Studying Smaller and Neglected Organisms in Modern Evolutionary Venomics Implementing RNASeq (Transcriptomics)—A Critical Guide

Abstract

Venoms are evolutionary key adaptations that species employ for defense, predation or competition. However, the processes and forces that drive the evolution of venoms and their toxin components remain in many aspects understudied. In particular, the venoms of many smaller, neglected (mostly invertebrate) organisms are not characterized in detail, especially with modern methods. For the majority of these taxa, even their biology is only vaguely known. Modern evolutionary venomics addresses the question of how venoms evolve by applying a plethora of -omics methods. These recently became so sensitive and enhanced that smaller, neglected organisms are now more easily accessible to comparatively study their venoms. More knowledge about these taxa is essential to better understand venom evolution in general. The methodological core pillars of integrative evolutionary venomics are genomics, transcriptomics and proteomics, which are complemented by functional morphology and the field of protein synthesis and activity tests. This manuscript focuses on transcriptomics (or RNASeq) as one toolbox to describe venom evolution in smaller, neglected taxa. It provides a hands-on guide that discusses a generalized RNASeq workflow, which can be adapted, accordingly, to respective projects. For neglected and small taxa, generalized recommendations are difficult to give and conclusions need to be made individually from case to case. In the context of evolutionary venomics, this overview highlights critical points, but also promises of RNASeq analyses. Methodologically, these concern the impact of read processing, possible improvements by perfoming multiple and merged assemblies, and adequate quantification of expressed transcripts. Readers are guided to reappraise their hypotheses on venom evolution in smaller organisms and how robustly these are testable with the current transcriptomics toolbox. The complementary approach that combines particular proteomics but also genomics with transcriptomics is discussed as well. As recently shown, comparative proteomics is, for example, most important in preventing false positive identifications of possible toxin transcripts. Finally, future directions in transcriptomics, such as applying 3rd generation sequencing strategies to overcome difficulties by short read assemblies, are briefly addressed.

Proteo-Transcriptomic Characterization of the Venom from the Endoparasitoid Wasp Pimpla turionellae with Aspects on Its Biology and Evolution

“…Subsequently, we only discuss protein families with transcripts for which reads map back that derive from the venom gland tissue library and that were as well identified by at least one transcript via the proteome data (Mascot value ≥ 30 and number of fragments ≥ 2, search window of 0.002 Da, see Table S2). Applying this strict and extensive filtering we avoid the possible over-or mis-interpretation of the transcriptome data as recently discussed [46][47][48].…”

An Economic Dilemma between Molecular Weapon Systems May Explain an Arachno-Atypical Venom in Wasp Spiders (Argiope bruennichi)

“…This restricted analyses to members of the families Atracidae, Ctenidae, Theraphosidae, Sicariidae and Theridiidae, which represent a narrow sample of spider diversity [1,15]. More recently, the advent of high-throughput methods compatible with miniscule samples has provided the means to expand the scope of such studies from less-accessible species [12,29]. Combinations of genomics, transcriptomics, proteomics and microbiomics [30,31] now allow the analyses of venoms from previously neglected taxa [32] in an emerging field known as modern evolutionary venomics [29].…”

The Significance of Comparative Genomics in Modern Evolutionary Venomics

“…Another reason that venom systems of most taxa remain unstudied is that only in the last years the modern, methodological toolbox has been established to easily assess the composition of venoms and the mode of their delivery of so far neglected, in many cases very small and more difficult to access species (Sunagar et al, 2016;von Reumont, 2018). The research area that addresses all aspects of venom related research is nowadays called venomics, a term that was originally coined in 2004 for proteomic-based analyses on snake venoms (Juárez et al, 2004;Bazaa et al, 2005).…”

“…Today the term venomics reflects the combination of several new-omics technologies to conduct general research on venoms in an integrative approach, see also (Calvete, 2017). Besides new approaches to identify more effectively secreted proteins via proteomics, new sequencing technologies provide in unprecedented details the framework to study evolution of venoms and toxin expression (Gopalakrishnakone and Calvete, 2016;Sunagar et al, 2016;von Reumont, 2018). Particularly for small, so far neglected venomous species, transcriptomics and proteo-transcriptomics appear as the foremost methods to assess possible venom compositions and contribute to extend our comprehension of venom diversity in general.…”

“…Particularly for small, so far neglected venomous species, transcriptomics and proteo-transcriptomics appear as the foremost methods to assess possible venom compositions and contribute to extend our comprehension of venom diversity in general. Recent studies show however, that de novo transcriptome analyses need to be conducted carefully to avoid misinterpretation of the data, and a combination of both transcriptomics and proteomics is of the utmost importance (Holding et al, 2018;Smith and Undheim, 2018;von Reumont, 2018). Nevertheless, it should be kept in mind that most of these studies pursue the goal to describe the venom composition of previously neglected species.…”