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.

DNA barcodes are widely used for species identification and biogeographic studies. Here, we compare the use of full mitochondrial genomes versus DNA barcodes and other mitochondrial DNA fragments for biogeographic and ecological analyses. Our dataset comprised 120 mitochondrial genomes from the genus Clunio (Diptera: Chironomidae), comprising five populations from two closely related species (Clunio marinus and Clunio balticus) and three ecotypes. We extracted cytochrome oxidase c subunit I (COI) barcodes and partitioned the mitochondrial genomes into non-overlapping windows of 750 or 1500 bp. Haplotype networks and diversity indices were compared for these windows and full mitochondrial genomes (15.4 kb). Full mitochondrial genomes indicate complete geographic isolation between populations, but do not allow for conclusions on the separation of ecotypes or species. COI barcodes have comparatively few polymorphisms, ideal for species identification, but do not resolve geographic isolation. Many of the similarly sized 750 bp windows have higher nucleotide and haplotype diversity than COI barcodes, but still do not resolve biogeography. Only when increasing the window size to 1500 bp, two windows resolve biogeography reasonably well. Our results suggest that the design and use of DNA barcodes in biogeographic studies must be carefully evaluated for each investigated species.

The viviparous eelpout Zoarces viviparus is a common fish across the North Atlantic and has successfully colonized habitats across environmental gradients. Due to its wide distribution and predictable phenotypic responses to pollution, Z. viviparus is used as an ideal marine bioindicator organism and has been routinely sampled over decades by several countries to monitor marine environmental health. Additionally, this species is a promising model to study adaptive processes related to environmental change, specifically global warming. Here, we report the chromosome-level genome assembly of Z. viviparus, which has a size of 663 Mb and consists of 607 scaffolds (N50 = 26 Mb). The 24 largest represent the 24 chromosomes of the haploid Z. viviparus genome, which harbors 98% of the complete Benchmarking Universal Single-Copy Orthologues defined for ray-finned fish, indicating that the assembly is highly contiguous and complete. Comparative analyses between the Z. viviparus assembly and the chromosome-level genomes of two other eelpout species revealed a high synteny, but also an accumulation of repetitive elements in the Z. viviparus genome. Our reference genome will be an important resource enabling future in-depth genomic analyses of the effects of environmental change on this important bioindicator species.