Exotic myxozoan parasites establish complex life cycles in farm pond aquaculture.

Myxozoans are obligate parasites with complex life cycles, typically infecting fish and annelids. Here, we examined annelids from fish farm pond sediments in the Beit Shean Valley, in the Syrian-African Rift Valley, Israel, for myxozoan infections. We examined 1486 oligochaetes, and found 74 (5 %) were infected with actinospore stages. We used mitochondrial 16S sequencing to infer identity of 25 infected annelids as species of Potamothrix, Psammoryctides, Tubifex and Dero. We identified 7 myxozoan types from collective groups Neoactinomyxum and Sphaeractinomyxon, and characterized them by small subunit ribosomal DNA sequencing. The Neoactinomyxum type was genetically most similar (∼93 %) to cyprinid fish-infecting Myxobolus spp. The six Sphaeractinomyxon types were genetically similar (93-100 %) to Mugilid-infecting Myxobolus spp.; with one being the previously unknown actinospore stage of a myxospore that infects mullet from aquaculture from the Israeli coast of the Mediterranean Sea. As the farm pond system is artificial and geographically isolated from the Mediterranean, the presence of at least seven myxozoans in their annelid hosts demonstrates introduction and establishment of these parasites in a novel, brackish environment.

The myxozoan parasite Myxobolus bejeranoi (Cnidaria: Myxozoa) infection dynamics and host specificity in hybrid tilapia aquaculture.

Nile × blue tilapia hybrid (Oreochromis niloticus × O. aureus) has become an important food fish in intensive freshwater aquaculture. Recently, the parasite Myxobolus bejeranoi (Cnidaria: Myxozoa) was found to infect hybrid tilapia gills at high prevalence, causing immune suppression and high mortality. Here, we explored additional characteristics of M. bejeranoi­tilapia interaction, which enable efficient proliferation of this parasite inside its specific host. Highly sensitive quantitative polymerase chain reaction (qPCR) and in situ hybridization analyses of fry collected from fertilization ponds provided evidence to an early-life infection of fish by a myxozoan parasite, occurring less than 3 weeks post-fertilization. Because Myxobolus species are highly host-specific, we next compared infection rates in hybrid tilapia and in both its parental species following a 1-week exposure to infectious pond water. Analysis by qPCR and histological sections showed that while blue tilapia was as susceptible to M. bejeranoi as the hybrid, Nile tilapia appeared to be resistant. This is the first report of differential susceptibility of a hybrid fish vs its parental purebreds to a myxozoan parasite. These findings advance our understanding of the relationship between M. bejeranoi and tilapia fish and raise important questions regarding the mechanisms that allow the parasite to distinguish between very closely related species and to infect a specific organ at very early-life stages.

The Molecular Mechanisms Employed by the Parasite Myxobolus bejeranoi (Cnidaria: Myxozoa) from Invasion through Sporulation for Successful Proliferation in Its Fish Host.

Myxozoa is a unique group of obligate endoparasites in the phylum Cnidaria that can cause emerging diseases in wild and cultured fish populations. Recently, we identified a new myxozoan species, Myxobolus bejeranoi, which infects the gills of cultured tilapia while suppressing host immunity. To uncover the molecular mechanisms underlying this successful parasitic strategy, we conducted transcriptomics analysis of M. bejeranoi throughout the infection. Our results show that histones, which are essential for accelerated cell division, are highly expressed even one day after invasion. As the infection progressed, conserved parasitic genes that are known to modulate the host immune reaction in different parasitic taxa were upregulated. These genes included energy-related glycolytic enzymes, as well as calreticulin, proteases, and miRNA biogenesis proteins. Interestingly, myxozoan calreticulin formed a distinct phylogenetic clade apart from other cnidarians, suggesting a possible function in parasite pathogenesis. Sporogenesis was in its final stages 20 days post-exposure, as spore-specific markers were highly expressed. Lastly, we provide the first catalog of transcription factors in a Myxozoa species, which is minimized compared to free-living cnidarians and is dominated by homeodomain types. Overall, these molecular insights into myxozoan infection support the concept that parasitic strategies are a result of convergent evolution.

