Are Fireworms Venomous? Evidence for the Convergent Evolution of Toxin Homologs in Three Species of Fireworms (Annelida, Amphinomidae)

Amphinomids, more commonly known as fireworms, are a basal lineage of marine annelids characterized by the presence of defensive dorsal calcareous chaetae, which break off upon contact. It has long been hypothesized that amphinomids are venomous and use the chaetae to inject a toxic substance. However, studies investigating fireworm venom from a morphological or molecular perspective are scarce and no venom gland has been identified to date, nor any toxin characterized at the molecular level. To investigate this question, we analyzed the transcriptomes of three species of fireworms— Eurythoe complanata, Hermodice carunculata, andParamphinome jeffreysii—following a venomics approach to identify putative venom compounds. Our venomics pipeline involved de novo transcriptome assembly, open reading frame, and signal sequence prediction, followed by three different homology search strategies: BLAST, HMMER sequence, and HMMER domain. Following this pipeline, we identified 34 clusters of orthologous genes, representing 13 known toxin classes that have been repeatedly recruited into animal venoms. Specifically, the three species share a similar toxin profile with C-type lectins, peptidases, metalloproteinases, spider toxins, and CAP proteins found among the most highly expressed toxin homologs. Despite their great diversity, the putative toxins identified are predominantly involved in three major biological processes: hemostasis, inflammatory response, and allergic reactions, all of which are commonly disrupted after fireworm stings. Although the putative fireworm toxins identified here need to be further validated, our results strongly suggest that fireworms are venomous animals that use a complex mixture of toxins for defense against predators

Molluscs from deep bioclastic gravel bottoms of the Alborán Island (LIFE+ INDEMARES Project)

Resumen de posterDurante el proyecto LIFE+ INDEMARES Alborán (2011-2013), se tomaron diez muestras mediante arte de arrastre (bou de vara) en los fondos de cascajo profundo que aparecen entre 80 y 200 m en la plataforma insular de la isla de Alborán. Estos fondos están formados por restos calcáreos biógenos (bioclastos). En las muestras se han encontrado un total de 2.713 ejemplares vivos de moluscos pertenecientes a 147 especies (2 solenogastros, 2 poliplacóforos, 97 gasterópodos, 45 bivalvos, 1 cefalópodo). Los muestreos realizados han puesto de manifiesto la presencia de una alta riqueza de especies de invertebrados. En el caso de los moluscos, la riqueza específica de las muestras fue alta (entre 24 y 55 especies), mientras que las abundancias fueron entre moderadas y altas (entre 154 y 846 individuos), oscilando la diversidad de Shannon-Wiener (H’) entre 2,20 y 3,30. En las muestras estudiadas destaca la presencia de pocas especies comunes y de un elevado número de especies raras. Estas últimas pueden ser accidentales en estos fondos y comunes en otros hábitats próximos, pero otras, como Rugulina monterosatoi Aartsen y Bogi, 1987, Epitonium linctum (de Boury y Monterosato, 1890), Epitonium pseudonanum Bouchet y Warén, 1986, Strobiligera flammulata Bouchet y Warén, 1993, Cerithiopsis atalaya Watson, 1885, Cerithiopsis nofronii Amati, 1987 y Pseudobabylonella minima (Reeve, 1856), se consideran raras ya que no son frecuentes en ningún otro hábitat. La mayoría de las especies de moluscos encontradas en los fondos de cascajo de la isla de Alborán tienen una distribución atlántico-mediterránea, y están presentes normalmente en el Mediterráneo y en el Atlántico ibero-marroquí o más allá de esta zona. Se han encontrado dos especies de afinidad biogeográfica subtropical (costa occidental africana), cuya presencia es uno de los rasgos característicos del mar de Alborán: Mitrella pallaryi (Dautzenberg, 1891) y Nassarius denticulatus (A. Adams, 1852). Además, se ha recolectado viva una especie de reciente descripción, Trophonopsis alboranensis (Smriglio, Mariottini y Bonfitto, 1997), probablemente endémica de esta zona al no haberse encontrado fuera de la isla de Alborán. Los gasterópodos Anatoma aspera (Philippi, 1844), Xenophora crispa (König, 1825), Schilderia achatidea (Gray en G. B. Sowerby I, 1837), Trophonopsis alboranensis y Fusiturris similis (Bivona Ant. en Bivona And., 1838), así como los bivalvos Bathyarca pectunculoides (Scacchi, 1834), Similipecten similis (Laskey, 1811) y Parvamussium fenestratum (Forbes, 1844), entre otras especies, caracterizan el cascajo profundo en la plataforma de Alborán, y lo diferencian de los fondos coralígenos o de “maërl” adyacentes. Por su elevada diversidad y por ser un hábitat esencial para varias especies vulnerables (no sólo de moluscos), consideramos que los fondos de cascajo profundo deben ser incluidos en el Anexo I (Hábitats naturales de interés comunitario cuya conservación requiere la designación de zonas de especial conservación) de la Directiva 92/43/CEE de Hábitats, para lo cual se ha elaborado la correspondiente propuesta en el marco del proyecto LIFE+ INDEMARES.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

