Venom proteome of the monophagous spider Ammoxenus amphalodes: a potential source of termite-specific toxins

Venoms are complex chemical arsenals that have evolved across various animal lineages for predation, defence, and other ecological functions. In predatory species, venom composition is often shaped by diet, potentially leading to unique toxin profiles. Spiders are the most diverse group of terrestrial venomous predators, yet the venoms of most species, including highly specialized stenophagous species with narrow trophic niches, remain poorly understood. In this study, we investigated the venom of Ammoxenus amphalodes , a spider that preys exclusively on termites, to explore its molecular composition and functional adaptations. Using a proteotranscriptomic approach, we identified 116 putative toxin sequences. Half of these were cysteine-rich peptides with distinct structural motifs, most of which diverge from known spider toxins containing an inhibitor-cystine-knot (ICK) motif. The venom proteome is dominated by two ammoxotoxin families, which together account for more than half of all transcripts, supporting the hypothesis that spiders with specialized diets have lower toxin diversity. We also identified a low proportion of larger venom proteins, including cholinesterases, phospholipases, immune proteins, carbonic anhydrases, and EF-hand proteins, which may serve housekeeping functions or play auxiliary roles in prey incapacitation. Pilot bioassays with selected lab-produced peptides revealed one peptide, U-AXTX(9)-Aa15, a member of the waprin family, which induced stronger paralysis in termites than in flies. However, the other tested peptides showed similar or higher paralysis in flies, challenging assumptions of venom specialization in A. amphalodes . These findings provide new insights into the evolution of venom in dietary specialists and broaden the search for novel bioactive compounds with potential biotechnological applications. • First characterization of venom from the termite-eating spider Ammoxenus amphalodes. • Venom comprises 116 components, many cysteine-rich toxins with novel scaffolds. • Ammoxotoxin families 1 and 3 dominate the venom composition. • Five selected toxins were tested; one induced reversible moderate paralysis in termites. • Ammoxotoxins merit further investigation for their prey-specific potential.