Highly conserved and extremely variable: the paradoxical pattern of toxin expression revealed by comparative venom-gland transcriptomics of Phalotris (Serpentes: Dipsadidae).

Although non-front fanged snakes account for almost two-thirds of snake diversity, most studies on venom composition and evolution focus exclusively on front-fanged species, which comprise most of the clinically relevant accidents. Comprehensive reports on venom composition of non-front fanged snakes are still scarce for several groups. In this study, we address such shortage of knowledge by providing new insights about the venom composition among species of Phalotris, a poorly studied Neotropical dipsadid genus. Phalotris are known for their specialized venom delivery system and toxic venoms, which can cause life-threatening accidents in humans. We evaluate the venom-gland transcriptome of Phalotris, comparing the following three South American species: P. reticulatus for the Araucaria Pine forests, P. lemniscatus for the Pampa grasslands, and P. mertensi for the Brazilian Cerrado. Our results indicate similar venom profiles, in which they share a high expression level of Kunitz-type inhibitors (KUNZ). On the other hand, comparative analyses revealed substantial differences in the expression levels of C-type lectins (CTL) and snake venom metalloproteinases (SVMP). The diverse set of SVMP and CTL isoforms shows signals of positive selection, and we also identified truncated forms of type III SVMPs, which resemble type II and type I SVMPs of viperids. Additionally, we identified a CNP precursor hosting a proline-rich region containing a BPP motif resembling those commonly detected in viperid venoms with hypotensive activity. Altogether, our results suggest an evolutionary history favoring high expression levels of few KUNZ isoforms in Phalotris venoms, contrasting with a highly diverse set of SVMP and CTL isoforms. Such diversity can be comparable with the venom variability observed in some viperids. Our findings highlight the extreme phenotypic diversity of non-front fanged snakes and the importance to allocate greater effort to study neglected groups of Colubroidea.

ToxCodAn-Genome: an automated pipeline for toxin-gene annotation in genome assembly of venomous lineages.

BACKGROUND: The rapid development of sequencing technologies resulted in a wide expansion of genomics studies using venomous lineages. This facilitated research focusing on understanding the evolution of adaptive traits and the search for novel compounds that can be applied in agriculture and medicine. However, the toxin annotation of genomes is a laborious and time-consuming task, and no consensus pipeline is currently available. No computational tool currently exists to address the challenges specific to toxin annotation and to ensure the reproducibility of the process. RESULTS: Here, we present ToxCodAn-Genome, the first software designed to perform automated toxin annotation in genomes of venomous lineages. This pipeline was designed to retrieve the full-length coding sequences of toxins and to allow the detection of novel truncated paralogs and pseudogenes. We tested ToxCodAn-Genome using 12 genomes of venomous lineages and achieved high performance on recovering their current toxin annotations. This tool can be easily customized to allow improvements in the final toxin annotation set and can be expanded to virtually any venomous lineage. ToxCodAn-Genome is fast, allowing it to run on any personal computer, but it can also be executed in multicore mode, taking advantage of large high-performance servers. In addition, we provide a guide to direct future research in the venomics field to ensure a confident toxin annotation in the genome being studied. As a case study, we sequenced and annotated the toxin repertoire of Bothrops alternatus, which may facilitate future evolutionary and biomedical studies using vipers as models. CONCLUSIONS: ToxCodAn-Genome is suitable to perform toxin annotation in the genome of venomous species and may help to improve the reproducibility of further studies. ToxCodAn-Genome and the guide are freely available at https://github.com/pedronachtigall/ToxCodAn-Genome.

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

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.