From Mollusks to Medicine: A Venomics Approach for the Discovery and Characterization of Therapeutics from Terebridae Peptide Toxins

Animal venoms comprise a diversity of peptide toxins that manipulate molecular targets such as ion channels and receptors, making venom peptides attractive candidates for the development of therapeutics to benefit human health. However, identifying bioactive venom peptides remains a significant challenge. In this review we describe our particular venomics strategy for the discovery, characterization, and optimization of Terebridae venom peptides, teretoxins. Our strategy reflects the scientific path from mollusks to medicine in an integrative sequential approach with the following steps: (1) delimitation of venomous Terebridae lineages through taxonomic and phylogenetic analyses; (2) identification and classification of putative teretoxins through omics methodologies, including genomics, transcriptomics, and proteomics; (3) chemical and recombinant synthesis of promising peptide toxins; (4) structural characterization through experimental and computational methods; (5) determination of teretoxin bioactivity and molecular function through biological assays and computational modeling; (6) optimization of peptide toxin affinity and selectivity to molecular target; and (7) development of strategies for effective delivery of venom peptide therapeutics. While our research focuses on terebrids, the venomics approach outlined here can be applied to the discovery and characterization of peptide toxins from any venomous taxa

Transcriptome Sequencing and Annotation of the Polychaete Hermodice Carunculata (Annelida, Amphinomidae)

Background: The amphinomid polychaete Hermodice carunculata is a cosmopolitan and ecologically important omnivore in coral reef ecosystems, preying on a diverse suite of reef organisms and potentially acting as a vector for coral disease. While amphinomids are a key group for determining the root of the Annelida, their phylogenetic position has been difficult to resolve, and their publically available genomic data was scarce. Results: We performed deep transcriptome sequencing (Illumina HiSeq) and profiling on Hermodice carunculata collected in the Western Atlantic Ocean. We focused this study on 58,454 predicted Open Reading Frames (ORFs) of genes longer than 200 amino acids for our homology search, and Gene Ontology (GO) terms and InterPro IDs were assigned to 32,500 of these ORFs. We used this de novo assembled transcriptome to recover major signaling pathways and housekeeping genes. We also identify a suite of H. carunculata genes related to reproduction and immune response. Conclusions: We provide a comprehensive catalogue of annotated genes for Hermodice carunculata and expand the knowledge of reproduction and immune response genes in annelids, in general. Overall, this study vastly expands the available genomic data for H. carunculata, of which previously consisted of only 279 nucleotide sequences in NCBI. This underscores the utility of Illumina sequencing for de novo transcriptome assembly in non-model organisms as a cost-effective and efficient tool for gene discovery and downstream applications, such as phylogenetic analysis and gene expression profiling

The cuticle modulates ultraviolet reflectance of avian eggshells

ABSTRACT Avian eggshells are variedly coloured, yet only two pigments, biliverdin and protoporphyrin IX, are known to contribute to the dramatic diversity of their colours. By contrast, the contributions of structural or other chemical components of the eggshell are poorly understood. For example, unpigmented eggshells, which appear white to the human eye, vary in their ultraviolet (UV) reflectance, which may be detectable by birds. We investigated the proximate mechanisms for the variation in UV-reflectance of unpigmented bird eggshells using spectrophotometry, electron microscopy, chemical analyses, and experimental manipulations. We specifically tested how UV-reflectance is affected by the eggshell cuticle, the outermost layer of most avian eggshells. The chemical dissolution of the outer eggshell layers, including the cuticle, increased UV-reflectance for only eggshells that contained a cuticle. Our findings demonstrate that the outer eggshell layers, including the cuticle, absorb UV-light, probably because they contain higher levels of organic components and other chemicals, such as calcium phosphates, compared to the predominantly calcite-based eggshell matrix. These data highlight the need to examine factors other than the known pigments in studies of avian eggshell colour

