Gene duplications are extensive and contribute significantly to the toxic proteome of nematocysts isolated from Acropora digitifera (Cnidaria: Anthozoa: Scleractinia)

Background: Gene duplication followed by adaptive selection is a well-accepted process leading to toxin diversification in venoms. However, emergent genomic, transcriptomic and proteomic evidence now challenges this role to be at best equivocal to other processess . Cnidaria are arguably the most ancient phylum of the extant metazoa that are venomous and such provide a definitive ancestral anchor to examine the evolution of this trait.\ud Methods: Here we compare predicted toxins from the translated genome of the coral Acropora digitifera to putative toxins revealed by proteomic analysis of soluble proteins discharged from nematocysts, to determine the extent to which gene duplications contribute to venom innovation in this reef-building coral species. A new bioinformatics tool called HHCompare was developed to detect potential gene duplications in the genomic data, which is made freely available (https://github.com/rgacesa/HHCompare).\ud Results: A total of 55 potential toxin encoding genes could be predicted from the A. digitifera genome, of which 36 (65 %) had likely arisen by gene duplication as evinced using the HHCompare tool and verified using two standard phylogeny methods. Surprisingly, only 22 % (12/55) of the potential toxin repertoire could be detected\ud following rigorous proteomic analysis, for which only half (6/12) of the toxin proteome could be accounted for as peptides encoded by the gene duplicates. Biological activities of these toxins are dominatedby putative phospholipases and toxic peptidases.\ud Conclusions: Gene expansions in A. digitifera venom are the most extensive yet described in any venomous animal, and gene duplication plays a significant role leading to toxin diversification in this coral species. Since such low numbers of toxins were detected in the proteome, it is unlikely that the venom is evolving rapidly by preydriven positive natural selection. Rather we contend that the venom has a defensive role deterring predation or\ud harm from interspecific competition and overgrowth by fouling organisms. Factors influencing translation of toxin encoding genes perhaps warrants more profound experimental consideration.United Kingdom Medical Research Council (MRC grant G82144A to R. Gacesa, D. Hranueli and P. F. Long)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP grants 2010/50174-7 to A. C. Morandini and 2011/50242-5 to A. C. Marques)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq grant 301039/2013-5 to A. C. Morandini)Universidade de São Paulo (USP grant 13.1.1502.9.8)NP-BioMar program at the Universidade de São Paul

Evolution of venoms in cnidarians

A ampla gama de animais peçonhentos, de medusas flutuantes a serpentes coleantes, tem provocado fascínio na história da humanidade. A evolução do veneno, cuja compreensão é um dos grandes desafios da biologia, seguramente teve um papel essencial na diversificação de diversas linhagens de animais. O veneno é um coquetel diverso de peptídeos farmacologicamente ativos, sais e moléculas orgânicas. A injeção de algumas dessas proteínas tóxicas, mesmo em quantidades ínfimas, pode paralisar e aniquilar presas em poucos minutos. Em Cnidaria, os nematocistos têm o papel de injetar o veneno, representando a mais antiga estrutura especializada de inoculação de veneno no reino animal. Porém, o conhecimento sobre o veneno de cnidários, sua composição e, especialmente, seu contexto evolutivo, é incipiente. Este estudo teve, como objetivo geral, ampliar a amostragem de táxons do filo Cnidaria nos quais seus venenos sejam caracterizados a partir de dados de transcriptomas e proteomas, e usar essas informações em um contexto comparativo para inovar na compreensão da evolução do veneno em Cnidaria. O capítulo 1, Beyond primary sequence Proteomic data reveal complex toxins in cnidarian venoms é um estudo comparativo sobre a composição do veneno de 11 espécies de cnidários gerados a partir de proteomas, inclusive um contexto mais amplo com venenos de animais peçonhentos em geral. O capítulo 2, Transcriptomic analyses and molecular evolution of cnidarian venoms traz uma análise transcriptômica de oito espécies de Medusozoa que caracteriza suas famílias de toxinas, além inferir as relações evolutivas e os padrões de seleção em cinco famílias de toxinas dentre as mais diversas do filo. O capítulo 3, Recruitment of toxin-like proteins with ancestral venom function supports endoparasitic lifestyles of Myxozoa, expande a compreensão sobre a evolução das toxinas em cnidários como um todo, enfocando o contexto evolutivo de reposicionamento das toxinas em um clado com modo de vida endoparasita. Por fim, o Capítulo 4, Reciprocal transplantation of the heterotrophic coral Tubastraea coccinea (Scleractinia: Dendrophylliidae) between distinct habitats did not alter its venom toxin composition, é um estudo experimental sobre a plasticidade de resposta intraespecífica, com origem ecológica e adaptativa, sobre a composição do veneno e dos nematocistos em um clones vivendo em diferentes ambientes.The wide range of venomous animals, from floating jellyfish to slithering snakes, has fascinated human history. The evolution of venom, whose understanding is one of the great challenges of biology, certainly played an essential role in the diversification of several animal lineages. Venom is a diverse cocktail of pharmacologically active peptides, salts and organic molecules. Injecting some of these toxic proteins, even in low amounts, can paralyze and annihilate prey within minutes. In Cnidaria, nematocysts have the role of injecting venom, representing the oldest specialized structure of venom inoculation in the animal kingdom. However, knowledge about the cnidarian venom, its composition and, especially, its evolutionary context, is incipient. This study aimed to broaden the sampling of Cnidaria phylum taxa in which their venoms are characterized from transcriptome and proteome data, and to use this information in a comparative context to innovate in understanding the evolution of the venom in Cnidaria. Chapter 1, \'Beyond primary sequence\' Proteomic data reveal complex toxins in cnidarian venoms is a comparative study of the venom composition of 11 species of cnidarians generated from proteomes, including a broader context with venoms from other venomous animal lineages. Chapter 2, \"Transcriptomic analyses and molecular evolution of cnidarian venoms\" provides a transcriptomic analysis of eight species of Medusozoa that characterizes their toxin families, in addition to inferring the evolutionary relationships and selection patterns in five toxin families, which are the most diverse in the phylum. Chapter 3, Recruitment of toxin-like proteins with ancestral venom function supports endoparasitic lifestyles of Myxozoa, expands our understanding of the evolution of toxins in cnidarians as a whole, focusing on the evolutionary context of repositioning toxins in a clade with mode of endoparasitic life. Finally, Chapter 4, \"Reciprocal transplantation of the heterotrophic coral Tubastraea coccinea (Scleractinia: Dendrophylliidae) between distinct habitats did not alter its venom toxin composition\", is an experimental study on the plasticity of the intraspecific response, with ecological and adaptive origin, on the composition of venom and nematocysts in clones living in different environment

