Functional and Proteomic Insights into Aculeata Venoms

Abstract

Aculeate hymenopterans use their venom for a variety of different purposes. The venom of solitary aculeates paralyze and preserve prey without killing it, whereas social aculeates utilize their venom in defence of their colony. These distinct applications of venom suggest that its components and their functions are also likely to differ. This study investigates a range of solitary and social species across Aculeata. We combined electrophoretic, mass spectrometric, and transcriptomic techniques to characterize the compositions of venoms from an incredibly diverse taxon. In addition, in vitro assays shed light on their biological activities. Although there were many common components identified in the venoms of species with different social behavior, there were also significant variations in the presence and activity of enzymes such as phospholipase A₂s and serine proteases and the cytotoxicity of the venoms. Social aculeate venom showed higher presence of peptides that cause damage and pain in victims. The venom-gland transcriptome from the European honeybee (Apis mellifera) contained highly conserved toxins which match those identified by previous investigations. In contrast, venoms from less-studied taxa returned limited results from our proteomic databases, suggesting that they contain unique toxins.

Keywords: Aculeata; cytotoxicity; proteomics; sociality; venom.

Figure 1

Schematic overview of the key aculeate groups sampled in this study, the samples derived from them, and the data generated. Photo species and credit (left to right): Vespa mandarinia (Asian giant hornet) by Gregory Mihaich under CC-BY-NC-SA, Dasymutilla gloriosa (thistledown velvet ant) by mrwood under CC-BY-NC, Scolia dubia (blue-winged flower wasp) by Thomas Shahan under CC-BY-NC, Xylocopa californica (western carpenter bee) Arman Moreno under CC-BY-NC, Apis mellifera (honeybee) Sandy Rae under CC-BY-SA, Paraponera clavata (bullet ant) by manimiranda under CC-BY-NC. All images were retrieved from iNaturalist (https://www.inaturalist.org/).

Figure 2

(A–D) Alignment of translated toxin CDS sequences with the sites with UniProt references. Residues identical to the reference are replaced by , and amino acids are colored according to the default settings of AliView [79]. Toxin families include: (A) icarapins, (B) phospholipase A₂, (C) anthophilins such as apamin [78], (D) carboxylesterases. (E) Relative length-normalized expression of these toxin families in the transcriptome, measured as total RPK for each family.

Figure 3

1D SDS-PAGE (12% acrylamide with Coomassie brilliant blue staining) of venom from bees and wasps: (A) social bees (reduced); 1 = Apis mellifera (European); 2 = A. mellifera (Africanised); 3 = A. andreniformis; 4 = A. cerana; 5 = A. dorsata; 6 = A. florea; 7 = A. koschevnikovi; 8 = Bombus huntii; 9 = B. impatiens. (B) Solitary bees (reduced); 1 = Centris aethycetra; 2 = C. rhodipus; 3 = Diadasia rinconis; 4 = Peponapis pruinosa; 5 = Xylocopa rufa; 6 = X. californica; 7 = Crawfordapis sp.; 8 = Lasioglossum kinabalueuse; 9 = X. veripuncta. (C) Epiponini wasps (reduced); 1 = Agelaia myrmecophila; 2 = Brachygastra mellifica; 3 = Polistes flavus; 4 = Polybia rejecta; 5 = Polybia sericea; 6 = Polybia simillima; 7 = Synoeca septentrionalis. (D) Polistes, Ropalidini, and Mischocyttarini wasps (reduced); 1 = Belonogaser juncea colonialis; 2 = Mischocyttarus flavitarsus; 3 = Polistes canadensis; 4 = Polistes comanchus navajoe; 5 = Polistes dorsalis; 6 = Parachartergus fraternus; 7 = Polistes major castaneocolor. (E) Vespinae wasps (reduced); 1 = Dolichovespula arenaria; 2 = D. maculata; 3 = Vespula pensylvanica; 4 = Vespula vulgaris; 5 = Vespa luctuosa; 6 = Vespa simillima; 7 = Vespa tropica. (F) Solitary wasps (reduced); 1 = Dasymutilla chiron; 2 = D. gloriosa; 3 = Scoliidae; 4 = Stictia.