Molecular Diversity and Gene Evolution of the Venom Arsenal of Terebridae Predatory Marine Snails

Venom peptides from predatory organisms are a resource for investigating evolutionary processes such as adaptive radiation or diversification, and exemplify promising targets for biomedical drug development. Terebridae are an understudied lineage of conoidean snails,which also includes cone snails and turrids. Characterization of cone snail venompeptides, conotoxins, has revealed a cocktail of bioactive compounds used to investigate physiological cellular function, predator-prey interactions, and to develop novel therapeutics. However, venom diversity of other conoidean snails remains poorly understood. The present research applies a venomics approach to characterize novel terebrid venom peptides, teretoxins, from the venom gland transcriptomes of Triplostephanus anilis and Terebra subulata. Next-generation sequencing and de novo assembly identified 139 putative teretoxins that were analyzed for the presence of canonical peptide features as identified in conotoxins. To meet the challenges of de novo assembly, multiple approaches for cross validation of findings were performed to achieve reliable assemblies of venom duct transcriptomes and to obtain a robust portrait of Terebridae venom. Phylogenetic methodology was used to identify 14 teretoxin gene superfamilies for the first time, 13 of which are unique to the Terebridae. Additionally, basic local algorithm search tool homologybased searches to venom-related genes and posttranslational modification enzymes identified a convergence of certain venom proteins, suchas actinoporin, commonly foundinvenoms. This research provides novel insights intovenomevolutionandrecruitment in Conoidean predatory marine snails and identifies a plethora of terebrid venom peptides that can be used to investigate fundamental questions pertaining to gene evolution

Modern venomics--Current insights, novel methods, and future perspectives in biological and applied animal venom research

Venoms have evolved >100 times in all major animal groups, and their components, known as toxins, have been fine-tuned over millions of years into highly effective biochemical weapons. There are many outstanding questions on the evolution of toxin arsenals, such as how venom genes originate, how venom contributes to the fitness of venomous species, and which modifications at the genomic, transcriptomic, and protein level drive their evolution. These questions have received particularly little attention outside of snakes, cone snails, spiders, and scorpions. Venom compounds have further become a source of inspiration for translational research using their diverse bioactivities for various applications. We highlight here recent advances and new strategies in modern venomics and discuss how recent technological innovations and multi-omic methods dramatically improve research on venomous animals. The study of genomes and their modifications through CRISPR and knockdown technologies will increase our understanding of how toxins evolve and which functions they have in the different ontogenetic stages during the development of venomous animals. Mass spectrometry imaging combined with spatial transcriptomics, in situ hybridization techniques, and modern computer tomography gives us further insights into the spatial distribution of toxins in the venom system and the function of the venom apparatus. All these evolutionary and biological insights contribute to more efficiently identify venom compounds, which can then be synthesized or produced in adapted expression systems to test their bioactivity. Finally, we critically discuss recent agrochemical, pharmaceutical, therapeutic, and diagnostic (so-called translational) aspects of venoms from which humans benefit.This work is funded by the European Cooperation in Science and Technology (COST, www.cost.eu) and based upon work from the COST Action CA19144 – European Venom Network (EUVEN, see https://euven-network.eu/). This review is an outcome of EUVEN Working Group 2 (“Best practices and innovative tools in venomics”) led by B.M.v.R. As coordinator of the group Animal Venomics until end 2021 at the Institute for Insectbiotechnology, JLU Giessen, B.M.v.R. acknowledges the Centre for Translational Biodiversity Genomics (LOEWE-TBG) in the programme “LOEWE – Landes-Offensive zur Entwicklung Wissenschaftlich-ökonomischer Exzellenz” of Hesse's Ministry of Higher Education, Research, and the Arts. B.M.v.R. and I.K. further acknowledge funding on venom research by the German Science Foundation to B.M.v.R. (DFG RE3454/6-1). A.C., A.V., and G.Z. were supported by the European Union's Horizon 2020 Research and Innovation program through Marie Sklodowska-Curie Individual Fellowships (grant agreements No. A.C.: 896849, A.V.: 841576, and G.Z.: 845674). M.P.I. is supported by the TALENTO Program by the Regional Madrid Government (2018-T1/BIO-11262). T.H.'s venom research is funded by the DFG projects 271522021 and 413120531. L.E. was supported by grant No. 7017-00288 from the Danish Council for Independent Research (Technology and Production Sciences). N.I. acknowledges funding on venom research by the Research Fund of Nevsehir Haci Bektas Veli University (project Nos. ABAP20F28, BAP18F26). M.I.K. and A.P. acknowledge support from GSRT National Research Infrastructure structural funding project INSPIRED (MIS 5002550). G.A. acknowledges support from the Slovenian Research Agency grants P1-0391, J4-8225, and J4-2547. G.G. acknowledges support from the Institute for Medical Research and Occupational Health, Zagreb, Croatia. E.A.B.U. is supported by a Norwegian Research Council FRIPRO-YRT Fellowship No. 287462

