Molecular Insights into the Low Complexity Secreted Venom of Calliactis polypus.

Sea anemones are venomous animals that rely on their venom for prey capture, defense against predators, and intraspecific competition. Currently, comprehensive molecular and evolutionary analyses of the toxin repertoire for sea anemones are limited by a lack of proteomic data for most species. In this study, proteo-transcriptomic analysis was used to expand our knowledge of the proteinaceous components of sea anemone venom by determining the secreted venom proteome of Calliactis polypus. Electromechanical stimulation was used to obtain the secreted venom of C. polypus. We identified a low complexity proteome that was dominated by toxins with similarity to known neurotoxins, as well as six novel toxin candidates. The novel putative toxin candidates were found to be taxonomically restricted to species from the superfamily Metridioidea. Furthermore, the secreted venom of C. polypus had only three putative toxins in common with the venom of acontia from the same species and little similarity with the secreted venom of closely related species. Overall, this demonstrates that regionalized and lineage-specific variability in toxin abundance is common among sea anemone species. Moreover, the limited complexity of the toxin repertoire found in C. polypus supports the idea that peptide neurotoxins make up the dominant toxin arsenal found in the venom of sea anemones.

Proteotransciptomics of the Most Popular Host Sea Anemone Entacmaea quadricolor Reveals Not All Toxin Genes Expressed by Tentacles Are Recruited into Its Venom Arsenal.

While the unique symbiotic relationship between anemonefishes and sea anemones is iconic, it is still not fully understood how anemonefishes can withstand and thrive within the venomous environment of their host sea anemone. In this study, we used a proteotranscriptomics approach to elucidate the proteinaceous toxin repertoire from the most common host sea anemone, Entacmaea quadricolor. Although 1251 different toxin or toxin-like RNA transcripts were expressed in E. quadricolor tentacles (0.05% of gene clusters, 1.8% of expression) and 5375 proteins were detected in milked venom, only 4% of proteins detected in venom were putative toxins (230), and they only represent on average 14% of the normalised protein expression in the milked venom samples. Thus, most proteins in milked venom do not appear to have a toxin function. This work raises the perils of defining a dominant venom phenotype based on transcriptomics data alone in sea anemones, as we found that the dominant venom phenotype differs between the transcriptome and proteome abundance data. E. quadricolor venom contains a mixture of toxin-like proteins of unknown and known function. A newly identified toxin protein family, Z3, rich in conserved cysteines of unknown function, was the most abundant at the RNA transcript and protein levels. The venom was also rich in toxins from the Protease S1, Kunitz-type and PLA2 toxin protein families and contains toxins from eight venom categories. Exploring the intricate venom toxin components in other host sea anemones will be crucial for improving our understanding of how anemonefish adapt to the venomous environment.

Structure-function relationships in ShKT domain peptides: ShKT-Ts1 from the sea anemone Telmatactis stephensoni.

Diverse structural scaffolds have been described in peptides from sea anemones, with the ShKT domain being a common scaffold first identified in ShK toxin from Stichodactyla helianthus. ShK is a potent blocker of voltage-gated potassium channels (KV 1.x), and an analog, ShK-186 (dalazatide), has completed Phase 1 clinical trials in plaque psoriasis. The ShKT domain has been found in numerous other species, but only a tiny fraction of ShKT domains has been characterized functionally. Despite adopting the canonical ShK fold, some ShKT peptides from sea anemones inhibit KV 1.x, while others do not. Mutagenesis studies have shown that a Lys-Tyr (KY) dyad plays a key role in KV 1.x blockade, although a cationic residue followed by a hydrophobic residue may also suffice. Nevertheless, ShKT peptides displaying an ShK-like fold and containing a KY dyad do not necessarily block potassium channels, so additional criteria are needed to determine whether new ShKT peptides might show activity against potassium channels. In this study, we used a combination of NMR and molecular dynamics (MD) simulations to assess the potential activity of a new ShKT peptide. We determined the structure of ShKT-Ts1, from the sea anemone Telmatactis stephensoni, examined its tissue localization, and investigated its activity against a range of ion channels. As ShKT-Ts1 showed no activity against KV 1.x channels, we used MD simulations to investigate whether solvent exposure of the dyad residues may be informative in rationalizing and potentially predicting the ability of ShKT peptides to block KV 1.x channels. We show that either a buried dyad that does not become exposed during MD simulations, or a partially exposed dyad that becomes buried during MD simulations, correlates with weak or absent activity against KV 1.x channels. Therefore, structure determination coupled with MD simulations, may be used to predict whether new sequences belonging to the ShKT family may act as potassium channel blockers.

