Global Transcriptome Analysis of the Scorpion Centruroides noxius: New Toxin Families and Evolutionary Insights from an Ancestral Scorpion Species

Herrera-Estrella A (2012) Global Transcriptome Analysis of the Scorpion Centruroides noxius: New Toxin Families and Evolutionary Insights from an Ancestral Scorpion Species. PLoS ONE 7(8): e43331. doi:10.1371/journal.pone.0043331 Global Transcriptome Analysis of the Scorpion Centruroides noxius : New Toxin Families and Evolutionary Insights from an Ancestral Scorpion Species Martha Rendo n-Anaya 0 Luis Delaye 0 Lourival D. Possani 0 Alfredo Herrera-Estrella 0 Cynthia Gibas, University of North Carolina at Charlotte, United States of America 0 1 Laboratorio Nacional de Gen o mica para la Biodiversidad, Centro de Investigacio n y de Estudios Avanzados (CINVESTAV) , Irapuato, Guanajuato , Me xico, 2 Departamento de Ingenier a Gene tica, Centro de Investigacio n y de Estudios Avanzados (CINVESTAV) , Irapuato, Guanajuato , Me xico, 3 Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnolog a, Universidad Nacional Auto noma de Me xico (UNAM) , Cuernavaca, Morelos, Me xico Scorpion venoms have been studied for decades, leading to the identification of hundreds of different toxins with medical and pharmacological implications. However, little emphasis has been given to the description of these arthropods from cellular and evolutionary perspectives. In this report, we describe a transcriptomic analysis of the Mexican scorpion Centruroides noxius Hoffmann, performed with a pyrosequencing platform. Three independent sequencing experiments were carried out, each including three different cDNA libraries constructed from RNA extracted from the whole body of the scorpion after telson removal, and from the venom gland before and after venom extraction. Over three million reads were obtained and assembled in almost 19000 isogroups. Within the telson-specific sequences, 72 isogroups (0.4% of total unique transcripts) were found to be similar to toxins previously reported in other scorpion species, spiders and sea anemones. The annotation pipeline also revealed the presence of important elements of the small non-coding RNA processing machinery, as well as microRNA candidates. A phylogenomic analysis of concatenated essential genes evidenced differential evolution rates in this species, particularly in ribosomal proteins and proteasome components. Additionally, statistical comparison of transcript abundance before and after venom extraction showed that 3% and 2% of the assembled isogroups had higher expression levels in the active and replenishing gland, respectively. Thus, our sequencing and annotation strategies provide a general view of the cellular and molecular processes that take place in these arthropods, allowed the discovery of new pharmacological and biotechnological targets and uncovered several regulatory and metabolic responses behind the assembly of the scorpion venom. The results obtained in this report represent the first high-throughput study that thoroughly describes the universe of genes that are expressed in the scorpion Centruroides noxius Hoffmann, a highly relevant organism from medical and evolutionary perspectives. - Funding: This work was partially supported by grant number IN204110 from Direccio n General del Personal Academico of National Autonomous University of Mexico (UNAM) (http://www.unam.mx/), and the support of Institute Bioclon S.A. de C.V. (http://www.bioclon.com.mx) to LDP. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Co-author Alfredo Herrera-Estrella is a PLOS ONE Editorial Board member. This does not alter the authors adherence to all the PLoS ONE policies on sharing data and materials. For decades, the study of venomous animals has focused on the isolation and biochemical characterization of specific venom components that have medical or biotechnological importance. Indeed, scorpions have been extensively studied under this optic, which has lead to the identification of hundreds of different transcripts encoding toxic peptides, and peptides with neurotoxic activities that are important not only in medical terms, but have been used as pharmacological tools to understand the function of different ion channels [1]. However, scorpions are not only interesting organisms only because of their toxin diversity, but also because they represent the most ancient terrestrial animals that fossil records have identified. About 2000 species have been described around the world, which also implies that scorpions are extremely well adapted arthropods that have managed to survive in different environmental conditions. Buthidae, the largest family of extant scorpions, has been proposed to be one of the most ancient or basal families [2]. Cladistic and phylogenetic analysis suggested that they arose ,350 million years ago [3,4] and, after a physical separation upon the partition of the Africa-South America continents (,150 My ago), several speciation events gave rise to different genus such as Buthus, Mesobuthus, Parabuthus, Hottentotta, Leiurus and Androctonus in Africa and Asia, and Tityus and Centruroides in South and North America, respectively [5]. In Mexico, the medically important scorpions that belong to the genus Centruroides are distributed along the states of the Pacific coast; among these, Centruroides noxius Hoffmann (from and then, simply abbreviated C. noxius) has been identified as the most dangerous species in the country (LD50 of 3.8 mg venom per 20 g mouse wt) [6]. In this context, it becomes clear that at the molecular and morphological levels, the venom glands of these arthropods have evolved over the past 400 million years giving as a result, a complex toxic arsenal that is efficiently used for prey and defence. Unfortunately, the evolutionary relevance of scorpions has been scarcely analyzed over the years. Different omic approaches have become a very powerful strategy for understanding the complexity of venomous animals; transcriptomics in particular, has been widely used to explore the transcriptional diversity of venom glands of several scorpion species. Except for the analysis of ESTs derived from resting or replete venom glands of the buthid scorpion Hottentota judaicus [7], the rest of the reported transcriptomes of scorpions from the Buthidae, Scorpionidae, Euscorpiidae, Caraboctonidae, Liochelidae and Iuridae families have shared a methodological principle: the RNA was collected from the venom glands 2 to 5 days after venom extraction by electric stimulation, implying that the gland is engaged in regenerating its venom, which can be referred to as active or replenishing state [812]. The high proportion of toxin-like transcripts identified in these reports (from 30 to 78% of the total ESTs) has emphasized the specialized role of the venom glands in the production of toxin peptides, unfortunately, the small number of tags generated by Sanger sequencing as well as the use of telson-specific RNA for random EST (...truncated)