Direct Electricity Production from Nematostella and Arthemia's Eggs in a Bio-Electrochemical Cell.

In recent years, extensive efforts have been made to develop clean energy technologies to replace fossil fuels to assist the struggle against climate change. One approach is to exploit the ability of bacteria and photosynthetic organisms to conduct external electron transport for electricity production in bio-electrochemical cells. In this work, we first show that the sea anemones Nematostella vectensis and eggs of Artemia (brine shrimp) secrete redox-active molecules that can reduce the electron acceptor Cytochrome C. We applied 2D fluorescence spectroscopy and identified NADH or NADPH as secreted species. Finally, we broaden the scope of living organisms that can be integrated with a bio-electrochemical cell to the sea anemones group, showing for the first time that Nematostella and eggs of Artemia can produce electrical current when integrated into a bio-electrochemical cell.

Morphological and molecular characterization of a novel myxosporean parasite Myxobolus bejeranoi n. sp. (Cnidaria: Myxosporea) from hybrid tilapia in Israel.

Myxosporean infections can cause severe damage to commercially grown tilapia. Here, we report a novel myxosporean that was found in gills of Oreochromis aureus male × Oreochromis niloticus female, which is an important aquaculture tilapia hybrid in Israel. Three-month-old fish were found to have cysts located in gill muscle tissue, which were filled with both immature and mature spores. Affected fish displayed higher mortality rate. Spore dimensions (10.8 ± 0.7 µm length × 6.8 ± 0.6 µm width) and molecular characterization using 18S ribosomal DNA revealed that the unknown parasite belongs in the Myxobolus clade. Based on the infection site, spore morphology and molecular characterization, we describe this parasite as Myxobolus bejeranoi n. sp. (MF401455). Phylogenetic analysis showed that the new species is most closely related to two Myxobolus spp. from O. niloticus in Egypt and Ghana.

Regulation of AP-1 by MAPK Signaling in Metal-Stressed Sea Anemone.

BACKGROUND/AIMS: AP-1 transcription factor plays a conserved role in the immediate response to stress. Activation of AP-1 members jun and fos is mediated by complex signaling cascades to control cell proliferation and survival. To understand the evolution of this broadly-shared pathway, we studied AP-1 regulation by MAPK signaling in a basal metazoan. METHODS: Metal- stressed cnidarian Nematostella vectensis anemones were tested with kinase inhibitors and analyzed for gene expression levels and protein phosphorylation. RESULTS: We show that in cnidarian, AP-1 is regulated differently than in bilaterian models. ERK2 and ERK5, the main MAPK drivers of AP-1 activation in Bilateria, down-regulated fos1 and jun1 transcription in anemones exposed to metal stress, whereas p38 MAPK, triggered transcription of jun1 but not fos1. Furthermore, our results reveal that GSK3-ß is the main driver of the immediate stress response in Nematostella. GSK3-ß triggered transcription of AP-1 and two other stress-related genes, egr1 and hsp70. Finally, phylogenetic analysis and protein characterization show that while MAPKs and GSK3-ß are evolutionarily conserved, Fos and Jun proteins in Nematostella and other cnidarians lack important regulatory and phosphorylation sites found in Bilateria. CONCLUSION: These findings reveal alternative network interactions of conserved signaling kinases, providing insight into the evolutionary plasticity of immediate stress response mechanisms.

The dynamically evolving nematocyst content of an anthozoan, a scyphozoan, and a hydrozoan.

