylogenetic grade that includes L arizonica αIB2a, αIB2b, and αIB2c.

Panning out to a broad description of the history of duplications and losses in this lineage, reconciliation analyses (Notung) of a species tree (subset of fig. 1) with an SMase D gene tree (NJ) that includes all unique amino acid sequences (all taxa in fig. 1 except Ixodes) estimated 110 duplications and 84 losses (D/L score = 249.0). This likely overestimates duplications because of the inclusion of allelic variants in monophyletic terminal intraspecific groups. Repeating the analysis including only terminal paralogs defined by minimum amino acid divergence thresholds of 2% (105 sequences) and 5% (75 sequences) estimated 44 duplication events and 71 losses (D/L score = 137.0), and 30 duplications and 74 losses (D/L score = 119.0), respectively. The 5% threshold resulted in monophyletic, terminal intraspecific sets of sequences being reduced to single representatives and therefore eliminates the possibility of allelic variants but also will exclude very recent duplications.

It has been hypothesized that gene families encoding spider venom toxins are evolving via mechanisms similar to those driving Conus toxin diversification in which there is rapid duplication followed by adaptive divergence (Sollod et al. 2005; Escoubas 2006). In spiders, rapid duplication rates in toxin gene families are assumed based on large numbers of paralogs expressed in venoms (King et al. 2002; Sollod et al. 2005; Escoubas 2006; Jiang et al. 2008). Little work in any toxin lineage has attempted to quantify duplication rates, and reasonably so, given a long list of limitations including 1) lack of comprehensive knowledge of gene family size that would require a sequenced genome; 2) the complicating influences of concerted evolution and the evolution of expression patterns (Duda and Remigio 2008); and 3) few analyses with dense taxon sampling of genes among closely related species of known relationships and with known divergence times. Of these, our work only overcomes the limitation of having a species tree and broad SicTox gene sampling. Thus, although preliminary, estimates from reconciliation serve as a starting point for discussion of general patterns of duplication rates in venom evolution.

To translate our reconciliation results to inferred duplication rates, if duplications were evenly distributed across the 25 taxa in our analysis, the average species would have undergone 1.2 (5% threshold) to 4.4 (all data included) duplications in the evolutionary time frame encompassed. This assumption is unlikely to be true given the wide range of paralog numbers we recovered across taxa. Thus, these estimates are conservative. Molecular dating analyses and biogeographic patterns support the MRCA of Sicariidae predating the separation of South America and Africa meaning a conservative estimate of minimum age of the MRCA is 95 Ma and a maximum estimate is 157 Ma (Binford et al. 2008). This translates to 0.0126–0.0463 duplications per million years for the minimum age of sicariids and 0.0076–0.0280 duplications per million years for the maximum age.

These estimates of duplication rates are within ranges of estimates under the best of circumstances using comparisons of whole sequenced genomes, but minimizing the confounding influence of gene conversion by constraining analyses to include only pairs of duplicate genes. These include 0.028, 0.0014, and 0.024 duplications per million years in yeast, Drosophila and Caenorhabditis elegans, respectively (Gu et al. 2002 assuming a molecular clock), 0.01–0.06 duplications per billion years in yeast (not assuming molecular clock, Gao and Innan 2004), and 0.009 duplications per million years in humans (Lynch and Conery 2003). We can estimate duplication rates from a venom toxin in the four-loop conotoxin lineage. At least seven duplications have occurred since the divergence of Conus abbreviatus and Conus lividus (Duda and Palumbi 1999) whose MRCA is estimated to have lived approximately 27 Ma (Duda and Kohn 2005), which results in an average duplication rate of 0.269 duplications per million years. Within the spadecia group described above, estimated duplication rates of SicTox would be between 0.041 and 0.071 per million years, which is faster than any of our estimates from reconciliation and faster than rigorous estimates from model systems but slower than the estimates for Conus four-loop toxins.

A final notable pattern is that single species (L. intermedia, L. boneti, and L. laeta) have SicTox paralogs in both the α and β clades. These are descendents of a duplication event that must predate the MRCA of Sicariidae and thus the putative origin of SMase D as a venom toxin in sicariids (Binford and Wells 2003).

Evidence of Purifying Selection, Episodic Positive Directional Selection and Positively Selected Sites

LRTs indicate significant improvements in log likelihood values under the free-ratios model relative to the fixed ratios model for both the α and the β clades (table 5). They also support significant deviation of ω from 1. Estimated ω values under the fixed model (constrained to a single value across the lineage) were considerably less than 1 (0.250 and 0.193 for the α and β clades, respectively), consistent with predominantly purifying selection in the lineage. Reconstructed estimates of ω along branches under the free-ratios model are generally less than 1; however, scattered branches within both the α and the β clades have ω values greater than 1 (fig. 2). The significantly better fit of the free-ratios model and branches with ω > 1 is consistent with a pattern of episodic positive selection acting in this lineage. Branches with estimated ω > 1 are particularly concentrated in the αIB and the βIA and B clades.