Structural and functional characterisation of Tst2, a novel TRPV1 inhibitory peptide from the Australian sea anemone Telmatactis stephensoni.

Sea anemone venoms are complex mixtures of biologically active compounds, including disulfide-rich peptides, some of which have found applications as research tools, and others as therapeutic leads. Our recent transcriptomic and proteomic studies of the Australian sea anemone Telmatactis stephensoni identified a transcript for a peptide designated Tst2. Tst2 is a 38-residue peptide showing sequence similarity to peptide toxins known to interact with a range of ion channels (NaV, TRPV1, KV and CaV). Recombinant Tst2 (rTst2, which contains an additional Gly at the N-terminus) was produced by periplasmic expression in Escherichia coli, enabling the production of both unlabelled and uniformly 13C,15N-labelled peptide for functional assays and structural studies. The LC-MS profile of the recombinant Tst2 showed a pure peak with molecular mass 6 Da less than that of the reduced form of the peptide, indicating the successful formation of three disulfide bonds from its six cysteine residues. The solution structure of rTst2 was determined using multidimensional NMR spectroscopy and revealed that rTst2 adopts an inhibitor cystine knot (ICK) structure. rTst2 was screened using various functional assays, including patch-clamp electrophysiological and cytotoxicity assays. rTst2 was inactive against voltage-gated sodium channels (NaV) and the human voltage-gated proton (hHv1) channel. rTst2 also did not possess cytotoxic activity when assessed against Drosophila melanogaster flies. However, the recombinant peptide at 100 nM showed >50% inhibition of the transient receptor potential subfamily V member 1 (TRPV1) and slight (∼10%) inhibition of transient receptor potential subfamily A member 1 (TRPA1). Tst2 is thus a novel ICK inhibitor of the TRPV1 channel.

Multiscale landscape genetic analysis identifies major waterways as a barrier to dispersal of feral pigs in north Queensland, Australia.

Feral pigs (Sus scrofa) are a destructive and widespread invasive pest in Australia. An understanding of feral pig movement is required to develop management strategies to control feral pigs in Australia. Because landscape structure can have a strong influence on animal movement, it is important to determine how landscape features facilitate or impede the movement of feral pigs. Consequently, we conducted a landscape genetic analysis of feral pig populations in the Herbert region of far north Queensland, Australia, to determine management units and provide recommendations to better inform feral pig population control strategies. Using microsatellite data obtained from 256 feral pig samples from 44 sites, we examined feral pig population structure at multiple spatial scales for univariate and multivariate landscape resistance surfaces to determine the optimal spatial scale and to identify which of the nine landscape features tested impede or facilitate feral pig gene flow. Only weak genetic structure was found among the 44 sampling sites, but major waterways were identified as a minor barrier to gene flow, and an isolation by distance model was supported. We also found that highways facilitated gene flow across the study area, and this suggests that they may act as movement corridors or indicate translocation of feral pigs. Additionally, incorporating a second spatial scale enhanced the ability of our landscape genetics analysis to detect the influence of landscape structure on gene flow. We identified three management units based on natural barriers to gene flow and future targeted control should be undertaken in these management units to deliver sustained reduction of feral pig populations in the Herbert region. This study demonstrates how a landscape genetic approach can be used to gain insight into the ecology of an invasive pest species and be used to develop population control strategies which utilise natural barriers to movement.