Venom evolution in cnidarians based on genomes and proteomes data

A evolução do veneno, uma das misturas mais complexas da natureza, tem sustentado o sucesso da diversificação de inúmeras linhagens de animais. Serpentes deslizantes ou medusas flutuantes utilizam o veneno, um coquetel de peptídeos farmacologicamente ativos, sais e moléculas orgânicas. Esses animais surpreendentes têm provocado grande fascínio ao longo da história humana. Nesta dissertação propomos um estudo da evolução dos venenos no filo Cnidaria, englobando dados proteômicos e genômicos. Este projeto teve como objetivos: (1) caracterizar e elucidar a evolução da composição do veneno em Cnidaria por meio da comparação de listas de proteínas; (2) testar a hipótese de que a variação na família de toxinas específica de cnidários tem sido o resultado de um regime de seleção positiva; e (3) determinar a extensão em que a duplicação de genes pode ser considerada como a principal razão para a diversificação de toxinas em Cnidaria. O capítulo \"Comparative proteomics reveals common components of a powerful arsenal in the earliest animal venomous lineage, the cnidarians\" propõe o estudo comparado mais completo sobre a composição do veneno de cnidários e uma hipótese sobre a montagem evolutiva do complexo arsenal bioquímico de cnidários e do veneno ancestral desse grupo basal. Vinte e oito famílias de proteínas foram identificadas. Destas, 13 famílias foram registradas pela primeira vez no proteoma de Cnidaria. Pelo menos 15 famílias de toxinas foram recrutadas no proteoma de veneno de cnidários antes da diversificação dos grupos Anthozoa e Medusozoa. Nos capítulos \"Evidence of episodic positive selection in the evolution of jellyfish toxins of the cnidarian venom\" e \"Gene duplications are extensive and contribute significantly to the toxic proteome of nematocysts isolated from Acropora digitifera (Cnidaria: Anthozoa: Scleractinia)\", nossas análises demonstram que as famílias de toxinas nos cnidários se diversificam amplamente mediante a duplicação de genes. Além disso, em contraste com as famílias de toxinas do veneno na maioria das linhagens animais; nós identificamos um padrão diferente na família de toxinas específica de cnidários, em que há uma seleção purificadora por longos períodos seguindo longos tempos de diversificação ou vice-versaThe evolution of venom, nature\'s most complex concoction, has underpinned the success and diversification of numerous animal lineages. Slithering serpents or buoyant jellyfishes employ venom, a cocktail of pharmacologically active peptides, salts, and organic molecules. These astonishing animals have generated a great fascination throughout human history. In this dissertation, we propose a study of the evolution of venoms in the phylum Cnidaria, encompassing proteomic and genomic data. This project aimed: (1) to characterize and elucidate the evolution of venom composition in Cnidaria by comparing protein lists; (2) to test the hypothesis that the variation in specific family of cnidarians toxins has been the result of a positive selection regime; and (3) to determine the extent to which the genes duplication may be regarded as the main reason for the diversity of toxins in Cnidaria. The chapter \"Comparative proteomics reveals common components of a powerful arsenal in the earliest animal venomous lineage, the cnidarians\" presents the most comprehensive comparative study on the cnidarians venom composition and a hypothesis about the evolutionary assembly of the complex biochemical arsenal of cnidarians and of the ancestral venom of this basal group. Twenty eight protein families were identified. Of these, 13 families were described for the first time in the proteome of Cnidaria. At least 15 types of toxin families were recruited in cnidarians venom proteome before the diversification of Anthozoa and Medusozoa groups. In the chapters \"Evidence of episodic positive selection in the evolution of jellyfish toxins of the cnidarian venom\" and \"Gene duplications are extensive and contribute significantly to the toxic proteome of nematocysts isolated from Acropora digitifera (Cnidaria: Anthozoa: Scleractinia)\", our analyses indicate that the families of toxins in cnidarians diversify broadly through gene duplication. Besides, in contrast to the families of venom toxins in most animals lineages, we identified a different pattern in the specific family of cnidarians toxins, where there is a purifying selection for periods long, followed by long periods of diversification or vice vers