44 Current Challenges in miRNomics.

Mature microRNAs (miRNAs) are short RNA sequences about 18-24 nucleotide long, which provide the recognition key within RISC for the posttranscriptional regulation of target RNAs. Considering the canonical pathway, mature miRNAs are produced via a multistep process. Their transcription (pri-miRNAs) and first processing step via the microprocessor complex (pre-miRNAs) occur in the nucleus. Then they are exported into the cytosol, processed again by Dicer (dsRNA) and finally a single strand (mature miRNA) is incorporated into RISC (miRISC). The sequence of the incorporated miRNA provides the function of RNA target recognition via hybridization. Following binding of the target, the mRNA is either degraded or translation is inhibited, which ultimately leads to less protein production. Conversely, it has been shown that binding within the 5' UTR of the mRNA can lead to an increase in protein product. Regulation of homeostasis is very important for a cell; therefore, all steps in the miRNA-based regulation pathway, from transcription to the incorporation of the mature miRNA into RISC, are under tight control. While much research effort has been exerted in this area, the knowledgebase is not sufficient for accurately modelling miRNA regulation computationally. The computational prediction of miRNAs is, however, necessary because it is not feasible to investigate all possible pairs of a miRNA and its target, let alone miRNAs and their targets. We here point out open challenges important for computational modelling or for our general understanding of miRNA-based regulation and show how their investigation is beneficial. It is our hope that this collection of challenges will lead to their resolution in the near future.

ToxCodAn: a new toxin annotator and guide to venom gland transcriptomics.

MOTIVATION: Next-generation sequencing has become exceedingly common and has transformed our ability to explore nonmodel systems. In particular, transcriptomics has facilitated the study of venom and evolution of toxins in venomous lineages; however, many challenges remain. Primarily, annotation of toxins in the transcriptome is a laborious and time-consuming task. Current annotation software often fails to predict the correct coding sequence and overestimates the number of toxins present in the transcriptome. Here, we present ToxCodAn, a python script designed to perform precise annotation of snake venom gland transcriptomes. We test ToxCodAn with a set of previously curated transcriptomes and compare the results to other annotators. In addition, we provide a guide for venom gland transcriptomics to facilitate future research and use Bothrops alternatus as a case study for ToxCodAn and our guide. RESULTS: Our analysis reveals that ToxCodAn provides precise annotation of toxins present in the transcriptome of venom glands of snakes. Comparison with other annotators demonstrates that ToxCodAn has better performance with regard to run time ($>20x$ faster), coding sequence prediction ($>3x$ more accurate) and the number of toxins predicted (generating $>4x$ less false positives). In this sense, ToxCodAn is a valuable resource for toxin annotation. The ToxCodAn framework can be expanded in the future to work with other venomous lineages and detect novel toxins.

A comparative analysis of heart microRNAs in vertebrates brings novel insights into the evolution of genetic regulatory networks.

BACKGROUND: During vertebrate evolution, the heart has undergone remarkable changes that lead to morphophysiological differences in the fully formed heart of these species, such as chamber septation, heart rate frequency, blood pressure, and cardiac output volume. Despite these differences, the heart developmental process is guided by a core gene set conserved across vertebrates. Nonetheless, the regulatory mechanisms controlling the expression of genes involved in heart development and maintenance are largely uncharted. MicroRNAs (miRNAs) have been described as important regulatory elements in several biological processes, including heart biology. These small RNA molecules are broadly conserved in sequence and genomic context in metazoans. Mutations may occur in miRNAs and/or genes that contribute to the establishment of distinct repertoires of miRNA-target interactions, thereby favoring the differential control of gene expression and, consequently, the origin of novel phenotypes. In fact, several studies showed that miRNAs are integrated into genetic regulatory networks (GRNs) governing specific developmental programs and diseases. However, studies integrating miRNAs in vertebrate heart GRNs under an evolutionary perspective are still scarce. RESULTS: We comprehensively examined and compared the heart miRNome of 20 species representatives of the five major vertebrate groups. We found 54 miRNA families with conserved expression and a variable number of miRNA families with group-specific expression in fishes, amphibians, reptiles, birds, and mammals. We also detected that conserved miRNAs present higher expression levels and a higher number of targets, whereas the group-specific miRNAs present lower expression levels and few targets. CONCLUSIONS: Both the conserved and group-specific miRNAs can be considered modulators orchestrating the core and peripheral genes of heart GRNs of vertebrates, which can be related to the morphophysiological differences and similarities existing in the heart of distinct vertebrate groups. We propose a hypothesis to explain evolutionary differences in the putative functional roles of miRNAs in the heart GRNs analyzed. Furthermore, we present new insights into the molecular mechanisms that could be helping modulate the diversity of morphophysiology in the heart organ of vertebrate species.

