Abstract
Venom is a critical evolutionary innovation enabling venomous snakes to become successful limbless predators; it is therefore vital that venomous snakes possess a highly efficient venom production and delivery system to maintain their predatory arsenal. Here, we exploit the unusual stability of messenger RNA in venom to conduct, for the first time, quantitative PCR to characterise the dynamics of gene expression of newly synthesised venom proteins following venom depletion. Quantitative PCR directly from venom enables real-time dynamic studies of gene expression in the same animals because it circumvents the conventional requirement to sacrifice snakes to extract mRNA from dissected venom glands. Using qPCR and proteomic analysis, we show that gene expression and protein re-synthesis triggered by venom expulsion peaks between days 3–7 of the cycle of venom replenishment, with different protein families expressed in parallel. We demonstrate that venom re-synthesis occurs very rapidly following depletion of venom stores, presumably to ensure venomous snakes retain their ability to efficiently predate and remain defended from predators. The stability of mRNA in venom is biologically fascinating, and could significantly empower venom research by expanding opportunities to produce transcriptomes from historical venom stocks and rare or endangered venomous species, for new therapeutic, diagnostic and evolutionary studies.
Highlights
Snake venom is an evolutionary innovation contributing to the success of venomous snakes as proficient limbless predators
PCR primer efficiency was tested by conventional PCR using standard samples including (i) venom gland cDNA from a B. arietans venom gland cDNA library constructed using methods described in Wagstaff et al [14] and (ii) cDNA synthesised from messenger RNA (mRNA) isolated from 10 mg of pooled mature venom
More than adequate amounts of mRNA were recovered from each venom sample for downstream qPCR analysis: a representative cDNA synthesis reaction yielded 22.74 mg (63.03 mg std. dev) of cDNA (20 ml) per reaction from 18.5 ng of mRNA (8 ml) - amounts sufficient for 20 qPCR reactions
Summary
Snake venom is an evolutionary innovation contributing to the success of venomous snakes as proficient limbless predators. Early studies suggest total RNA and total protein levels peak at day 3 and days 4 to 8 days post venom expulsion respectively [7], little is known about the expression dynamics of individual venom components This has been historically problematic because of the unpalatable need to sacrifice snakes (often rare, difficult to capture and CITES listed) to isolate mRNA from dissected venom glands. The observation of Chen et al [8], demonstrating that intact mRNA can be recovered, and toxins can be PCR-amplified and cloned from snake venoms and the venoms/skin secretions of other animals including Heloderma lizard [9], scorpion [10] and fire-bellied toads [11,12,13], has potential to resolve this research bottleneck Exploiting these unusual observations, we have used venom as a resource and developed qPCR techniques as a tool to monitor the expression dynamics of mRNA encoding multiple venom toxin genes. We demonstrate that mRNA is a remarkably stable component of venom and discuss how this unusual phenomenon has potential to significantly empower venom research
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