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.

Acontia, a Specialised Defensive Structure, Has Low Venom Complexity in Calliactis polypus.

Phylum Cnidaria represents a unique group among venomous taxa, with its delivery system organised as individual organelles, known as nematocysts, heterogeneously distributed across morphological structures rather than packaged as a specialised organ. Acontia are packed with large nematocysts that are expelled from sea anemones during aggressive encounters with predatory species and are found in a limited number of species in the superfamily Metridioidea. Little is known about this specialised structure other than the commonly accepted hypothesis of its role in defence and a rudimentary understanding of its toxin content and activity. This study utilised previously published transcriptomic data and new proteomic analyses to expand this knowledge by identifying the venom profile of acontia in Calliactis polypus. Using mass spectrometry, we found limited toxin diversity in the proteome of acontia, with an abundance of a sodium channel toxin type I, and a novel toxin with two ShK-like domains. Additionally, genomic evidence suggests that the proposed novel toxin is ubiquitous across sea anemone lineages. Overall, the venom profile of acontia in Calliactis polypus and the novel toxin identified here provide the basis for future research to define the function of acontial toxins in sea anemones.

Advancing tree genomics to future proof next generation orchard production.

The challenges facing tree orchard production in the coming years will be largely driven by changes in the climate affecting the sustainability of farming practices in specific geographical regions. Identifying key traits that enable tree crops to modify their growth to varying environmental conditions and taking advantage of new crop improvement opportunities and technologies will ensure the tree crop industry remains viable and profitable into the future. In this review article we 1) outline climate and sustainability challenges relevant to horticultural tree crop industries, 2) describe key tree crop traits targeted for improvement in agroecosystem productivity and resilience to environmental change, and 3) discuss existing and emerging genomic technologies that provide opportunities for industries to future proof the next generation of orchards.

Cellular adaptations leading to coral fragment attachment on artificial substrates in Acropora millepora (Am-CAM).

Reproductive propagation by asexual fragmentation in the reef-building coral Acropora millepora depends on (1) successful attachment to the reef substrate through modification of soft tissues and (2) a permanent bond with skeletal encrustation. Despite decades of research examining asexual propagation in corals, the initial response, cellular reorganisation, and development leading to fragment substrate attachment via a newly formed skeleton has not been documented in its entirety. Here, we establish the first "coral attachment model" for this species ("Am-CAM") by developing novel methods that allow correlation of fluorescence and electron microscopy image data with in vivo microscopic time-lapse imagery. This multi-scale imaging approach identified three distinct phases involved in asexual propagation: (1) the contact response of the coral fragment when contact with the substrate, followed by (2) fragment stabilisation through anchoring by the soft tissue, and (3) formation of a "lappet-like appendage" structure leading to substrate bonding of the tissue for encrustation through the onset of skeletal calcification. In developing Am-CAM, we provide new biological insights that can enable reef researchers, managers and coral restoration practitioners to begin evaluating attachment effectiveness, which is needed to optimise species-substrate compatibility and achieve effective outplanting.

Fruit Fly Larval Survival in Picked and Unpicked Tomato Fruit of Differing Ripeness and Associated Gene Expression Patterns.