Nematocytes, the stinging cells of cnidarians, are the most evolutionarily ancient venom apparatus. These nanosyringe-like weaponry systems reach pressures of approximately 150 atmospheres before discharging and punching through the outer layer of the prey or predator at accelerations of more than 5 million g, making them one of the fastest biomechanical events known. To gain better understanding of the function of the complex, phylum-specific nematocyst organelle, and its venom payload, we compared the soluble nematocyst's proteome from the sea anemone Anemonia viridis, the jellyfish Aurelia aurita, and the hydrozoan Hydra magnipapillata, each belonging to one of the three basal cnidarian lineages which diverged over 600 Ma. Although the basic morphological and functional characteristics of the nematocysts of the three organisms are similar, out of hundreds of proteins identified in each organism, only six are shared. These include structural proteins, a chaperone which may help maintain venon activity over extended periods, and dickkopf, an enigmatic Wnt ligand which may also serve as a toxin. Nevertheless, many protein domains are shared between the three organisms' nematocyst content suggesting common proteome functionalities. The venoms of Hydra and Aurelia appear to be functionally similar and composed mainly of cytotoxins and enzymes, whereas the venom of the Anemonia is markedly unique and based on peptide neurotoxins. Cnidarian venoms show evidence for functional recruitment, yet evidence for diversification through positive selection, common to other venoms, is lacking. The final injected nematocyst payload comprises a mixture of dynamically evolving proteins involved in the development, maturation, maintenance, and discharge of the nematocysts, which is unique to each organism and potentially to each nematocyst type.

The making of an embryo in a basal metazoan: Proteomic analysis in the sea anemone Nematostella vectensis.

Cnidarians are widely distributed basal metazoans that play an important role in the marine ecosystem. Their genetic diversity and dispersal depends on successful oogenesis, fertilization and embryogenesis. To understand the processes that lead to successful embryogenesis in these basal organisms, we conducted comparative proteomics on the model sea anemone Nematostella vectensis. We examined four developmental stages from oocyte maturation through early embryogenesis, as well as the oocyte jelly sac in which fertilization and embryogenesis take place. Our analysis revealed 37 stage-specifically expressed proteins, including cell cycle, cellular energy related and DNA replication proteins and transcription regulators. Using in situ hybridization, we show that within the mesenteria, two cell types support successful oocyte development and embryogenesis. Large somatic supporting cells synthesize vitellogenin, the most abundant egg yolk protein within the oocyte, whereas mesenteria gland cells synthesize mucin 5B, which was found to be the main component of the jelly sac. These findings shed light on the sexual reproduction program in cnidarians and suggest a high conservation with proteins governing oogenesis in Bilateria.

Transcriptome profiling of the dynamic life cycle of the scypohozoan jellyfish Aurelia aurita.

BACKGROUND: The moon jellyfish Aurelia aurita is a widespread scyphozoan species that forms large seasonal blooms. Here we provide the first comprehensive view of the entire complex life of the Aurelia Red Sea strain by employing transcriptomic profiling of each stage from planula to mature medusa. RESULTS: A de novo transcriptome was assembled from Illumina RNA-Seq data generated from six stages throughout the Aurelia life cycle. Transcript expression profiling yielded clusters of annotated transcripts with functions related to each specific life-cycle stage. Free-swimming planulae were found highly enriched for functions related to cilia and microtubules, and the drastic morphogenetic process undergone by the planula while establishing the future body of the polyp may be mediated by specifically expressed Wnt ligands. Specific transcripts related to sensory functions were found in the strobila and the ephyra, whereas extracellular matrix functions were enriched in the medusa due to high expression of transcripts such as collagen, fibrillin and laminin, presumably involved in mesoglea development. The CL390-like gene, suggested to act as a strobilation hormone, was also highly expressed in the advanced strobila of the Red Sea species, and in the medusa stage we identified betaine-homocysteine methyltransferase, an enzyme that may play an important part in maintaining equilibrium of the medusa's bell. Finally, we identified the transcription factors participating in the Aurelia life-cycle and found that 70% of these 487 identified transcription factors were expressed in a developmental-stage-specific manner. CONCLUSIONS: This study provides the first scyphozoan transcriptome covering the entire developmental trajectory of the life cycle of Aurelia. It highlights the importance of numerous stage-specific transcription factors in driving morphological and functional changes throughout this complex metamorphosis, and is expected to be a valuable resource to the community.

Early and late response of Nematostella vectensis transcriptome to heavy metals.