MITGARD: an automated pipeline for mitochondrial genome assembly in eukaryotic species using RNA-seq data.

MOTIVATION: Over the past decade, the field of next-generation sequencing (NGS) has seen dramatic advances in methods and a decrease in costs. Consequently, a large expansion of data has been generated by NGS, most of which have originated from RNA-sequencing (RNA-seq) experiments. Because mitochondrial genes are expressed in most eukaryotic cells, mitochondrial mRNA sequences are usually co-sequenced within the target transcriptome, generating data that are commonly underused or discarded. Here, we present MITGARD, an automated pipeline that reliably recovers the mitochondrial genome from RNA-seq data from various sources. The pipeline identifies mitochondrial sequence reads based on a phylogenetically related reference, assembles them into contigs, and extracts a complete mtDNA for the target species. RESULTS: We demonstrate that MITGARD can reconstruct the mitochondrial genomes of several species throughout the tree of life. We noticed that MITGARD can recover the mitogenomes in different sequencing schemes and even in a scenario of low-sequencing depth. Moreover, we showed that the use of references from congeneric species diverging up to 30 million years ago (MYA) from the target species is sufficient to recover the entire mitogenome, whereas the use of species diverging between 30 and 60 MYA allows the recovery of most mitochondrial genes. Additionally, we provide a case study with original data in which we estimate a phylogenetic tree of snakes from the genus Bothrops, further demonstrating that MITGARD is suitable for use on biodiversity projects. MITGARD is then a valuable tool to obtain high-quality information for studies focusing on the phylogenetic and evolutionary aspects of eukaryotes and provides data for easily identifying a sample using barcoding, and to check for cross-contamination using third-party tools.

CodAn: predictive models for precise identification of coding regions in eukaryotic transcripts.

MOTIVATION: Characterization of the coding sequences (CDSs) is an essential step in transcriptome annotation. Incorrect identification of CDSs can lead to the prediction of non-existent proteins that can eventually compromise knowledge if databases are populated with similar incorrect predictions made in different genomes. Also, the correct identification of CDSs is important for the characterization of the untranslated regions (UTRs), which are known to be important regulators of the mRNA translation process. Considering this, we present CodAn (Coding sequence Annotator), a new approach to predict confident CDS and UTR regions in full or partial transcriptome sequences in eukaryote species. RESULTS: Our analysis revealed that CodAn performs confident predictions on full-length and partial transcripts with the strand sense of the CDS known or unknown. The comparative analysis showed that CodAn presents better overall performance than other approaches, mainly when considering the correct identification of the full CDS (i.e. correct identification of the start and stop codons). In this sense, CodAn is the best tool to be used in projects involving transcriptomic data. AVAILABILITY: CodAn is freely available at https://github.com/pedronachtigall/CodAn. CONTACT: aland@usp.br. SUPPLEMENTARY INFORMATION: Supplementary data are available at Briefings in Bioinformatics online.

Size Matters: An Evaluation of the Molecular Basis of Ontogenetic Modifications in the Composition of Bothrops jararacussu Snake Venom.

