Sea Anemone Membrane Attack Complex/Perforin Superfamily Demonstrates an Evolutionary Transitional State between Venomous and Developmental Functions.

Gene duplication is a major force driving evolutionary innovation. A classic example is generating new animal toxins via duplication of physiological protein-encoding genes and recruitment into venom. While this process drives the innovation of many animal venoms, reverse recruitment of toxins into nonvenomous cells remains unresolved. Using comparative genomics, we find members of the Membrane Attack Complex and Perforin Family (MAC) have been recruited into venom-injecting cells (cnidocytes), in soft and stony corals and sea anemones, suggesting that the ancestral MAC was a cnidocyte expressed toxin. Further investigation into the model sea anemone Nematostella vectensis reveals that three members have undergone Nematostella-specific duplications leading to their reverse recruitment into endomesodermal cells. Furthermore, simultaneous knockdown of all three endomesodermally expressed MACs leads to mis-development, supporting that these paralogs have nonvenomous function. By resolving the evolutionary history and function of MACs in Nematostella, we provide the first proof for reverse recruitment from venom to organismal development.

Venom trade-off shapes interspecific interactions, physiology, and reproduction.

The ability of an animal to effectively capture prey and defend against predators is pivotal for survival. Venom is often a mixture of many components including toxin proteins that shape predator-prey interactions. Here, we used the sea anemone Nematostella vectensis to test the impact of toxin genotypes on predator-prey interactions. We developed a genetic manipulation technique to demonstrate that both transgenically deficient and a native Nematostella strain lacking a major neurotoxin (Nv1) have a reduced ability to defend themselves against grass shrimp, a native predator. In addition, secreted Nv1 can act indirectly in defense by attracting mummichog fish, which prey on grass shrimp. Here, we provide evidence at the molecular level of an animal-specific tritrophic interaction between a prey, its antagonist, and a predator. Last, this study reveals an evolutionary trade-off, as the reduction of Nv1 levels allows for faster growth and increased reproductive rates.

An ancient pan-cnidarian microRNA regulates stinging capsule biogenesis in Nematostella vectensis.

An ancient evolutionary innovation of a novel cell type, the stinging cell (cnidocyte), appeared >600 million years ago in the phylum Cnidaria (sea anemones, corals, hydroids, and jellyfish). A complex bursting nano-injector of venom, the cnidocyst, is embedded in cnidocytes and enables cnidarians to paralyze their prey and predators, contributing to this phylum's evolutionary success. In this work, we show that post-transcriptional regulation by a pan-cnidarian microRNA, miR-2022, is essential for biogenesis of these cells in the sea anemone Nematostella vectensis. By manipulation of miR-2022 levels in a transgenic reporter line of cnidocytes, followed by transcriptomics, single-cell data analysis, prey paralysis assays, and cell sorting of transgenic cnidocytes, we reveal that miR-2022 enables cnidocyte biogenesis in Nematostella, while exhibiting a conserved expression domain with its targets in cnidocytes of other cnidarian species. Thus, here we revealed a functional basis to the conservation of one of nature's most ancient microRNAs.

Genomic, functional and structural analyses elucidate evolutionary innovation within the sea anemone 8 toxin family.

