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. 2021 Dec 22;27(1):47.
doi: 10.3390/molecules27010047.

De Novo Transcriptome Analysis of the Venom of Latrodectus geometricus with the Discovery of an Insect-Selective Na Channel Modulator

Pornsawan Khamtorn  1 , Steve Peigneur  2 , Fernanda Gobbi Amorim  3 , Loïc Quinton  3 , Jan Tytgat  2 , Sakda Daduang  4   5   6
Affiliations

De Novo Transcriptome Analysis of the Venom of Latrodectus geometricus with the Discovery of an Insect-Selective Na Channel Modulator

Pornsawan Khamtorn et al. Molecules. .

Abstract

The brown widow spider, Latrodectus geometricus, is a predator of a variety of agricultural insects and is also hazardous for humans. Its venom is a true pharmacopeia representing neurotoxic peptides targeting the ion channels and/or receptors of both vertebrates and invertebrates. The lack of transcriptomic information, however, limits our knowledge of the diversity of components present in its venom. The purpose of this study was two-fold: (1) carry out a transcriptomic analysis of the venom, and (2) investigate the bioactivity of the venom using an electrophysiological bioassay. From 32,505 assembled transcripts, 8 toxin families were classified, and the ankyrin repeats (ANK), agatoxin, centipede toxin, ctenitoxin, lycotoxin, scorpion toxin-like, and SCP families were reported in the L. geometricus venom gland. The diversity of L. geometricus venom was also uncovered by the transcriptomics approach with the presence of defensins, chitinases, translationally controlled tumor proteins (TCTPs), leucine-rich proteins, serine proteases, and other important venom components. The venom was also chromatographically purified, and the activity contained in the fractions was investigated using an electrophysiological bioassay with the use of a voltage clamp on ion channels in order to find if the neurotoxic effects of the spider venom could be linked to a particular molecular target. The findings show that U24-ctenitoxin-Pn1a involves the inhibition of the insect sodium (Nav) channels, BgNav and DmNav. This study provides an overview of the molecular diversity of L. geometricus venom, which can be used as a reference for the venom of other spider species. The venom composition profile also increases our knowledge for the development of novel insecticides targeting voltage-gated sodium channels.

Keywords: Latrodectus geometricus; insect sodium channels; ion channel; spider; toxin; transcriptome; venom.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Size distributions of (A) contig and (B) unigene in the transcriptome of the Latrodectus geometricus venom gland. The sequence size is represented on the x-axis, while the numbers of contig and unigene are represented on the y-axis.
Figure 2
Figure 2
Venn diagram of unigenes annotated with InterPro, NR, KEGG, KOG, and SwissProt databases.
Figure 3
Figure 3
Species distribution showing the proportions of different species associated with the unigene annotations according to the results of the NR annotations.
Figure 4
Figure 4
Gene ontology classifications of assembled unigenes Latrodectus geometricus venom gland. The figure shows a summary classification in three categories: biological process, cellular component, and molecular function.
Figure 5
Figure 5
The expressed ion channels of the Latrodectus geometricus venom gland.
Figure 6
Figure 6
Toxinome of L. geometricus venom gland. All 212 unigenes were clustered using sequence homology and domain prediction.
Figure 7
Figure 7
Domain architecture of Latrodectus geometricus toxins was predicted by the simple modular architecture research tool (SMART) and the protein family database (Pfam) servers (http://www.smart.embl-heidelberg.de, accessed on 4 April 2021 and http://pfam.janelia.org, accessed date 4 April 2021) [42,43]. All toxins were grouped based on the specific family classification. This scheme presents only domain matching among all unigenes. The abbreviations of domain names are as follows: ANK (SMART ID: SM00248); TY (SMART ID: SM00211); toxin 35 (Pfam ID: PF10530); toxin 9 (Pfam ID: PF02819); SCP (SMART ID: SM00198); trypsin (Pfam ID: PF00089).
Figure 8
Figure 8
The relative abundance expressed in FPKM of the venom component of the Latrodectus geometricus venom gland. Unigene sequences were classified into known toxin subfamilies according to the UniProt database. Bars represent the sum of FPKM for each transcript belonging to the described groups.
Figure 9
Figure 9
Reverse-phase HPLC separation on Vydac C18 of Latrodectus geometricus venom. The chromatogram presents the separated fractions from the crude venom separated at a flow rate of 1 mL/min. The red line indicates the gradient of acetonitrile in 0.085% (v/v) aqueous trifluoroacetic acid.
Figure 10
Figure 10
Electrophysiological profiles of L. geometricus venom fraction 14 on Nav and Kv channels. Panels show superimposed current traces of 0.4 µg/µL fraction 14. Black indicates a current trace in control conditions; red or blue indicates a current trace in toxin situation. The dotted line indicates zero current level. The asterisk marks steady-state current trace after application of toxin.
Figure 11
Figure 11
The effects on the insect BgNav and DmNav channels induced by fraction 14 on currents of the cloned insect BgNav channels (A) and DmNav channels (B), expressed in Xenopus oocytes. Current traces were elicited by 5 mV step depolarizations in control (black) and the presence of 0.4 µg/μL fraction 14 (red). The dotted line indicates zero current level.
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