Convergent Evolution of Biochemical Innovations in Polychaetes: Characterizing the Molecular Basis of Bioluminescence and Venom Production

Tesis doctoral inédita cotutelada por la City University of New York y la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología. Fecha de lectura: 26-01-2018Esta tesis tiene embargado el acceso al texto completo hasta el 26-07-2019La presente tesis doctoral investiga las bases moleculares de dos innovaciones bioquímicas convergentes en anélidos poliquetos, la bioluminiscencia y la producción de veneno. Se usa transcriptómica comparativa y filogenia para investigar las bases moleculares y la evolución de estas dos adaptaciones. En el Capítulo 1 hacemos una introducción a la bioluminiscencia y veneno en Annelida. En el Capítulo 2, investigamos la filogenia molecular de Odontosyllis y proporcionamos datos sobre el papel del cortejo bioluminiscente en su evolución y patrones de especiación. En el Capítulo 3, usamos una metodología de transcriptómica comparativa para identificar genes candidatos relacionados con la producción de luz en dos especies de Odontosyllis, O. phosphorea y O. enopla. En el Capítulo 4, llevamos a cabo un análisis de expresión génica diferencial en la especie bioluminiscente Harmothoe areolata para identificar genes candidatos involucrados en la producción de luz. Finalmente, en los Capítulos 5 y 6 utilizamos una aproximación venómica para identificar homológos de toxinas en los transcriptomas de tres especies de gusanos de fuego (Hermodice carunculata, Eurythoe complanata and Paramphinome jeffreysii) y tres polinoidos (Harmothoe areolata, Harmothoe imbricata and Lepidonotopodium sp.

Distinct patterns of gene expression during regeneration and asexual reproduction in the annelid Pristina leidyi

Regeneration, the ability to replace lost body parts, is a widespread phenomenon in the animal kingdom often connected to asexual reproduction or fission, since the only difference between the two appears to be the stimulus that triggers them. Both developmental processes have largely been characterized; however, the molecular toolkit and genetic mechanisms underlying these events remain poorly unexplored. Annelids, in particular the oligochaete Pristina leidyi, provide a good model system to investigate these processes as they show diverse ways to regenerate, and can reproduce asexually through fission under laboratory conditions. Here, we used a comparative transcriptomics approach based on RNA-sequencing and differential gene expression analyses to understand the molecular mechanisms involved in anterior regeneration and asexual reproduction. We found 291 genes upregulated during anterior regeneration, including several regeneration-related genes previously reported in other annelids such as frizzled, paics, and vdra. On the other hand, during asexual reproduction, 130 genes were found upregulated, and unexpectedly, many of them were related to germline development during sexual reproduction. We also found important differences between anterior regeneration and asexual reproduction, with the latter showing a gene expression profile more similar to that of control individuals. Nevertheless, we identified 35 genes that were upregulated in both conditions, many of them related to cell pluripotency, stem cells, and cell proliferation. Overall, our results shed light on the molecular mechanisms that control anterior regeneration and asexual reproduction in annelids and reveal similarities with other animals, suggesting that the genetic machinery controlling these processes is conserved across metazoansH2020 Marie Sklodowska‐Curie Actions, Grant/Award Number: 841576; Comunidad de Madrid, Grant/Award Number: SI1/PJI/ 2019‐00532; Universidad Autónoma de Madrid, Grant/Award Number: 3931‐326F‐ 726DP6A38‐4F77; European Molecular Biology Organization, Grant/Award Number: ALTF‐217‐201