BACKGROUND: The ShK toxin from Stichodactyla helianthus has established the therapeutic potential of sea anemone venom peptides, but many lineage-specific toxin families in Actiniarians remain uncharacterised. One such peptide family, sea anemone 8 (SA8), is present in all five sea anemone superfamilies. We explored the genomic arrangement and evolution of the SA8 gene family in Actinia tenebrosa and Telmatactis stephensoni, characterised the expression patterns of SA8 sequences, and examined the structure and function of SA8 from the venom of T. stephensoni. RESULTS: We identified ten SA8-family genes in two clusters and six SA8-family genes in five clusters for T. stephensoni and A. tenebrosa, respectively. Nine SA8 T. stephensoni genes were found in a single cluster, and an SA8 peptide encoded by an inverted SA8 gene from this cluster was recruited to venom. We show that SA8 genes in both species are expressed in a tissue-specific manner and the inverted SA8 gene has a unique tissue distribution. While the functional activity of the SA8 putative toxin encoded by the inverted gene was inconclusive, its tissue localisation is similar to toxins used for predator deterrence. We demonstrate that, although mature SA8 putative toxins have similar cysteine spacing to ShK, SA8 peptides are distinct from ShK peptides based on structure and disulfide connectivity. CONCLUSIONS: Our results provide the first demonstration that SA8 is a unique gene family in Actiniarians, evolving through a variety of structural changes including tandem and proximal gene duplication and an inversion event that together allowed SA8 to be recruited into the venom of T. stephensoni.

Micro and macroevolution of sea anemone venom phenotype.

Venom is a complex trait with substantial inter- and intraspecific variability resulting from strong selective pressures acting on the expression of many toxic proteins. However, understanding the processes underlying toxin expression dynamics that determine the venom phenotype remains unresolved. By interspecific comparisons we reveal that toxin expression in sea anemones evolves rapidly and that in each species different toxin family dictates the venom phenotype by massive gene duplication events. In-depth analysis of the sea anemone, Nematostella vectensis, revealed striking variation of the dominant toxin (Nv1) diploid copy number across populations (1-24 copies) resulting from independent expansion/contraction events, which generate distinct haplotypes. Nv1 copy number correlates with expression at both the transcript and protein levels with one population having a near-complete loss of Nv1 production. Finally, we establish the dominant toxin hypothesis which incorporates observations in other venomous lineages that animals have convergently evolved a similar strategy in shaping their venom.

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.

Functional characterization of a 'plant-like' HYL1 homolog in the cnidarian Nematostella vectensis indicates a conserved involvement in microRNA biogenesis.

While the biogenesis of microRNAs (miRNAs) in both animals and plants depends on the RNase III Dicer, its partner proteins are considered distinct for each kingdom. Nevertheless, recent discovery of homologs of Hyponastic Leaves1 (HYL1), a 'plant-specific' Dicer partner, in the metazoan phylum Cnidaria, challenges the view that miRNAs evolved convergently in animals and plants. Here, we show that the HYL1 homolog Hyl1-like a (Hyl1La) is crucial for development and miRNA biogenesis in the cnidarian model Nematostella vectensis. Inhibition of Hyl1La by morpholinos resulted in metamorphosis arrest in Nematostella embryos and a significant reduction in levels of most miRNAs. Further, meta-analysis of morphants of miRNA biogenesis components, like Dicer1, shows clustering of their miRNA profiles with Hyl1La morphants. Strikingly, immunoprecipitation of Hyl1La followed by quantitative PCR revealed that in contrast to the plant HYL1, Hyl1La interacts only with precursor miRNAs and not with primary miRNAs. This was complemented by an in vitro binding assay of Hyl1La to synthetic precursor miRNA. Altogether, these results suggest that the last common ancestor of animals and plants carried a HYL1 homolog that took essential part in miRNA biogenesis and indicate early emergence of the miRNA system before plants and animals separated.