Site model analyses support significant improvement of log likelihood values in the α clade under the M2a and M8 models relative to their nested models. In this clade, 1.8% and 5.1% of amino acids are identified as evolving under positive selection by M2a and M8 models, respectively (table 5). Thirteen amino acids were identified by Bayes Empirical Bayes (BEB) analyses (Yang et al. 2005) (table 5) as candidates for positively selected sites, three with posterior probabilities > 0.95 (18, 151, and 204, numbering according to PDB 1xx1 L laeta H17, αIII1i). Some of the weakly supported amino acids are near active sites in the protein; however, they all appear to be solvent-exposed positions that are neither in the active site nor in the plug region. We are investigating possible functional roles for these residues.

Analyses of the β clade did not detect significant improvement in models that support amino acids that are under positive selection across the lineage (table 5). However, BEB analyses under the M8 model identified three sites, 39, 60, and 259 as candidates for evolving under positive selection in this clade but with low posterior probabilities (ranging from 0.55 to 0.63) (table 5). Like the residues in the α clade, these are all solvent exposed, and their functional relevance is unclear. As we fill in our data set with more thorough taxon sampling and more resolution in functional diversity, we will pursue branch-site analyses that may be more sensitive at detecting amino acids that are under selection in particular lineages.

Major Species Lineages Vary in SMase D-Sized Proteins in Venoms and in SMase D Activity

Qualitative comparative protein composition data and quantitative SMase D enzyme activity data allow a preliminarily view of how differences in expression of SicTox gene family members may influence differences in excreted venoms. They also add to a growing body of comparative data of whole crude venoms (table 1 and references therein).

Expressed proteins from members of this gene family are between 31 and 35 kDa (see table 1 references). Using comparative SDS-PAGE, we detected proteins in this size range from all Loxosceles and Sicarius but not in either African or Argentine Drymusa (fig. 3b). Variation among taxa that have proteins in this size region correlates to some degree with species relatedness and patterns of relationships of SicTox gene family members. Among venoms from North America, L. reclusa has a concentrated region of proteins around 32 kDa, whereas other reclusa species group members from the desert southwest of the United States (L. arizonica, L. apachea, Loxosceles sabina, and Loxosceles deserta), have a broader range of proteins in that region (∼31–34 kDa) (fig. 3a). Banding patterns of L. laeta are strikingly different from all other New World venoms with the most dense banding at 31 kDa and a reduction in proteins larger than that (fig. 3a and c). The differences in banding between L. intermedia and L. hirsuta are notable given the putative close relationship between these species (fig. 3a). Banding patterns for the African species Loxosceles vonwredei are more similar to New World species than they are to the African spinulosa group (fig. 3a), which is consistent with patterns of species relationships (Binford et al. 2008, fig. 1). Venoms from the African spinulosa clade (from which we have only isolated SicTox genes in the β clade) tend to have higher molecular weight proteins than other Loxosceles venoms. Venom profiles from Sicarius, again with isolated SicTox genes only from the β clade, have notable differences between New World and Old World species (fig. 3b and c). Venoms from African Sicarius have a higher concentration of larger molecular weight proteins, comparable with the African Loxosceles venoms, whereas the South American Sicarius have a region of dense proteins at ∼31 kDa. This apparent size variation could be influenced by posttranslational modification (glycosylation), but it likely also reflects differences in the expressed SicTox genes. Disentangling these relative influences on protein variation will require paralog-specific expressions and Westerns, and a better understanding of posttranslational modifications.

We assayed crude venoms from 36 taxa for SMase D activity (table 2). Sicarius species from Africa, all Loxosceles from the Americas, and Loxosceles rufescens have high SMase D activity using 0.5 or 1.0 μg of crude venom, whereas activity from venoms from African Loxosceles is generally lower (fig. 4a), and there is no activity in Drymusa venom. All of these patterns are consistent with previous work (Binford and Wells 2003). However, we detected little or no activity at these concentrations in venoms from South and Central American Sicarius (fig. 4a). When we repeated the experiment for South American Sicarius with concentrations of crude venom that were increased by two orders of magnitude (50 μg) enzyme activity increased but was never as high as in North American Loxosceles or South African Sicarius assayed at the original low concentrations (fig. 4b). The increase in SMase D activity with increasing amounts of venom from Argentine Sicarius terrosus (Quijades) and Sicarius patagonicus may reflect differences in substrate specificity of expressed SicTox members that results in a reduced binding efficacy for sphingomyelin. The fact that African Sicarius have strong SMase D signals and homologs for these species have only been recovered from the β clade suggests that at least some proteins in the β clade have strong efficacy for hydrolyzing sphingomyelin.