The larvae of frugivorous tephritid fruit flies feed within fruit and are global pests of horticulture. With the reduced use of pesticides, alternative control methods are needed, of which fruit resistance is one. In the current study, we explicitly tested for phenotypic evidence of induced fruit defences by running concurrent larval survival experiments with fruit on or off the plant, assuming that defence induction would be stopped or reduced by fruit picking. This was accompanied by RT-qPCR analysis of fruit defence and insect detoxification gene expression. Our fruit treatments were picking status (unpicked vs. picked) and ripening stage (colour break vs. fully ripe), our fruit fly was the polyphagous Bactrocera tryoni, and larval survival was assessed through destructive fruit sampling at 48 and 120 h, respectively. The gene expression study targeted larval and fruit tissue samples collected at 48 h and 120 h from picked and unpicked colour-break fruit. At 120 h in colour-break fruit, larval survival was significantly higher in the picked versus unpicked fruit. The gene expression patterns in larval and plant tissue were not affected by picking status, but many putative plant defence and insect detoxification genes were upregulated across the treatments. The larval survival results strongly infer an induced defence mechanism in colour-break tomato fruit that is stronger/faster in unpicked fruits; however, the gene expression patterns failed to provide the same clear-cut treatment effect. The lack of conformity between these results could be related to expression changes in unsampled candidate genes, or due to critical changes in gene expression that occurred during the unsampled periods.

Venoms for all occasions: The functional toxin profiles of different anatomical regions in sea anemones are related to their ecological function.

The phylum Cnidaria is the oldest extant venomous group and is defined by the presence of nematocysts, specialized organelles responsible for venom production and delivery. Although toxin peptides and the cells housing nematocysts are distributed across the entire animal, nematocyte and venom profiles have been shown to differ across morphological structures in actiniarians. In this study, we explore the relationship between patterns of toxin expression and the ecological roles of discrete anatomical structures in Telmatactis stephensoni. Specifically, using a combination of proteomic and transcriptomic approaches, we examined whether there is a direct correlation between the functional similarity of regions and the similarity of their associated toxin expression profiles. We report that the regionalization of toxin production is consistent with the partitioning of the ecological roles of venom across envenomating structures, and that three major functional regions are present in T. stephensoni: tentacles, epidermis and gastrodermis. Additionally, we find that most structures that serve similar functions not only have comparable putative toxin profiles but also similar nematocyst types. There was no overlap in the putative toxins identified using proteomics and transcriptomics, but the expression patterns of specific milked venom peptides were conserved across RNA-sequencing and mass spectrometry imaging data sets. Furthermore, based on our data, it appears that acontia of T. stephensoni may be transcriptionally inactive and only mature nematocysts are present in the distal portions of the threads. Overall, we find that the venom profile of different anatomical regions in sea anemones varies according to its ecological functions.

Gene selection for studying frugivore-plant interactions: a review and an example using Queensland fruit fly in tomato.

Fruit production is negatively affected by a wide range of frugivorous insects, among them tephritid fruit flies are one of the most important. As a replacement for pesticide-based controls, enhancing natural fruit resistance through biotechnology approaches is a poorly researched but promising alternative. The use of quantitative reverse transcription PCR (RT-qPCR) is an approach to studying gene expression which has been widely used in studying plant resistance to pathogens and non-frugivorous insect herbivores, and offers a starting point for fruit fly studies. In this paper, we develop a gene selection pipe-line for known induced-defense genes in tomato fruit, Solanum lycopersicum, and putative detoxification genes in Queensland fruit fly, Bactrocera tryoni, as a basis for future RT-qPCR research. The pipeline started with a literature review on plant/herbivore and plant/pathogen molecular interactions. With respect to the fly, this was then followed by the identification of gene families known to be associated with insect resistance to toxins, and then individual genes through reference to annotated B. tryoni transcriptomes and gene identity matching with related species. In contrast for tomato, a much better studied species, individual defense genes could be identified directly through literature research. For B. tryoni, gene selection was then further refined through gene expression studies. Ultimately 28 putative detoxification genes from cytochrome P450 (P450), carboxylesterase (CarE), glutathione S-transferases (GST), and ATP binding cassette transporters (ABC) gene families were identified for B. tryoni, and 15 induced defense genes from receptor-like kinase (RLK), D-mannose/L-galactose, mitogen-activated protein kinase (MAPK), lipoxygenase (LOX), gamma-aminobutyric acid (GABA) pathways and polyphenol oxidase (PPO), proteinase inhibitors (PI) and resistance (R) gene families were identified from tomato fruit. The developed gene selection process for B. tryoni can be applied to other herbivorous and frugivorous insect pests so long as the minimum necessary genomic information, an annotated transcriptome, is available.