In both animals and plants, small molecules known as micro ribonucleic acids (or miRNAs for short) control the amount of proteins cells make from instructions encoded in their DNA. Cells make mature miRNA molecules by cutting and modifying newly-made RNA molecules in two stages. Some of the components animals and plants utilize to make and use miRNAs are similar, but most are completely different. For example, in plants an enzyme known as Dicer cuts newly made RNAs into mature miRNAs with the help of a protein called HYL1, whereas humans and other animals do not have HYL1 and Dicer works with alternative partner proteins, instead. Therefore, it is generally believed that miRNAs evolved separately in animals and plants after they split from a common ancestor around 1.6 billion years ago. Recent studies on sea anemones and other primitive animals challenge this idea. Proteins similar to HYL1 in plants have been discovered in sea anemones and sponges, and sea anemone miRNAs show several similarities to plant miRNAs including their mode of action. However, it is not clear whether these HYL1-like proteins work in the same way as their plant counterparts. Here, Tripathi, Admoni et al. investigated the role of the HYL1-like protein in sea anemones. The experiments found that this protein was essential for the sea anemones to make miRNAs and to grow and develop properly. Unlike HYL1 in plants ­ which is involved in both stages of processing newly-made miRNAs into mature miRNAs ­ the sea anemone HYL1-like protein only helped in the second stage to make mature miRNAs from intermediate molecules known as precursor miRNAs. These findings demonstrate that some of the components plants use to make miRNAs also perform similar roles in sea anemones. This suggests that the miRNA system evolved before the ancestors of plants and animals separated from each other. Questions for future studies will include investigating how plants and animals evolved different miRNA machinery, and why sponges and jellyfish have HYL1-like proteins, whereas humans and other more complex animals do not.

Transposons Increase Transcriptional Complexity: The Good Parasite?

A recent study by Cosby et al. sheds light on the role of transposons in the adaptive evolution of their hosts. These genetic elements were thought to be largely deleterious. However, when coupled with alternative splicing, there appears to be an exponential increase in the diversity of proteins encoded, which display novel functions and are conserved by natural selection.

Insights into how development and life-history dynamics shape the evolution of venom.

Venomous animals are a striking example of the convergent evolution of a complex trait. These animals have independently evolved an apparatus that synthesizes, stores, and secretes a mixture of toxic compounds to the target animal through the infliction of a wound. Among these distantly related animals, some can modulate and compartmentalize functionally distinct venoms related to predation and defense. A process to separate distinct venoms can occur within and across complex life cycles as well as more streamlined ontogenies, depending on their life-history requirements. Moreover, the morphological and cellular complexity of the venom apparatus likely facilitates the functional diversity of venom deployed within a given life stage. Intersexual variation of venoms has also evolved further contributing to the massive diversity of toxic compounds characterized in these animals. These changes in the biochemical phenotype of venom can directly affect the fitness of these animals, having important implications in their diet, behavior, and mating biology. In this review, we explore the current literature that is unraveling the temporal dynamics of the venom system that are required by these animals to meet their ecological functions. These recent findings have important consequences in understanding the evolution and development of a convergent complex trait and its organismal and ecological implications.

Toxin-like neuropeptides in the sea anemone Nematostella unravel recruitment from the nervous system to venom.

The sea anemone Nematostella vectensis (Anthozoa, Cnidaria) is a powerful model for characterizing the evolution of genes functioning in venom and nervous systems. Although venom has evolved independently numerous times in animals, the evolutionary origin of many toxins remains unknown. In this work, we pinpoint an ancestral gene giving rise to a new toxin and functionally characterize both genes in the same species. Thus, we report a case of protein recruitment from the cnidarian nervous to venom system. The ShK-like1 peptide has a ShKT cysteine motif, is lethal for fish larvae and packaged into nematocysts, the cnidarian venom-producing stinging capsules. Thus, ShK-like1 is a toxic venom component. Its paralog, ShK-like2, is a neuropeptide localized to neurons and is involved in development. Both peptides exhibit similarities in their functional activities: They provoke contraction in Nematostella polyps and are toxic to fish. Because ShK-like2 but not ShK-like1 is conserved throughout sea anemone phylogeny, we conclude that the two paralogs originated due to a Nematostella-specific duplication of a ShK-like2 ancestor, a neuropeptide-encoding gene, followed by diversification and partial functional specialization. ShK-like2 is represented by two gene isoforms controlled by alternative promoters conferring regulatory flexibility throughout development. Additionally, we characterized the expression patterns of four other peptides with structural similarities to studied venom components and revealed their unexpected neuronal localization. Thus, we employed genomics, transcriptomics, and functional approaches to reveal one venom component, five neuropeptides with two different cysteine motifs, and an evolutionary pathway from nervous to venom system in Cnidaria.

