Venom-gland transcriptomic, venomic, and antivenomic profiles of the spine-bellied sea snake (Hydrophis curtus) from the South China Sea

Abstract

Background: A comprehensive evaluation of the -omic profiles of venom is important for understanding the potential function and evolution of snake venom. Here, we conducted an integrated multi-omics-analysis to unveil the venom-transcriptomic and venomic profiles in a same group of spine-bellied sea snakes (Hydrophis curtus) from the South China Sea, where the snake is a widespread species and might generate regionally-specific venom potentially harmful to human activities. The capacity of two heterologous antivenoms to immunocapture the H. curtus venom was determined for an in-depth evaluation of their rationality in treatment of H. curtus envenomation. In addition, a phylogenetic analysis by maximum likelihood was used to detect the adaptive molecular evolution of full-length toxin-coding unigenes.

Results: A total of 90,909,384 pairs of clean reads were generated via Illumina sequencing from a pooled cDNA library of six specimens, and yielding 148,121 unigenes through de novo assembly. Sequence similarity searching harvested 63,845 valid annotations, including 63,789 non-toxin-coding and 56 toxin-coding unigenes belonging to 22 protein families. Three protein families, three-finger toxins (3-FTx), phospholipase A₂ (PLA₂), and cysteine-rich secretory protein, were detected in the venom proteome. 3-FTx (27.15% in the transcriptome/41.94% in the proteome) and PLA₂ (59.71%/49.36%) were identified as the most abundant families in the venom-gland transcriptome and venom proteome. In addition, 24 unigenes from 11 protein families were shown to have experienced positive selection in their evolutionary history, whereas four were relatively conserved throughout evolution. Commercial Naja atra antivenom exhibited a stronger capacity than Bungarus multicinctus antivenom to immunocapture H. curtus venom components, especially short neurotoxins, with the capacity of both antivenoms to immunocapture short neurotoxins being weaker than that for PLA₂s.

Conclusions: Our study clarified the venom-gland transcriptomic and venomic profiles along with the within-group divergence of a H. curtus population from the South China Sea. Adaptive evolution of most venom components driven by natural selection appeared to occur rapidly during evolutionary history. Notably, the utility of commercial N. atra and B. multicinctus antivenoms against H. curtus toxins was not comprehensive; thus, the development of species-specific antivenom is urgently needed.

Keywords: Antivenomic; Hydrophis curtus; Omics; Positive selection; Proteome; Snake venom; Transcriptome.

Fig. 1

Venom-gland transcriptomic profiles of H. curtus. The details are listed in Additional Tables S1 and S2. FPKM, expected number of fragments per kilobase of transcript sequence per millions base pairs sequenced. The percentage of annotated (toxin and non-toxin) and unannotated (unidentified) unigenes in the whole transcriptome was listed in the inserted pie graph. 3-FTx, three finger toxin; PLA₂, phospholipase A₂; CRISP, cysteine-rich secretory protein; CTL, C-type lectin; SVMP, snake venom metalloproteinase; SVSP, snake venom serine proteinase; PLB, phospholipase B; PDE, phosphodiesterase; HA, hyaluronidase; 5′NT, 5′ nucleotidase; CREGF, cysteine-rich EGF-like domain; VEGF, vascular endothelial growth factor; VF, venom factor; AP, aminopeptidase; NGF, nerve growth factor; AchE, acetylcholinesterase; QC, glutaminyl-peptide cyclotransferases; LAAO, l-amino acid oxidase

Fig. 2

Characterization of the venom proteins of H. curtus from South China Sea. Three milligrams of total venom were applied to a C18 column, and separated as described on Materials and methods. Fractions were collected manually and submitted to molecular determination by SDS-PAGE under reduced conditions (original images were listed in Figure S1). Protein bands were excised, tryptic digested and analyzed by MALDI-TOF/TOF or nESI-MS/MS for their assignment to known protein families. The results are shown in Table 1

Fig. 3

Venom proteomic profiles of H. curtus. The details are listed in Table 1. 3-FTx, three finger toxin; PLA₂, phospholipase A₂; CRISP, cysteine-rich secretory protein; LNX, long chain α-neurotoxin; SNX, short chain α-neurotoxin

Fig. 4

Correlation between mRNA and protein abundances of individual gene for each toxin family. SNX was excluded from the analysis. All data were centered log-ratio (clr) transformed. N, number of toxin transcripts; ρ, Spearman’s rank correlation coefficient; R, Pearson’s correlation coefficient

Fig. 5

Antivenomics analysis of H. curtus venom using commercial antivenom by RP-HPLC. Chromatographic profiles of 50, 100, 150, 300 and 600 μg whole venom components (panels A, F K, P and U) and, of immunocaptured venom components (panels B, D, G, I, L, N, Q, S, V and X) and non-immunocaptured venom components (panels C, E, H, J, M, O, R, T, W and Y) recovered from the affinity columns after incubation with the corresponding amounts of venom. Bm, B. multicinctus; Na, N. atra

Fig. 6

Cross-reaction between H. curtus venom and commercial antivenom determined by ELISA. Normal horse serum was used as negative control. Data are expressed as mean ± SD (n = 3)

Fig. 7

Cross-reaction between H. curtus venom and commercial antivenom determined by western blotting. A SDS-PAGE profiles of venom protein (identified proteins were listed in Table S4); B B. multicinctus antivenom; C N. atra antivenom (original images were listed in Figure S2). PLA₂, phospholipase A₂; CRISP, cysteine-rich secretory protein; LNX, long chain α-neurotoxin; SNX, short chain α-neurotoxin