A growing body of work is indicating that defining the activities of venom-expressed members of this gene family as SMase D is inappropriately narrow, and there is variation in substrate specificity among homologs (Tambourgi et al. 1998; Lee and Lynch 2005; Murakami et al. 2005, da Silveira et al. 2006). Thus, our SMase D assay is a narrow assessment of the important biological activity in these venoms. Nonetheless, these assays may help identify patterns of variation in crude venom function that can focus further work to identify features of the molecules that are associated with the evolution of functional specificity. One pattern that emerges from the integration of comparative analyses of crude venoms, the gene family analysis, and published characterizations of expression products of SicTox genes is evidence of evolution of functional specificity in members of the SicTox β clade. For identifying specific changes in the molecule responsible for functional evolution, it may be particularly fruitful to investigate functional differences among paralogs from Sicarius. Given the striking difference in SMase D activity between African and American Sicarius, it is somewhat surprising that for every paralog we recovered from African Sicarius, we found orthologs in American Sicarius. In fact, our methods recovered one paralog in American Sicarius (the βIB lineage) for which we did not find a candidate ortholog in African Sicarius. Given the support for the sister–taxon relationship with βIA, and the reduced/low SMase D activity in American Sicarius, S. peruensis and S. terrosus βIB genes are good candidates of genes that have a function other than SMase D. Moreover, high levels of SMase D activity in African Sicarius suggest that, unless our method has missed capturing mRNAs of expressed α clade genes in these species, one of the other β clade paralogs must be SMase D active.

Patterns of Sequence and Structural Conservation

The structure solved for SicTox gene family member L. laeta αIII1 (H17) (Murakami et al. 2005) allows us to analyze sequence conservation in the context of structural position and proposed active sites (fig. 5). The most conserved solvent-exposed residues are at the two openings of the barrel, either in the vicinity of the active site at the top of the barrel, or in and around a previously described “plug motif” (Cordes and Binford 2006) that caps the bottom. In the active site, all residues proposed by Murakami et al. (2005, 2006) to be directly involved in phosphate or Mg²⁺ binding (His 12, Glu 32, Asp 34, His 47, Asp 91, Lys 93, and Trp 230; fig.5b in green) are >98% conserved across all homologs. An additional set of surface residues near the putative phosphate binding site (Pro 50, Cys 51, Asp 52, and Asn 252; fig. 5b in orange) also shows >98% conservation. Two of these (Asp 52 and Asn 252) are directly hydrogen bonded to His 12. Met 250 and Tyr 228 (fig. 5b in yellow), which line a deep cleft beneath the phosphate site, are also >98% conserved. Many of the residues listed above have been proposed to play some significant role in chemical catalysis; the remainder could be important for general aspects of substrate binding. The role of residues in the plug motif remains unknown, but their conservation across all SicTox homologs as well as in Ixodes and Corynebacterium relatives (Cordes and Binford 2006) suggests some vital role either in function or maintenance of structural integrity and stability.

The distal side of the active site pocket (to the right of the Mg²⁺ and sulfate ions in fig 5a) contains a deep cleft whose role in substrate binding and/or catalysis is unclear at this point. Murakami et al. (2006) proposed that certain residues in this area, such as Pro 134, might contribute to substrate specificity. Interestingly, there is a contiguous set of residues in this cleft that shows >95% conservation within the α clade (Val 89, Ser 132, Pro 134, Asp 164, Ser 166, and Ser 195; see fig. 5b in salmon and fig. 5c), but subclade-specific sequence variation within the β clade. We hypothesize that these residues are involved in specific aspects of substrate binding, and consequently, we predict that members of the α clade will show relatively conserved substrate specificity profiles; by contrast, variation in these residues within the β clade may lead to more variable substrate specificity profiles among this group. It must be noted, however, that most of these residues also differ between the α clade and homologs in Corynebacterium (fig. 5c) even though both proteins are known to hydrolyze sphingomyelin substrates.

