Not so Dangerous After All? Venom Composition and Potency of the Pholcid (Daddy Long-Leg) Spider Physocyclus mexicanus

Pholcid spiders (Araneae: Pholcidae), officially "cellar spiders" but popularly known as "daddy long-legs," are renown for the potential of deadly toxic venom, even though venom composition and potency has never formally been studied. Here we detail the venom composition of male Physocyclus mexicanus using proteomic analyses and venom-gland transcriptomes ("venomics"). We also analyze the venom's potency on insects, and assemble available evidence regarding mammalian toxicity. The majority of the venom (51% of tryptic polypeptides and 62% of unique tryptic peptides) consists of proteins homologous to known venom toxins including enzymes (astacin metalloproteases, serine proteases and metalloendopeptidases, particularly neprilysins) and venom peptide neurotoxins. We identify 17 new groups of peptides (U1-17-PHTX) most of which are homologs of known venom peptides and are predicted to have an inhibitor cysteine knot fold; of these, 13 are confirmed in the proteome. Neprilysins (M13 peptidases), and astacins (M12 peptidases) are the most abundant venom proteins, respectively representing 15 and 11% of the individual proteins and 32 and 20% of the tryptic peptides detected in crude venom. Comparative evidence suggests that the neprilysin gene family is expressed in venoms across a range of spider taxa, but has undergone an expansion in the venoms of pholcids and may play a central functional role in these spiders. Bioassays of crude venoms on crickets resulted in an effective paralytic dose of 3.9 mu g/g, which is comparable to that of crude venoms of Plectreurys tristis and other Synspermiata taxa. However, crickets exhibit flaccid paralysis and regions of darkening that are not observed after P. tristis envenomation. Documented bites on humans make clear that while these spiders can bite, the typical result is a mild sting with no long-lasting effects. Together, the evidence we present indicates pholcid venoms are a source of interesting new peptides and proteins, and effects of bites on humans and other mammals are inconsequential.National Institute of Health [R15-GM-097696-01]; Lewis Clark College; Lewis & Clark students SophiaOpen access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

MALDI-TOF mass spectra for unlabeled S64

<p>(<b>left</b>) <b>and S67</b> (<b>right</b>)<b>.</b> The predicted monoisotopic masses for fully oxidized S64 are 3558.3 Da for the [M+H]⁺ ion and 1779.69 Da for the [M+2H]²⁺ ion. Likewise, the predicted mass for fully oxidized S67 is 2121.41 Da for the [M+2H]²⁺ ion. The appearance of peaks at these masses indicates that the cysteines are oxidized for both S64 and S67. The additional peaks seen to the left of the [M+2H]²⁺ peak in the S67 mass spectrum correspond to ions that have dehydrated. These peaks are also present in the S64 spectrum but are difficult to discern due to the much wider scale used for this spectrum.</p

Sequence alignment for mature toxin sequences.

<p>This figure displays the mature toxin sequences for S64 and S67 aligned with representative toxins found using a BLASTp search using the Arachnoserver toxin peptide database. Sequences were aligned using ClustalX 2.1 and visualized using JalView 2.7. The coloring makes use of the default ClustalX color scheme, which is a function of sequence identity and amino acid type.</p

Sexually Dimorphic Venom Proteins in Long-Jawed Orb-Weaving Spiders ( Tetragnatha) Comprise Novel Gene Families

Venom has been associated with the ecological success of many groups of organisms, most notably reptiles, gastropods, and arachnids. In some cases, diversification has been directly linked to tailoring of venoms for dietary specialization. Spiders in particular are known for their diverse venoms and wide range of predatory behaviors, although there is much to learn about scales of variation in venom composition and function. The current study focuses on venom characteristics in different sexes within a species of spider. We chose the genus Tetragnatha (Tetragnathidae) because of its unusual courtship behavior involving interlocking of the venom delivering chelicerae (i.e., the jaws), and several species in the genus are already known to have sexually dimorphic venoms. Here, we use transcriptome and proteome analyses to identify venom components that are dimorphic in Tetragnatha versicolor. We present cDNA sequences including unique, male-specific high molecular weight proteins that have remote, if any, detectable similarity to known venom components in spiders or other venomous lineages and have no detectable homologs in existing databases. While the function of these proteins is not known, their presence in association with the cheliceral locking mechanism during mating together with the presence of prolonged male-male mating attempts in a related, cheliceral-locking species (Doryonychus raptor) lacking the dimorphism suggests potential for a role in sexual communication