Some like it hot: population-specific adaptations in venom production to abiotic stressors in a widely distributed cnidarian.

BACKGROUND: In cnidarians, antagonistic interactions with predators and prey are mediated by their venom, whose synthesis may be metabolically expensive. The potentially high cost of venom production has been hypothesized to drive population-specific variation in venom expression due to differences in abiotic conditions. However, the effects of environmental factors on venom production have been rarely demonstrated in animals. Here, we explore the impact of specific abiotic stresses on venom production of distinct populations of the sea anemone Nematostella vectensis (Actiniaria, Cnidaria) inhabiting estuaries over a broad geographic range where environmental conditions such as temperatures and salinity vary widely. RESULTS: We challenged Nematostella polyps with heat, salinity, UV light stressors, and a combination of all three factors to determine how abiotic stressors impact toxin expression for individuals collected across this species' range. Transcriptomics and proteomics revealed that the highly abundant toxin Nv1 was the most downregulated gene under heat stress conditions in multiple populations. Physiological measurements demonstrated that venom is metabolically costly to produce. Strikingly, under a range of abiotic stressors, individuals from different geographic locations along this latitudinal cline modulate differently their venom production levels. CONCLUSIONS: We demonstrate that abiotic stress results in venom regulation in Nematostella. Together with anecdotal observations from other cnidarian species, our results suggest this might be a universal phenomenon in Cnidaria. The decrease in venom production under stress conditions across species coupled with the evidence for its high metabolic cost in Nematostella suggests downregulation of venom production under certain conditions may be highly advantageous and adaptive. Furthermore, our results point towards local adaptation of this mechanism in Nematostella populations along a latitudinal cline, possibly resulting from distinct genetics and significant environmental differences between their habitats.

The Rapid Regenerative Response of a Model Sea Anemone Species Exaiptasia pallida Is Characterised by Tissue Plasticity and Highly Coordinated Cell Communication.

Regeneration of a limb or tissue can be achieved through multiple different pathways and mechanisms. The sea anemone Exaiptasia pallida has been observed to have excellent regenerative proficiency, but this has not yet been described transcriptionally. In this study, we examined the genetic expression changes during a regenerative timecourse and reported key genes involved in regeneration and wound healing. We found that the major response was an early (within the first 8 h) upregulation of genes involved in cellular movement and cell communication, which likely contribute to a high level of tissue plasticity resulting in the rapid regeneration response observed in this species. We find the immune system was only transcriptionally active in the first 8 h post-amputation and conclude, in accordance with previous literature, that the immune system and regeneration have an inverse relationship. Fifty-nine genes (3.8% of total) differentially expressed during regeneration were identified as having no orthologues in other species, indicating that regeneration in E. pallida may rely on the activation of species-specific novel genes. Additionally, taxonomically restricted novel genes, including species-specific novels, and highly conserved genes were identified throughout the regenerative timecourse, showing that both may work in concert to achieve complete regeneration.

A Versatile and Robust Serine Protease Inhibitor Scaffold from Actinia tenebrosa.

Serine proteases play pivotal roles in normal physiology and a spectrum of patho-physiological processes. Accordingly, there is considerable interest in the discovery and design of potent serine protease inhibitors for therapeutic applications. This led to concerted efforts to discover versatile and robust molecular scaffolds for inhibitor design. This investigation is a bioprospecting study that aims to isolate and identify protease inhibitors from the cnidarian Actinia tenebrosa. The study isolated two Kunitz-type protease inhibitors with very similar sequences but quite divergent inhibitory potencies when assayed against bovine trypsin, chymostrypsin, and a selection of human sequence-related peptidases. Homology modeling and molecular dynamics simulations of these inhibitors in complex with their targets were carried out and, collectively, these methodologies enabled the definition of a versatile scaffold for inhibitor design. Thermal denaturation studies showed that the inhibitors were remarkably robust. To gain a fine-grained map of the residues responsible for this stability, we conducted in silico alanine scanning and quantified individual residue contributions to the inhibitor's stability. Sequences of these inhibitors were then used to search for Kunitz homologs in an A. tenebrosa transcriptome library, resulting in the discovery of a further 14 related sequences. Consensus analysis of these variants identified a rich molecular diversity of Kunitz domains and expanded the palette of potential residue substitutions for rational inhibitor design using this domain.

