Differences in PLA2 Constitution Distinguish the Venom of Two Endemic Brazilian Mountain Lanceheads, Bothrops cotiara and Bothrops fonsecai

Abstract

Interspecific differences in snake venom compositions can result from distinct regulatory mechanisms acting in each species. However, comparative analyses focusing on identifying regulatory elements and patterns that led to distinct venom composition are still scarce. Among venomous snakes, Bothrops cotiara and Bothrops fonsecai represent ideal models to complement our understanding of the regulatory mechanisms of venom production. These recently diverged species share a similar specialized diet, habitat, and natural history, but each presents a distinct venom phenotype. Here, we integrated data from the venom gland transcriptome and miRNome and the venom proteome of B. fonsecai and B. cotiara to better understand the regulatory mechanisms that may be acting to produce differing venom compositions. We detected not only the presence of similar toxin isoforms in both species but also distinct expression profiles of phospholipases A2 (PLA2) and some snake venom metalloproteinases (SVMPs) and snake venom serine proteinases (SVSPs) isoforms. We found evidence of modular expression regulation of several toxin isoforms implicated in venom divergence and observed correlated expression of several transcription factors. We did not find strong evidence for miRNAs shaping interspecific divergence of the venom phenotypes, but we identified a subset of toxin isoforms whose final expression may be fine-tuned by specific miRNAs. Sequence analysis on orthologous toxins showed a high rate of substitutions between PLA2s, which indicates that these toxins may be under strong positive selection or represent paralogous toxins in these species. Our results support other recent studies in suggesting that gene regulation is a principal mode of venom evolution across recent timescales, especially among species with conserved ecotypes.

Venom Composition of Neglected Bothropoid Snakes from the Amazon Rainforest: Ecological and Toxinological Implications

“…The RNA was extracted as previously described [ 74 ]. RNA extraction, quantification, quality control, and library preparation were carried out as previously described [ 75 ]. Briefly, libraries were prepared with 1 µg of each sample with an Illumina TruSeq Stranded RNA HT kit consisting of TruSeq Stranded RNA HT/cDNA Synthesis PCR, TruSeq Stranded RNA HT/Adapter Plate Box, and TruSeq Stranded HT mRNA.…”

“…We used five different assemblers with different k-mer values and assembly parameters (Trinity: k-mer 31; rnaSPADES: k-mer 31, 75, and 127; Extender: default, overlap 150, and seed size 2000; SeqMan Ngen: k-mer 21; and Bridger: k-mer 30) [ 34 , 35 , 78 , 79 , 80 ]. Coverage analysis and annotation were performed as described in [ 75 ]. The coding sequences annotated from all individuals of the same species were grouped into a single file at 98% identity using cd-hit-est [ 35 , 81 ] and the expression of each transcript was estimated in transcripts per million (TPM) using the RSEM software v1.3.3 [ 81 ] after mapping the merged reads from each sample using Bowtie2.…”

“…Raw data were processed and searched using the search tool Peaks (Version 10.5) against an in-house database consisting of the protein sequences predicted from a nonredundant transcriptome master set of the same specimens used for venom extraction, using the same parameters as previously described [ 75 ]. Scaffold (version Scaffold_5.2.1) was used to validate MS/MS-based peptide and protein identifications.…”

Unveiling Novel Kunitz- and Waprin-Type Toxins in the Micrurus mipartitus Coral Snake Venom Gland: An In Silico Transcriptome Analysis

“…The assembly of the snake venom gland transcriptome was successfully achieved employing several assemblers [74][75][76]. Three assemblers were used, and the combined results were subjected to redundancy filtering for a final data set.…”