A proteomics and transcriptomics investigation of the venom from the barychelid spider Trittame loki (brush-foot trapdoor)

Abstract

Although known for their potent venom and ability to prey upon both invertebrate and vertebrate species, the Barychelidae spider family has been entirely neglected by toxinologists. In striking contrast, the sister family Theraphosidae (commonly known as tarantulas), which last shared a most recent common ancestor with Barychelidae over 200 million years ago, has received much attention, accounting for 25% of all the described spider toxins while representing only 2% of all spider species. In this study, we evaluated for the first time the venom arsenal of a barychelid spider, Trittame loki, using transcriptomic, proteomic, and bioinformatic methods. The venom was revealed to be dominated by extremely diverse inhibitor cystine knot (ICK)/knottin peptides, accounting for 42 of the 46 full-length toxin precursors recovered in the transcriptomic sequencing. In addition to documenting differential rates of evolution adopted by different ICK/knottin toxin lineages, we discovered homologues with completely novel cysteine skeletal architecture. Moreover, acetylcholinesterase and neprilysin were revealed for the first time as part of the spider-venom arsenal and CAP (CRiSP/Allergen/PR-1) were identified for the first time in mygalomorph spider venoms. These results not only highlight the extent of venom diversification in this neglected ancient spider lineage, but also reinforce the idea that unique venomous lineages are rich pools of novel biomolecules that may have significant applied uses as therapeutics and/or insecticides.

Figure 1

Magnetic resonance imaging of Trittame loki venom glands.

Figure 2

Phylogenetic reconstruction of Trittame loki and related inhibitor cystine knot (ICK)/knottin peptide toxins, conserved ancestral cysteines are shown in black, newly evolved cysteines are in red. Sequences obtained in this study are in green. Signal peptides are shown in lowercase.

Figure 3

Sequence alignment of spider venom colipase venom peptides: (1) Trittame loki COLIPASE-1; (2) D2Y2E5 Haplopelma hainanum; (3) Q5D233 Hadronyche infensa; (4) Q5D231 Hadronyche sp. (strain 20); (5) Q5D232 Hadronyche sp. (strain 20); (6) B1P1J0 Chilobrachys jingzhao; and (7) B1P1J2 Chilobrachys jingzhao. Signal peptides are shown in lowercase.

Figure 4

Sequence alignment of spider venom CAP (CRiSP/Allergen/PR-1) venom peptides: (1) Trittame loki CAP-1; and (2) A9QQ26 Lycosa singoriensis. Signal peptides are shown in lowercase.

Figure 5

Sequence alignment of spider venom kunitz venom peptides: (1) Trittame loki KUNITZ-1; and (2) E7D1N7 Latrodectus hesperus. Signal peptides are shown in lowercase.

Figure 6

Sequence alignment of the Trittame loki venom acetylcholinesterase and the non-venom homologue P56161 Anopheles stephensi. Signal peptides are shown in lowercase.

Figure 7

Sequence alignment of the Trittame loki venom neprilysin and the snake venom convergent neprilysin homologue T1E4Z0 Crotalus horridus. Signal peptides are shown in lowercase.