The draft genome of Actinia tenebrosa reveals insights into toxin evolution.

Sea anemones have a wide array of toxic compounds (peptide toxins found in their venom) which have potential uses as therapeutics. To date, the majority of studies characterizing toxins in sea anemones have been restricted to species from the superfamily, Actinioidea. No highly complete draft genomes are currently available for this superfamily, however, highlighting our limited understanding of the genes encoding toxins in this important group. Here we have sequenced, assembled, and annotated a draft genome for Actinia tenebrosa. The genome is estimated to be approximately 255 megabases, with 31,556 protein-coding genes. Quality metrics revealed that this draft genome matches the quality and completeness of other model cnidarian genomes, including Nematostella, Hydra, and Acropora. Phylogenomic analyses revealed strong conservation of the Cnidaria and Hexacorallia core-gene set. However, we found that lineage-specific gene families have undergone significant expansion events compared with shared gene families. Enrichment analysis performed for both gene ontologies, and protein domains revealed that genes encoding toxins contribute to a significant proportion of the lineage-specific genes and gene families. The results make clear that the draft genome of A. tenebrosa will provide insight into the evolution of toxins and lineage-specific genes, and provide an important resource for the discovery of novel biological compounds.

Structural and functional characterisation of a novel peptide from the Australian sea anemone Actinia tenebrosa.

Sea anemone venoms have long been recognised as a rich source of peptides with interesting pharmacological and structural properties. Our recent transcriptomic studies of the Australian sea anemone Actinia tenebrosa have identified a novel 13-residue peptide, U-AITx-Ate1. U-AITx-Ate1 contains a single disulfide bridge and bears no significant homology to previously reported amino acid sequences of peptides from sea anemones or other species. We have produced U-AITx-Ate1 using solid-phase peptide synthesis, followed by oxidative folding and purification of the folded peptide using reversed-phase high-performance liquid chromatography. The solution structure of U-AITx-Ate1 was determined based on two-dimensional nuclear magnetic resonance spectroscopic data. Diffusion-ordered NMR spectroscopy revealed that U-AITx-Ate1 was monomeric in solution. Perturbations in the 1D 1H NMR spectrum of U-AITx-Ate1 in the presence of dodecylphosphocholine micelles together with molecular dynamics simulations indicated an interaction of U-AITx-Ate1 with lipid membranes, although no binding was detected to 100% POPC and 80% POPC: 20% POPG lipid nanodiscs by isothermal titration calorimetry. Functional assays were performed to explore the biological activity profile of U-AITx-Ate1. U-AITx-Ate1 showed no activity in voltage-clamp electrophysiology assays and no change in behaviour and mortality rates in crustacea. Moderate cytotoxic activity was observed against two breast cancer cell lines.

A process of convergent amplification and tissue-specific expression dominates the evolution of toxin and toxin-like genes in sea anemones.