Spit and Venom from Scytodes Spiders: A Diverse and Distinct Cocktail

Spiders from the family Scytodidae have a unique prey capturing technique: they spit a zig-zagged silken glue to tether prey to a surface. Effectiveness of this sticky mixture is based on a combination of contraction and adhesion, trapping prey until the spider immobilizes it by envenomation and then feeds. We identify components expressed in Scytodes thoracica venom glands using combined transcriptomic and proteomic analyses. These include homologues of toxic proteins astacin metalloproteases and potentially toxic proteins including venom allergen, longistatin, and translationally controlled tumor protein (TCTP). We classify 19 distinct groups of candidate peptide toxins; 13 of these were detected in the venom, making up 35% of the proteome. Six have significant similarity to toxins from spider species spanning mygalomorph and nonhaplogyne araneomorph lineages, suggesting their expression in venom is phylogenetically widespread. Twelve peptide toxin groups have homologues in venom gland transcriptomes of other haplogynes. Of the transcripts, approximately 50% encode glycine-rich peptides that may contribute to sticky fibers in Scytodes spit. Fifty-one percent of the identified venom proteome is a family of proteins that is homologous to sequences from Drosophila sp. and Latrodectus hesperus with uncharacterized function. Characterization of these components holds promise for discovering new functional activity

HPLC chromatograms for the cleavage reaction products from S64

<p>(<b>top</b>) <b>and S67</b> (<b>bottom</b>)<b>.</b> The solid line corresponds to the left axis and represents the absorbance at 280 nm; the dashed line corresponds to the right axis and represents the solvent composition. In both chromatograms, the cleaved peptide is well-resolved from other peaks. The large peak that appears at high acetonitrile concentrations corresponds to a combination of the maltose binding domain (MBP) tag, uncleaved fusion protein (MBP:S64/MBP:S67) and TEV protease. Peak identities were confirmed by performing gel electrophoresis and mass spectrometry for select fractions. The flow rate was 4 mL/min (top) and 2 mL/min (bottom).</p

¹⁵N-HMQC spectra for S64

<p>(<b>top</b>) <b>and S67</b> (<b>bottom</b>)<b>.</b> The S64 spectrum includes some minor peaks that are not assigned. These peaks do not have discernable cross-peaks in the NOESY spectra and only very weak cross-peaks in the 3D assignment spectra, which suggest that they are due to unfolded peptide rather than a minor conformation with a different disulfide-bonding pattern.</p

Ensemble

<p>(<b>top</b>) <b>and cartoon</b> (<b>bottom</b>) <b>representations of S64</b> (<b>left</b>) <b>and S67</b> (<b>right</b>)<b>.</b> The N and C termini are labeled in both the ensemble (top) and cartoon (bottom) representations of the structures of S64 (left) and S67 (right). The three disulfide bonds in the cartoon representations are also labeled. The ensembles represents the 20 structures with the lowest total energy out of 100 calculated structures. The cartoon representations show the lowest energy structure from each ensemble. Note that in the cartoon representation for S64 the third disulfide bond connects to the β sheet (red), not the strand (teal) in front of it.</p

Sequence alignment for the translated sequences of S64 and S67.

<p>This sequence alignment for S64 and S67 illustrates the signal sequence, linker, and mature toxin. Small arrows show the predicted cleavage sites for the mature toxins. The experimentally determined disulfide bond connectivity shown applies to both peptides. Sequences were aligned using ClustalX 2.1 and visualized using JalView 2.7 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054401#pone.0054401-Waterhouse1" target="_blank">[36]</a>. The coloring makes use of the default ClustalX color scheme, which is a function of sequence identity and amino acid type.</p