Environmental contamination from heavy metals poses a global concern for the marine environment, as heavy metals are passed up the food chain and persist in the environment long after the pollution source is contained. Cnidarians play an important role in shaping marine ecosystems, but environmental pollution profoundly affects their vitality. Among the cnidarians, the sea anemone Nematostella vectensis is an advantageous model for addressing questions in molecular ecology and toxicology as it tolerates extreme environments and its genome has been published. Here, we employed a transcriptome-wide RNA-Seq approach to analyse N. vectensis molecular defence mechanisms against four heavy metals: Hg, Cu, Cd and Zn. Altogether, more than 4800 transcripts showed significant changes in gene expression. Hg had the greatest impact on up-regulating transcripts, followed by Cu, Zn and Cd. We identified, for the first time in Cnidaria, co-up-regulation of immediate-early transcription factors such as Egr1, AP1 and NF-κB. Time-course analysis of these genes revealed their early expression as rapidly as one hour after exposure to heavy metals, suggesting that they may complement or substitute for the roles of the metal-mediating Mtf1 transcription factor. We further characterized the regulation of a large array of stress-response gene families, including Hsp, ABC, CYP members and phytochelatin synthase, that may regulate synthesis of the metal-binding phytochelatins instead of the metallothioneins that are absent from Cnidaria genome. This study provides mechanistic insight into heavy metal toxicity in N. vectensis and sheds light on ancestral stress adaptations.

Continuous drug release by sea anemone Nematostella vectensis stinging microcapsules.

Transdermal delivery is an attractive option for drug delivery. Nevertheless, the skin is a tough barrier and only a limited number of drugs can be delivered through it. The most difficult to deliver are hydrophilic drugs. The stinging mechanism of the cnidarians is a sophisticated injection system consisting of microcapsular nematocysts, which utilize built-in high osmotic pressures to inject a submicron tubule that penetrates and delivers their contents to the prey. Here we show, for the first time, that the nematocysts of the starlet sea anemone Nematostella vectensis can be isolated and incorporated into a topical formulation for continuous drug delivery. We demonstrate quantitative delivery of nicotinamide and lidocaine hydrochloride as a function of microcapsular dose or drug exposure. We also show how the released submicron tubules can be exploited as a skin penetration enhancer prior to and independently of drug application. The microcapsules are non-irritant and may offer an attractive alternative for hydrophilic transdermal drug delivery.

Lysyl oxidase-like-2 promotes tumour angiogenesis and is a potential therapeutic target in angiogenic tumours.

Lysyl oxidase-like 2 (LOXL2), a secreted enzyme that catalyzes the cross-linking of collagen, plays an essential role in developmental angiogenesis. We found that administration of the LOXL2-neutralizing antibody AB0023 inhibited bFGF-induced angiogenesis in Matrigel plug assays and suppressed recruitment of angiogenesis promoting bone marrow cells. Small hairpin RNA-mediated inhibition of LOXL2 expression or inhibition of LOXL2 using AB0023 reduced the migration and network-forming ability of endothelial cells, suggesting that the inhibition of angiogenesis results from a direct effect on endothelial cells. To examine the effects of AB0023 on tumour angiogenesis, AB0023 was administered to mice bearing tumours derived from SKOV-3 ovarian carcinoma or Lewis lung carcinoma (LLC) cells. AB0023 treatment significantly reduced the microvascular density in these tumours but did not inhibit tumour growth. However, treatment of mice bearing SKOV-3-derived tumours with AB0023 also promoted increased coverage of tumour vessels with pericytes and reduced tumour hypoxia, providing evidence that anti-LOXL2 therapy results in the normalization of tumour blood vessels. In agreement with these data, treatment of mice bearing LLC-derived tumours with AB0023 improved the perfusion of the tumour-associated vessels as determined by ultrasonography. Improved perfusion and normalization of tumour vessels after treatment with anti-angiogenic agents were previously found to improve the delivery of chemotherapeutic agents into tumours and to result in an enhancement of chemotherapeutic efficiency. Indeed, treatment with AB0023 significantly enhanced the anti-tumourigenic effects of taxol. Our results suggest that inhibition of LOXL2 may prove beneficial for the treatment of angiogenic tumours.