Members of phylum Cnidaria are an ancient group of venomous animals and rely on a number of specialized tissues to produce toxins in order to fulfil a range of ecological roles including prey capture, defence against predators, digestion and aggressive encounters. However, limited comprehensive analyses of the evolution and expression of toxin genes currently exist for cnidarian species. In this study, we use genomic and transcriptomic sequencing data to examine gene copy number variation and selective pressure on toxin gene families in phylum Cnidaria. Additionally, we use quantitative RNA-seq and mass spectrometry imaging to understand expression patterns and tissue localization of toxin production in sea anemones. Using genomic data, we demonstrate that the first large-scale expansion and diversification of known toxin genes occurs in phylum Cnidaria, a process we also observe in other venomous lineages, which we refer to as convergent amplification. Our analyses of selective pressure on sea anemone toxin gene families reveal that purifying selection is the dominant mode of evolution for these genes and that phylogenetic inertia is an important determinant of toxin gene complement in this group. The gene expression and tissue localization data revealed that specific genes and proteins from toxin gene families show strong patterns of tissue and developmental-phase specificity in sea anemones. Overall, convergent amplification and phylogenetic inertia have strongly influenced the distribution and evolution of the toxin complement observed in sea anemones, while the production of venoms with different compositions across tissues is related to the functional and ecological roles undertaken by each tissue type.

Insights into the phylogenetic and molecular evolutionary histories of Fad and Elovl gene families in Actiniaria.

The biosynthesis of long-chain polyunsaturated fatty acids (LC-PUFAs, ≥ C20) is reliant on the action of desaturase and elongase enzymes, which are encoded by the fatty acyl desaturase (Fad) and elongation of very long-chain fatty acid (Elovl) gene families, respectively. In Metazoa, research investigating the distribution and evolution of these gene families has been restricted largely to Bilateria. Here, we provide insights into the phylogenetic and molecular evolutionary histories of the Fad and Elovl gene families in Cnidaria, the sister phylum to Bilateria. Four model cnidarian genomes and six actiniarian transcriptomes were interrogated. Analysis of the fatty acid composition of a candidate cnidarian species, Actinia tenebrosa, was performed to determine the baseline profile of this species. Phylogenetic analysis revealed lineage-specific gene duplication in actiniarians for both the Fad and Elovl gene families. Two distinct cnidarian Fad clades clustered with functionally characterized Δ5 and Δ6 proteins from fungal and plant species, respectively. Alternatively, only a single cnidarian Elovl clade clustered with functionally characterized Elovl proteins (Elovl4), while two additional clades were identified, one actiniarian-specific (Novel ElovlA) and the another cnidarian-specific (Novel ElovlB). In actiniarians, selection analyses revealed pervasive purifying selection acting on both gene families. However, codons in the Elovl gene family show patterns of nucleotide variation consistent with the action of episodic diversifying selection following gene duplication events. Significantly, these codons may encode amino acid residues that are functionally important for Elovl proteins to target and elongate different precursor fatty acids. In A. tenebrosa, the fatty acid analysis revealed an absence of LC-PUFAs > C20 molecules and implies that the Elovl enzymes are not actively contributing to the elongation of these LC-PUFAs. Overall, this study has revealed that actiniarians possess Fad and Elovl genes required for the biosynthesis of some LC-PUFAs, and that these genes appear to be distinct from bilaterians.

Evidence for a Large Expansion and Subfunctionalization of Globin Genes in Sea Anemones.

The globin gene superfamily has been well-characterized in vertebrates, however, there has been limited research in early-diverging lineages, such as phylum Cnidaria. This study aimed to identify globin genes in multiple cnidarian lineages, and use bioinformatic approaches to characterize the evolution, structure, and expression of these genes. Phylogenetic analyses and in silico protein predictions showed that all cnidarians have undergone an expansion of globin genes, which likely have a hexacoordinate protein structure. Our protein modeling has also revealed the possibility of a single pentacoordinate globin lineage in anthozoan species. Some cnidarian globin genes displayed tissue and development specific expression with very few orthologous genes similarly expressed across species. Our phylogenetic analyses also revealed that eumetazoan globin genes form a polyphyletic relationship with vertebrate globin genes. Overall, our analyses suggest that a Ngb-like and GbX-like gene were most likely present in the globin gene repertoire for the last common ancestor of eumetazoans. The identification of a large-scale expansion and subfunctionalization of globin genes in actiniarians provides an excellent starting point to further our understanding of the evolution and function of the globin gene superfamily in early-diverging lineages.

