Predatory robber flies (Diptera, Asilidae) have been suspected to be venomous due to their ability to overpower well-defended prey. However, details of their venom composition and toxin arsenal remained unknown. Here, we provide a detailed characterization of the venom system of robber flies through the application of comparative transcriptomics, proteomics and functional morphology. Our results reveal asilid venoms to be dominated by peptides and non-enzymatic proteins, and that the majority of components in the crude venom is represented by just ten toxin families, which we have named Asilidin1–10. Contrary to what might be expected for a liquid-feeding predator, the venoms of robber flies appear to be rich in novel peptides, rather than enzymes with a putative pre-digestive role. The novelty of these peptides suggests that the robber fly venom system evolved independently from hematophagous dipterans and other pancrustaceans. Indeed, six Asilidins match no other venom proteins, while three represent known examples of peptide scaffolds convergently recruited to a toxic function. Of these, members of Asilidin1 closely resemble cysteine inhibitor knot peptides (ICK), of which neurotoxic variants occur in cone snails, assassin bugs, scorpions and spiders. Synthesis of one of these putative ICKs, U-Asilidin₁-Mar1a, followed by toxicity assays against an ecologically relevant prey model revealed that one of these likely plays a role as a neurotoxin involved in the immobilization of prey. Our results are fundamental to address these insights further and to understand processes that drive venom evolution in dipterans as well as other arthropods.

Venoms evolved convergently in diverse animal lineages as key adaptations that increase the evolutionary fitness of species which are manifold employed for defense, predation, and competition. They constitute complex cocktails of various toxins that feature a broad range of bioactivities. The majority of described venom proteins belong to protein families that are known to comprise housekeeping genes or harbor protein-domains, which are present in genes with non-venom related functions. However, the evolutionary processes and mechanisms that foster the origin of these venom proteins and triggered their recruitment into the venom delivery system are still critically discussed. In most instances single or combined proteomic and transcriptomic approaches are applied to describe venom compositions and the biological context of venoms. For neglected species these studies represent crucial contributions to improve our understanding of venom diversity on a broader scale. Nonetheless, the inference of the evolutionary origin of putative toxins in these studies could be misleading without appropriate coverage of gene populations from different tissue samples (gene completeness) or complementary genome data. Providing a valid backbone to correctly map transcriptome and proteome data, whole genome sequences facilitate a clear distinction between variability of venom proteins or toxins due to posttranslational modifications, alternative splicing, and false-positive matches that stem from sequencing or read processing and assembly errors. High-quality whole genome sequence data of venomous species are still sparse and unevenly distributed within taxon lineages. However, to reveal the evolutionary pattern of putative toxins in venomous lineages and to identify ancestral variants of venom proteins, the appropriate sampling of genomes from venomous and non-venomous species is crucial. Nevertheless, larger comparative studies based on multiple whole genome data sets are still sparse to uncover processes of venom evolution. Here, we review the general potential of comparative genomics in venomics to unravel mechanisms and patterns of evolutionary origin of toxin genes. Finally, we discuss the benefit of whole genome data to improve transcriptomics and proteomics-only studies, in particular if datasets are applied to assess the evolutionary origin of venom proteins.

We present the first molecular characterization of glycerotoxin (GLTx), a potent neurotoxin found in the venom of the bloodworm Glycera tridactyla (Glyceridae, Annelida). Within the animal kingdom, GLTx shows a unique mode of action as it can specifically up-regulate the activity of Cav2.2 channels (N-type) in a reversible manner. The lack of sequence information has so far hampered a detailed understanding of its mode of action. Our analyses reveal three ~3.8 kb GLTx full-length transcripts, show that GLTx represents a multigene family, and suggest it functions as a dimer. An integrative approach using transcriptomics, quantitative real-time PCR, in situ hybridization, and immunocytochemistry shows that GLTx is highly expressed exclusively in four pharyngeal lobes, a previously unrecognized part of the venom apparatus. Our results overturn a century old textbook view on the glycerid venom system, suggesting that it is anatomically and functionally much more complex than previously thought. The herein presented GLTx sequence information constitutes an important step towards the establishment of GLTx as a versatile tool to understand the mechanism of synaptic function, as well as the mode of action of this novel neurotoxin.

The complete mitochondrial genome of Dasypogon diadema (Insecta: Diptera) was sequenced using the Illumina MiSeq` platform. Its mt-genome spans over 16,947 bp with a GC content of 26.6% containing 13 protein-coding genes, 22 tRNA genes, and 2 rRNA genes. The phylogenetic relationship of Dasypogon diadema and 11 other dipteran species was reconstructed and the phylogenetic position confirmed.