The Tentacular Spectacular: Evolution of Regeneration in Sea Anemones.

Sea anemones vary immensely in life history strategies, environmental niches and their ability to regenerate. While the sea anemone Nematostella vectensis is the starlet of many key regeneration studies, recent work is emerging on the diverse regeneration strategies employed by other sea anemones. This manuscript will explore current molecular mechanisms of regeneration employed by non-model sea anemones Exaiptasia diaphana (an emerging model species for coral symbiosis studies) and Calliactis polypus (a less well-studied species) and examine how these species compare to the model sea anemone N. vectensis. We summarize the field of regeneration within sea anemones, within the greater context of phylum Cnidaria and in other invertebrate models of regeneration. We also address the current knowledge on two key systems that may be implemented in regeneration: the innate immune system and developmental pathways, including future aspects of work and current limitations.

Tentacle Morphological Variation Coincides with Differential Expression of Toxins in Sea Anemones.

Phylum Cnidaria is an ancient venomous group defined by the presence of cnidae, specialised organelles that serve as venom delivery systems. The distribution of cnidae across the body plan is linked to regionalisation of venom production, with tissue-specific venom composition observed in multiple actiniarian species. In this study, we assess whether morphological variants of tentacles are associated with distinct toxin expression profiles and investigate the functional significance of specialised tentacular structures. Using five sea anemone species, we analysed differential expression of toxin-like transcripts and found that expression levels differ significantly across tentacular structures when substantial morphological variation is present. Therefore, the differential expression of toxin genes is associated with morphological variation of tentacular structures in a tissue-specific manner. Furthermore, the unique toxin profile of spherical tentacular structures in families Aliciidae and Thalassianthidae indicate that vesicles and nematospheres may function to protect branched structures that host a large number of photosynthetic symbionts. Thus, hosting zooxanthellae may account for the tentacle-specific toxin expression profiles observed in the current study. Overall, specialised tentacular structures serve unique ecological roles and, in order to fulfil their functions, they possess distinct venom cocktails.

Characterising Functional Venom Profiles of Anthozoans and Medusozoans within Their Ecological Context.

This review examines the current state of knowledge regarding toxins from anthozoans (sea anemones, coral, zoanthids, corallimorphs, sea pens and tube anemones). We provide an overview of venom from phylum Cnidaria and review the diversity of venom composition between the two major clades (Medusozoa and Anthozoa). We highlight that the functional and ecological context of venom has implications for the temporal and spatial expression of protein and peptide toxins within class Anthozoa. Understanding the nuances in the regulation of venom arsenals has been made possible by recent advances in analytical technologies that allow characterisation of the spatial distributions of toxins. Furthermore, anthozoans are unique in that ecological roles can be assigned using tissue expression data, thereby circumventing some of the challenges related to pharmacological screening.

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.

The effect of diet change and insulin dysregulation on the faecal microbiome of ponies.

The equine microbiome can change in response to dietary alteration and may play a role in insulin dysregulation. The aim of this study was to determine the effect of adding pasture to a hay diet on the faecal bacterial microbiome of both healthy and insulin-dysregulated ponies. Faecal samples were collected from 16 ponies before and after dietary change to enable bacterial 16S rRNA sequencing of the V3-V4 region. The dominant phyla in all samples were the Firmicutes and Bacteroidetes. The evenness of the bacterial populations decreased after grazing pasture, and when a pony was moderately insulin dysregulated (P=0.001). Evenness scores negatively correlated with post-prandial glucagon-like peptide-1 concentration after a hay-only diet (r²=-0.7, P=0.001). A change in diet explained 3% of faecal microbiome variability. We conclude that metabolically healthy ponies have greater microbial stability when challenged with a subtle dietary change, compared with moderately insulin-dysregulated ponies.

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.

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.