Selecting 16S rRNA Primers for Microbiome Analysis in a Host-Microbe System: The Case of the Jellyfish Rhopilema nomadica.

Amplicon sequencing of the 16S rRNA gene is extensively used to characterize bacterial communities, including those living in association with eukaryotic hosts. Deciding which region of the 16S rRNA gene to analyze and selecting the appropriate PCR primers remains a major decision when initiating any new microbiome study. Based on a detailed literature survey of studies focusing on cnidarian microbiomes, we compared three commonly used primers targeting different hypervariable regions of the 16S rRNA gene, V1V2, V3V4, and V4V5, using the jellyfish Rhopilema nomadica as a model. Although all primers exhibit a similar pattern in bacterial community composition, the performance of the V3V4 primer set was superior to V1V2 and V4V5. The V1V2 primers misclassified bacteria from the Bacilli class and exhibited low classification resolution for Rickettsiales, which represent the second most abundant 16S rRNA gene sequence in all the primers. The V4V5 primer set detected almost the same community composition as the V3V4, but the ability of these primers to also amplify the eukaryotic 18S rRNA gene may hinder bacterial community observations. However, after overcoming the challenges possessed by each one of those primers, we found that all three of them show very similar bacterial community dynamics and compositions. Nevertheless, based on our results, we propose that the V3V4 primer set is potentially the most suitable for studying jellyfish-associated bacterial communities. Our results suggest that, at least for jellyfish samples, it may be feasible to directly compare microbial community estimates from different studies, each using different primers but otherwise similar experimental protocols. More generally, we recommend specifically testing different primers for each new organism or system as a prelude to large-scale 16S rRNA gene amplicon analyses, especially of previously unstudied host-microbe associations.

Receptor activity modifying protein-3 mediates the protumorigenic activity of lysyl oxidase-like protein-2.

Lysyl oxidase-like protein-2 (LOXL2) induces epithelial to mesenchymal transition and promotes invasiveness. To understand the mechanisms involved, we examined the effect of LOXL2 overexpression in MCF-7 cells on gene expression. We found that LOXL2 up-regulated the expression of receptor activity modifying protein-3 (RAMP3). Expression of RAMP3 in MDA-MB-231 cells in which LOXL2 expression was inhibited restored vimentin expression, invasiveness, and tumor development. Inhibition of RAMP3 expression in MDA-MB-231 cells mimicked the effects produced by inhibition of LOXL2 expression and was accompanied by inhibition of p38 phosphorylation. LOXL2 overexpression in these cells did not restore invasiveness, suggesting that RAMP3 functions downstream to LOXL2. LOXL2 and RAMP3 are strongly coexpressed in human colon, breast, and gastric carcinomas but not in normal colon or gastric epithelial cells. RAMP3 associates with several G-protein-coupled receptors forming receptors for peptides, such as adrenomedullin and amylin. We hypothesized that RAMP3 could function as a transducer of autocrine signals induced by such peptides. However, the proinvasive effects of RAMP3 could not be abrogated following inhibition of the expression or activity of these peptides. Our experiments suggest that the protumorigenic effects of LOXL2 are partially mediated by RAMP3 and that RAMP3 inhibitors may function as antitumorigenic agents. -

Infection by the Parasite Myxobolus bejeranoi (Cnidaria: Myxozoa) Suppresses the Immune System of Hybrid Tilapia.

Myxozoa (Cnidaria) is a large group of microscopic obligate endoparasites that can cause emerging diseases, affecting wild fish populations and fisheries. Recently, the myxozoan Myxobolus bejeranoi was found to infect the gills of hybrid tilapia (Nile tilapia (Oreochromis niloticus) × Jordan/blue tilapia (O. aureus)), causing high morbidity and mortality. Here, we used comparative transcriptomics to elucidate the molecular processes occurring in the fish host following infection by M. bejeranoi. Fish were exposed to pond water containing actinospores for 24 h and the effects of minor, intermediate, and severe infections on the sporulation site, the gills, and on the hematopoietic organs, head kidney and spleen, were compared. Enrichment analysis for GO and KEGG pathways indicated immune system activation in gills at severe infection, whereas in the head kidney a broad immune suppression included deactivation of cytokines and GATA3 transcription factor responsible for T helper cell differentiation. In the spleen, the cytotoxic effector proteins perforin and granzyme B were downregulated and insulin, which may function as an immunomodulatory hormone inducing systemic immune suppression, was upregulated. These findings suggest that M. bejeranoi is a highly efficient parasite that disables the defense mechanisms of its fish host hybrid tilapia.