Ontogenetic changes in venom composition have been described in Bothrops snakes, but only a few studies have attempted to identify the targeted paralogues or the molecular mechanisms involved in modifications of gene expression during ontogeny. In this study, we decoded B. jararacussu venom gland transcripts from six specimens of varying sizes and analyzed the variability in the composition of independent venom proteomes from 19 individuals. We identified 125 distinct putative toxin transcripts, and of these, 73 were detected in venom proteomes and only 10 were involved in the ontogenetic changes. Ontogenetic variability was linearly related to snake size and did not correspond to the maturation of the reproductive stage. Changes in the transcriptome were highly predictive of changes in the venom proteome. The basic myotoxic phospholipases A2 (PLA2s) were the most abundant components in larger snakes, while in venoms from smaller snakes, PIII-class SVMPs were the major components. The snake venom metalloproteinases (SVMPs) identified corresponded to novel sequences and conferred higher pro-coagulant and hemorrhagic functions to the venom of small snakes. The mechanisms modulating venom variability are predominantly related to transcriptional events and may consist of an advantage of higher hematotoxicity and more efficient predatory function in the venom from small snakes.

Genome-wide microRNA screening in Nile tilapia reveals pervasive isomiRs' transcription, sex-biased arm switching and increasing complexity of expression throughout development.

MicroRNAs (miRNAs) are key regulators of gene expression in multicellular organisms. The elucidation of miRNA function and evolution depends on the identification and characterization of miRNA repertoire of strategic organisms, as the fast-evolving cichlid fishes. Using RNA-seq and comparative genomics we carried out an in-depth report of miRNAs in Nile tilapia (Oreochromis niloticus), an emergent model organism to investigate evo-devo mechanisms. Five hundred known miRNAs and almost one hundred putative novel vertebrate miRNAs have been identified, many of which seem to be teleost-specific, cichlid-specific or tilapia-specific. Abundant miRNA isoforms (isomiRs) were identified with modifications in both 5p and 3p miRNA transcripts. Changes in arm usage (arm switching) of nine miRNAs were detected in early development, adult stage and even between male and female samples. We found an increasing complexity of miRNA expression during ontogenetic development, revealing a remarkable synchronism between the rate of new miRNAs recruitment and morphological changes. Overall, our results enlarge vertebrate miRNA collection and reveal a notable differential ratio of miRNA arms and isoforms influenced by sex and developmental life stage, providing a better picture of the evolutionary and spatiotemporal dynamics of miRNAs.

Fishing Into the MicroRNA Transcriptome.

In the last decade, several studies have been focused on revealing the microRNA (miRNA) repertoire and determining their functions in farm animals such as poultry, pigs, cattle, and fish. These small non-protein coding RNA molecules (18-25 nucleotides) are capable of controlling gene expression by binding to messenger RNA (mRNA) targets, thus interfering in the final protein output. MiRNAs have been recognized as the main regulators of biological features of economic interest, including body growth, muscle development, fat deposition, and immunology, among other highly valuable traits, in aquatic livestock. Currently, the miRNA repertoire of some farmed fish species has been identified and characterized, bringing insights about miRNA functions, and novel perspectives for improving health and productivity. In this review, we summarize the current advances in miRNA research by examining available data on Neotropical and other key species exploited by fisheries and in aquaculture worldwide and discuss how future studies on Neotropical fish could benefit from this knowledge. We also make a horizontal comparison of major results and discuss forefront strategies for miRNA manipulation in aquaculture focusing on forward-looking ideas for forthcoming research.

Combining Results from Distinct MicroRNA Target Prediction Tools Enhances the Performance of Analyses.