Insights into the innate immunome of actiniarians using a comparative genomic approach.

BACKGROUND: Innate immune genes tend to be highly conserved in metazoans, even in early divergent lineages such as Cnidaria (jellyfish, corals, hydroids and sea anemones) and Porifera (sponges). However, constant and diverse selection pressures on the immune system have driven the expansion and diversification of different immune gene families in a lineage-specific manner. To investigate how the innate immune system has evolved in a subset of sea anemone species (Order: Actiniaria), we performed a comprehensive and comparative study using 10 newly sequenced transcriptomes, as well as three publically available transcriptomes, to identify the origins, expansions and contractions of candidate and novel immune gene families. RESULTS: We characterised five conserved genes and gene families, as well as multiple novel innate immune genes, including the newly recognised putative pattern recognition receptor CniFL. Single copies of TLR, MyD88 and NF-κB were found in most species, and several copies of IL-1R-like, NLR and CniFL were found in almost all species. Multiple novel immune genes were identified with domain architectures including the Toll/interleukin-1 receptor (TIR) homology domain, which is well documented as functioning in protein-protein interactions and signal transduction in immune pathways. We hypothesise that these genes may interact as novel proteins in immune pathways of cnidarian species. Novelty in the actiniarian immunome is not restricted to only TIR-domain-containing proteins, as we identify a subset of NLRs which have undergone neofunctionalisation and contain 3-5 N-terminal transmembrane domains, which have so far only been identified in two anthozoan species. CONCLUSIONS: This research has significance in understanding the evolution and origin of the core eumetazoan gene set, including how novel innate immune genes evolve. For example, the evolution of transmembrane domain containing NLRs indicates that these NLRs may be membrane-bound, while all other metazoan and plant NLRs are exclusively cytosolic receptors. This is one example of how species without an adaptive immune system may evolve innovative solutions to detect pathogens or interact with native microbiota. Overall, these results provide an insight into the evolution of the innate immune system, and show that early divergent lineages, such as actiniarians, have a diverse repertoire of conserved and novel innate immune genes.

Comparative Analysis and Distribution of Omega-3 lcPUFA Biosynthesis Genes in Marine Molluscs.

Recent research has identified marine molluscs as an excellent source of omega-3 long-chain polyunsaturated fatty acids (lcPUFAs), based on their potential for endogenous synthesis of lcPUFAs. In this study we generated a representative list of fatty acyl desaturase (Fad) and elongation of very long-chain fatty acid (Elovl) genes from major orders of Phylum Mollusca, through the interrogation of transcriptome and genome sequences, and various publicly available databases. We have identified novel and uncharacterised Fad and Elovl sequences in the following species: Anadara trapezia, Nerita albicilla, Nerita melanotragus, Crassostrea gigas, Lottia gigantea, Aplysia californica, Loligo pealeii and Chlamys farreri. Based on alignments of translated protein sequences of Fad and Elovl genes, the haeme binding motif and histidine boxes of Fad proteins, and the histidine box and seventeen important amino acids in Elovl proteins, were highly conserved. Phylogenetic analysis of aligned reference sequences was used to reconstruct the evolutionary relationships for Fad and Elovl genes separately. Multiple, well resolved clades for both the Fad and Elovl sequences were observed, suggesting that repeated rounds of gene duplication best explain the distribution of Fad and Elovl proteins across the major orders of molluscs. For Elovl sequences, one clade contained the functionally characterised Elovl5 proteins, while another clade contained proteins hypothesised to have Elovl4 function. Additional well resolved clades consisted only of uncharacterised Elovl sequences. One clade from the Fad phylogeny contained only uncharacterised proteins, while the other clade contained functionally characterised delta-5 desaturase proteins. The discovery of an uncharacterised Fad clade is particularly interesting as these divergent proteins may have novel functions. Overall, this paper presents a number of novel Fad and Elovl genes suggesting that many mollusc groups possess most of the required enzymes for the synthesis of lcPUFAs.