Integrative multi-omics analysis reveals the contribution of neoVTX genes to venom diversity of Synanceia verrucosa

Abstract

Background: Animal venom systems are considered as valuable model for investigating the molecular mechanisms underlying phenotypic evolution. Stonefish are the most venomous and dangerous fish because of severe human envenomation and occasionally fatalities, whereas the genomic background of their venom has not been fully explored compared with that in other venomous animals.

Results: In this study, we followed modern venomic pipelines to decode the Synanceia verrucosa venom components. A catalog of 478 toxin genes was annotated based on our assembled chromosome-level genome. Integrative analysis of the high-quality genome, the transcriptome of the venom gland, and the proteome of crude venom revealed mechanisms underlying the venom complexity in S. verrucosa. Six tandem-duplicated neoVTX subunit genes were identified as the major source for the neoVTX protein production. Further isoform sequencing revealed massive alternative splicing events with a total of 411 isoforms demonstrated by the six genes, which further contributed to the venom diversity. We then characterized 12 dominantly expressed toxin genes in the venom gland, and 11 of which were evidenced to produce the venom protein components, with the neoVTX proteins as the most abundant. Other major venom proteins included a presumed CRVP, Kuntiz-type serine protease inhibitor, calglandulin protein, and hyaluronidase. Besides, a few of highly abundant non-toxin proteins were also characterized and they were hypothesized to function in housekeeping or hemostasis maintaining roles in the venom gland. Notably, gastrotropin like non-toxin proteins were the second highest abundant proteins in the venom, which have not been reported in other venomous animals and contribute to the unique venom properties of S. verrucosa.

Conclusions: The results identified the major venom composition of S. verrucosa, and highlighted the contribution of neoVTX genes to the diversity of venom composition through tandem-duplication and alternative splicing. The diverse neoVTX proteins in the venom as lethal particles are important for understanding the adaptive evolution of S. verrucosa. Further functional studies are encouraged to exploit the venom components of S. verrucosa for pharmaceutical innovation.

Keywords: Synanceia verrucosa; Alternative splicing; Multi-omics; Tandem duplication; Venom diversity.

Fig. 1

Toxin genes in the genome of S. verrucosa a) Visualized circle plot of chromosomal level genome assembly of S. verrucosa, with N ratio, GC ratio, repeat content and gene density shown from outside to inside, corresponding to each pseudochromosomes. b) Summary of the genome assembly evaluation. c) Protein families of all the annotated toxin genes. d) Tandem-duplicated SNTX/VTX genes clustered together with sequence divergence

Fig. 2

Transcriptomic analysis of the venom gland a) A heatmap of the highest 50 toxin genes transcription with hierarchical clustering in the venom gland among all the 7 samples. The color represented the expression level based on TPM value. b) Categorizes of the dominantly expressed toxin genes (DETGs) with TPM > 500, the highest 10 non-toxin genes, and 5 unannotated genes with TPM > 500. The value was shown as mean ± SD, n = 7. c) Volcano plots showing the differentially expressed genes (DEGs) between the venom gland and the skin tissue, with log2 (fold change) thresholds of -1 and 1, and P < 0.05. d) GO enrichment bubble plot analysis of the DEGs from non-toxin genes set (with TPM > 500 in VG) between the VG and SK. The bubble size indicated the gene number, the color represented the adjusted p value. BP, biological process; CC, cellular components; MF, molecular function

Fig. 3

Proteomic analysis of the crude venom a) Pie charts illustrating the composition of toxin and non-toxin proteins. b) Protein abundance of the major toxins and non-toxins, n = 7. c) Pie charts showing the contribution of each neoVTX subunit genes at transcriptomic and proteomic level. d) Native SDS-PAGE of lyophilized crude venom. Lane 1, protein ladders; lane 2, lyophilized crude venom. The asterisk indicated neoVTX proteins with different molecular weight. e) Illustration of the major protein toxin genes (11) deciphering in S. verrucosa. The contribution (proportion in total) of the 11 major protein toxin genes to the whole expressed toxin genes (240) in the VG and toxin proteins (33) was mapped to the corresponding column with diagonal lines

Fig. 4

Massive alternative isoforms from the neovtx genes in the venom gland a) The work flow for sequencing and analysis for full length transcriptome construction. b) Distribution of the number of alternative splicing isoforms per annotated toxin gene. c) Percent of the AS isoforms from each DETGs in the total isoforms (856) mapped to the annotated toxin genes (476). d) The 6 tandem-repeated neovtx genes demonstrated the most isoforms. The isoforms were mapped to each gene with the exon usage shown. 5 trans-splicing isoforms were listed at the lower panel, showing a single isoform embraced exons from different genes. e) Expression level of 27 cis-splicing and 5 trans-splicing isoforms from the 6 neovtx genes, the value was illustrated as mean ± SD, n = 7