Summary of Inferred Evolutionary Mechanisms Influencing SicTox

Together our data indicate that evolutionary dynamics of the SicTox gene family are similar in some aspects and different in others from the emerging picture of evolutionary dynamics of other venom toxin lineages. Frequent duplications are consistent with a birth-and-death model of evolution (review in Nei and Rooney 2005) that has been described for other venom toxin families (e.g., Duda and Palumbi 1999; Espiritu et al. 2001; Fry et al. 2003; Lynch 2007); however, our estimates of SicTox duplications are lower than estimates for small peptide neurotoxins in Conus venoms. Once duplications occur, our data are consistent with purifying selection in the SicTox lineage with episodic directional selection. However, our data do not support the same level of high d_N/d_S values documented for other toxin lineages (e.g., Duda and Palumbi 1999; Kini and Chan 1999; Lynch 2007). In fact, recent estimates of average ω of 1.28 for the toxic enzyme PLA2s from snake venoms (Lynch 2007) are an order of magnitude higher than our estimates (table 5). The general pattern of SicTox genes having possibly lower duplication rates and lower levels of diversifying selection than other venom toxins is provocative with respect to understanding general principles that influence venom evolution. These patterns invite the consideration that some of the evolution of copy number may be best described under a model of genomic drift (Nogawa et al. 2007) in which the absolute number of paralogs is of little adaptive consequence. The little we know about the genome structure of SicTox is consistent with the genes lying in a region of high recombination within the genome. The coding region of SicTox genes in L. arizonica is compartmentalized into five exons, some of which are separated by large introns (Binford et al. 2005). Moreover, we have evidence that intron–exon boundaries are not conserved among paralogs (Binford GJ, unpublished data).

One limitation for understanding SicTox evolution is a lack of understanding of the functional role of these toxins in the complex dynamics of immobilization and/or digestion of arthropod prey. Recent expressed sequence tag analyses indicate that SicTox homologs are the most abundant transcripts in venoms of L. laeta (Fernandes-Pedrosa et al. 2008), consistent with an important functional role. Although a cytotoxic role is apparent, recent work indicates that SMase D and other phospholipase D can inhibit ion channels because of an interaction between the channel and the head groups of membrane phospholipids (Ramu et al. 2007; Xu et al. 2008). Thus, SicTox proteins may also have a neurotoxic effect on prey. As we gain insight into divergence in sequence, structure, and function more focused analyses of particular lineages and regions of the protein that have undergone directional selection will help clarify evolutionary dynamics in this lineage.

Relevance for Understanding Risks of Bites and Development of Treatments

Although the functional role of SicTox genes in prey capture remains unclear, the central role of SMase D activity in the pathology of human envenomation is well documented. The phylogenetic structure and diversity and insight into evolutionary dynamics of SicTox gene family members provides a framework for understanding the distribution of risks associated with bites from spiders in the sicariid lineage and for strategies of developing broadly effective treatments for bites.

The distribution of risks associated with bites of diverse species should be a function of the distribution of toxins in the venom of these species. The diverse set of SicTox homologs makes this pattern complex. It is clear that some SicTox gene family members are sufficient causative agents for causing dermonecrosis (table 1 and references therein), but there is much to learn about the role of the non-SMase D active members in the clinical syndrome of human envenomation. In particular, there is little understanding of the contribution of genes in the β clade. Bites from species of African Loxosceles from the spinulosa group and African Sicarius cause dermonecrotic lesions, and Sicarius envenomation can be particularly damaging (Newlands and Atkinson 1988; Van Aswegen et al. 1997). From both of these lineages, we only recovered genes in the β clade. There are no published records of the effects of bites from New World Sicarius; however, injection assays of venom into mice have yielded no lesions (Alegre et al. 1977). Comparative bioassays of crude venoms and expression products using well-established rabbit models will provide better estimates of whether or not reduced SMase D activity in South American Sicarius venoms correlates with potential risk of bites. Results of these analyses will be particularly informative about whether or not SMase D activity is necessary for causing dermonecrosis, and the contribution of other SicTox gene functions to the necrotic syndrome.

Much work has focused on patterns of antigenic cross-reactivity in venoms within Loxosceles with the goal of developing antibody-based treatments of bites (Barbaro et al. 1996, 2005; Gomez et al. 2001; Ramos-Cerrillo et al. 2004: Olvera et al. 2006; de Roodt et al. 2007). To date, these analyses have been restricted to a subset of New World Loxosceles. The data presented here may help direct future work to produce treatments that are broadly, perhaps even globally, effective for treating sicariid envenomations. One important example is the phylogenetic placement of the L. rufescens SicTox genes. Loxosceles rufescens is the most cosmopolitan of all Loxosceles species. They are native to Mediterranean Europe and have recently dispersed to all major continents. The placement of L. rufescens SicTox genes in the α clade make it reasonable to predict that antivenoms developed for North and South American Loxosceles (Barbaro et al. 2005; Olvera et al. 2006) may be effective for treating L. rufescens bites. This work may also help with the possibility of designing a treatment that is effective for bites of African Loxosceles spinulosa group members and Sicarius bites. The apparent lack of α clade genes in venoms of African spinulosa and Sicarius suggests that a divergent set of genes beyond those that have been well characterized (table 1) is responsible for the clinical effects of bites of these species. For any treatment to be globally effective, antibodies raised against a diverse set of β clade genes will likely also be necessary.

[Supplementary Data]

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