Figure 4

1D SDS-PAGE (12% acrylamide with Coomassie brilliant blue staining) of venom from ants (reduced): (A) 1 = Paraponera clavata; 2 = Diacamma; 3 = Euponera sennaaren; 4 = Leptogenys; 5 = Neoponera villosa; 6 = Odontomachus; 7 = Opthalmopone; 8 = Megaponera analis. (B) 1 = Pachycondyla crassinoda; 2 = Paltothyreus tarsatus; 3 = Platythyrea lamellosa; 4 = P. strigulosa; 5 = Streblognathus aethiopicus; 6 = Neoponera commutata; 7 = N. commutata (Queen); 8 = Odontoponera. (C) 1 = Ectatomma tuberculatum; 2 = Ectatomma; 3 = Gnaptogenys; 4 = Rhytidoponera metallica; 5 = Pogonomyrmex maricopa; 6 = P. occidentalis; 7 = P. rugosus; 8 = Diacamma. (D) 1 = Tetraponera sp.; 2 = Myrmecia browningii; 3 = M. gulosa; 4 = M. nigripes; 5 = M. pilosula; 6 = M. rufinodis; 7 = M. simillima; 8 = M. tarsata.

Figure 5

Representative LC-MS profiles of bee species: (A) Apis mellifera, (B) A. andreniformis, (C) Bombus impatiens, (D) B. sonorus, (E) Xylocopa californica, (F) Peponapis pruinosa. The x-axis is time (minutes); the y-axis is relative intensity (0–100%). Reconstructed mass in Daltons is shown above each peak.

Figure 6

Representative LC-MS profiles of wasp species. (A) Agelaia myrmecophila, (B) Polybia sericea, (C) Polistes major castaneocolor, (D) Vespula vulgaris, (E) Stictia sp., (F) Dasymutilla klugii. The x-axis is time (minutes); the y-axis is relative intensity (0–100%). Reconstructed mass in Daltons is shown above each peak.

Figure 7

Representative LC-MS profiles of Formicidae species. (A) Dinoponera gigantea, (B) Myrmecia rufinodis, (C) Pachycondyla crassinoda, (D) Platythyrea strigulosa, (E) Paltothyreus tarsatus, (F) Odontomachus sp. The x-axis is time (minutes); the y-axis is relative intensity (0–100%). Reconstructed mass in Daltons is shown above each peak.

Figure 8

A phylogeny of venom samples which were analyzed though LC-MS/MS and the toxins in the reference database which returned matches to peptides in those venoms. Phylogeny topology and branch lengths from TimeTree (https://timetree.org/) and other previously published phylogenies [81,82,83,84,85,86,87,88,89] were used to manually construct a combined phylogeny in Mesquite 3.7 [90].

Figure 9

Ancestral state reconstructions of PLA₂ activity (left) and serine protease activity (right). Activity was measured as relative percentage absorbance, and warmer colors represent higher activity. Grey boxes indicate social species. Phylogeny topologies and branch lengths from TimeTree (https://timetree.org/) and other previously published phylogenies [81,82,83,84,85,86,87,88,89] were used to manually construct a combined phylogeny in Mesquite 3.7 [90].

Figure 10

Ancestral state reconstructions of the cytotoxic effects of aculeate venoms against melanoma (MM96L) cancerous cells (left) and the non-transformed (NFF) cell line (right). Cytotoxicity was measured using the area under the curve of cell mortality over the course of the assay. Warmer colors represent greater toxicity. Grey boxes indicate social species. Phylogeny topologies and branch lengths from TimeTree (https://timetree.org/) and other previously published phylogenies [81,82,83,84,85,86,87,88,89] were used to manually construct a combined phylogeny in Mesquite 3.7 [90].