Target prediction is generally the first step toward recognition of bona fide microRNA (miRNA)-target interactions in living cells. Several target prediction tools are now available, which use distinct criteria and stringency to provide the best set of candidate targets for a single miRNA or a subset of miRNAs. However, there are many false-negative predictions, and consensus about the optimum strategy to select and use the output information provided by the target prediction tools is lacking. We compared the performance of four tools cited in literature-TargetScan (TS), miRanda-mirSVR (MR), Pita, and RNA22 (R22), and we determined the most effective approach for analyzing target prediction data (individual, union, or intersection). For this purpose, we calculated the sensitivity, specificity, precision, and correlation of these approaches using 10 miRNAs (miR-1-3p, miR-17-5p, miR-21-5p, miR-24-3p, miR-29a-3p, miR-34a-5p, miR-124-3p, miR-125b-5p, miR-145-5p, and miR-155-5p) and 1,400 genes (700 validated and 700 non-validated) as targets of these miRNAs. The four tools provided a subset of high-quality predictions and returned few false-positive predictions; however, they could not identify several known true targets. We demonstrate that union of TS/MR and TS/MR/R22 enhanced the quality of in silico prediction analysis of miRNA targets. We conclude that the union rather than the intersection of the aforementioned tools is the best strategy for maximizing performance while minimizing the loss of time and resources in subsequent in vivo and in vitro experiments for functional validation of miRNA-target interactions.

MicroRNA-499 expression distinctively correlates to target genes sox6 and rod1 profiles to resolve the skeletal muscle phenotype in Nile tilapia.

A class of small non-coding RNAs, the microRNAs (miRNAs), has been shown to be essential for the regulation of specific cell pathways, including skeletal muscle development, maintenance and homeostasis in vertebrates. However, the relative contribution of miRNAs for determining the red and white muscle cell phenotypes is far from being fully comprehended. To better characterize the role of miRNA in skeletal muscle cell biology, we investigated muscle-specific miRNA (myomiR) signatures in Nile tilapia fish. Quantitative (RT-qPCR) and spatial (FISH) expression analyses revealed a highly differential expression (forty-four-fold) of miR-499 in red skeletal muscle compared to white skeletal muscle, whereas the remaining known myomiRs were equally expressed in both muscle cell types. Detailed examination of the miR-499 targets through bioinformatics led us to the sox6 and rod1 genes, which had low expression in red muscle cells according to RT-qPCR, FISH, and protein immunofluorescence profiling experiments. Interestingly, we verified that the high expression of miR-499 perfectly correlates with a low expression of sox6 and rod1 target genes, as verified by a distinctive predominance of mRNA destabilization and protein translational decay to these genes, respectively. Through a genome-wide comparative analysis of SOX6 and ROD1 protein domains and through an in silico gene regulatory network, we also demonstrate that both proteins are essentially similar in vertebrate genomes, suggesting their gene regulatory network may also be widely conserved. Overall, our data shed light on the potential regulation of targets by miR-499 associated with the slow-twitch muscle fiber type phenotype. Additionally the results provide novel insights into the evolutionary dynamics of miRNA and target genes enrolled in a putative constrained molecular pathway in the skeletal muscle cells of vertebrates.

A streamlined DNA tool for global identification of heavily exploited coastal shark species (genus Rhizoprionodon).

Obtaining accurate species-specific landings data is an essential step toward achieving sustainable shark fisheries. Globally distributed sharpnose sharks (genus Rhizoprionodon) exhibit life-history characteristics (rapid growth, early maturity, annual reproduction) that suggests that they could be fished in a sustainable manner assuming an investment in monitoring, assessment and careful management. However, obtaining species-specific landings data for sharpnose sharks is problematic because they are morphologically very similar to one another. Moreover, sharpnose sharks may also be confused with other small sharks (either small species or juveniles of large species) once they are processed (i.e., the head and fins are removed). Here we present a highly streamlined molecular genetics approach based on seven species-specific PCR primers in a multiplex format that can simultaneously discriminate body parts from the seven described sharpnose shark species commonly occurring in coastal fisheries worldwide. The species-specific primers are based on nucleotide sequence differences among species in the nuclear ribosomal internal transcribed spacer 2 locus (ITS2). This approach also distinguishes sharpnose sharks from a wide range of other sharks (52 species) and can therefore assist in the regulation of coastal shark fisheries around the world.