"beyond Primary Sequence" - Proteomic Data Reveal Complex Toxins in Cnidarian Venoms

Venomous animals can deploy toxins for both predation and defense. These dual functions of toxins might be expected to promote the evolution of new venoms and alteration of their composition. Cnidarians are the most ancient venomous animals but our present understanding of their venom diversity is compromised by poor taxon sampling. New proteomic data were therefore generated to characterize toxins in venoms of a staurozoan, a hydrozoan, and an anthozoan. We then used a novel clustering approach to compare venom diversity in cnidarians to other venomous animals. Comparison of the presence or absence of 32 toxin protein families indicated venom composition did not vary widely among the 11 cnidarian species studied. Unsupervised clustering of toxin peptide sequences suggested that toxin composition of cnidarian venoms is just as complex as that in many venomous bilaterians, including marine snakes. The adaptive significance of maintaining a complex and relatively invariant venom remains unclear. Future study of cnidarian venom diversity, venom variation with nematocyst types and in different body regions are required to better understand venom evolution

Comparative proteomics reveals recruitment patterns of some protein families in the venoms of Cnidaria

Cnidarians are probably the oldest group of animals to be venomous, yet our current picture of cnidarian venom evolution is highly imbalanced due to limited taxon sampling. High-throughput tandem mass spectrometry was used to determine venom composition of the scyphozoan Chrysaora lactea and two cubozoans Tamoya haplonema and Chiropsalmus quadrumanus. Protein recruitment patterns were then compared against 5 other cnidarian venom proteomes taken from the literature. A total of 28 putative toxin protein families were identified, many for the first time in Cnidaria. Character mapping analysis revealed that 17 toxin protein families with predominantly cytolytic biological activities were likely recruited into the cnidarian venom proteome before the lineage split between Anthozoa and Medusozoa. Thereafter, venoms of Medusozoa and Anthozoa differed during subsequent divergence of cnidarian classes. Recruitment and loss of toxin protein families did not correlate with accepted phylogenetic patterns of Cnidaria. Selective pressures that drive toxin diversification independent of taxonomic positioning have yet to be identified in Cnidaria and now warrant experimental consideration

Heat stress increases immune cell function in Hexacorallia

Climate change induced heat stress has increased coral bleaching events worldwide. Differentially regulated immune genes are one of the primary responses to heat stress suggesting that immune activation is critical. However, the cellular immune mechanisms of coral bleaching is currently unknown, and it is still not known if the immune response documented during heat stress is a consequence of bleaching or is directly caused by the heat stress itself. To address this question, we have used two model system sea anemones (Order: Actiniaria): Exaiptasia diaphana and Nematostella vectensis . E. diaphana is an established sea anemone model for algal symbiont interaction, while N. vectensis is an established sea anemone model that lacks the algal symbiont. Here, we examined the effect of increased temperature on phagocytic activity, as an indication of immune function. Our data shows that immune cell activity increases during heat stress, while small molecule pinocytosis remains unaffected. We observed an increase in cellular production of reactive oxygen species with increasing temperatures. We also found that the cellular immune activity was not affected by the presence of the Symbiodiniaceae. Our results suggest that the immune activity observed in heat-stress induced bleaching in corals is a fundamental and basic response independent of the bleaching effect. These results establish a foundation for improving our understanding of hexacorallian immune cell biology, and its potential role in coral bleaching