Cellular pathways during spawning induction in the starlet sea anemone Nematostella vectensis.

In cnidarians, long-term ecological success relies on sexual reproduction. The sea anemone Nematostella vectensis, which has emerged as an important model organism for developmental studies, can be induced for spawning by temperature elevation and light exposure. To uncover molecular mechanisms and pathways underlying spawning, we characterized the transcriptome of Nematostella females before and during spawning induction. We identified an array of processes involving numerous receptors, circadian clock components, cytoskeleton, and extracellular transcripts that are upregulated upon spawning induction. Concurrently, processes related to the cell cycle, fatty acid metabolism, and other housekeeping functions are downregulated. Real-time qPCR revealed that light exposure has a minor effect on expression levels of most examined transcripts, implying that temperature change is a stronger inducer for spawning in Nematostella. Our findings reveal the potential mechanisms that may enable the mesenteries to serve as a gonad-like tissue for the developing oocytes and expand our understanding of sexual reproduction in cnidarians.

Ectopic activation of GABAB receptors inhibits neurogenesis and metamorphosis in the cnidarian Nematostella vectensis.

The metabotropic gamma-aminobutyric acid B receptor (GABABR) is a G protein-coupled receptor that mediates neuronal inhibition by the neurotransmitter GABA. While GABABR-mediated signalling has been suggested to play central roles in neuronal differentiation and proliferation across evolution, it has mostly been studied in the mammalian brain. Here, we demonstrate that ectopic activation of GABABR signalling affects neurogenic functions in the sea anemone Nematostella vectensis. We identified four putative Nematostella GABABR homologues presenting conserved three-dimensional extracellular domains and residues needed for binding GABA and the GABABR agonist baclofen. Moreover, sustained activation of GABABR signalling reversibly arrests the critical metamorphosis transition from planktonic larva to sessile polyp life stage. To understand the processes that underlie the developmental arrest, we combined transcriptomic and spatial analyses of control and baclofen-treated larvae. Our findings reveal that the cnidarian neurogenic programme is arrested following the addition of baclofen to developing larvae. Specifically, neuron development and neurite extension were inhibited, resulting in an underdeveloped and less organized nervous system and downregulation of proneural factors including NvSoxB(2), NvNeuroD1 and NvElav1. Our results thus point to an evolutionarily conserved function of GABABR in neurogenesis regulation and shed light on early cnidarian development.

A novel asymmetric 3D in-vitro assay for the study of tumor cell invasion.

BACKGROUND: The induction of tumor cell invasion is an important step in tumor progression. Due to the cost and slowness of in-vivo invasion assays, there is need for quantitative in-vitro invasion assays that mimic as closely as possible the tumor environment and in which conditions can be rigorously controlled. METHODS: We have established a novel asymmetric 3D in-vitro invasion assay by embedding a monolayer of tumor cells between two layers of collagen. The cells were then allowed to invade the upper and lower layers of collagen. To visualize invading cells the gels were sectioned perpendicular to the monolayer so that after seeding the monolayer appears as a thin line precisely defining the origin of invasion. The number of invading tumor cells, their proliferation rate, the distance they traverse and the direction of invasion could then be determined quantitatively. RESULTS: The assay was used to compare the invasive properties of several tumor cell types and the results compare well with those obtained by previously described assays. Lysyl-oxidase like protein-2 (Loxl2) is a potent inducer of invasiveness. Using our assay we show for the first time that inhibition of endogenous Loxl2 expression in several types of tumor cells strongly inhibits their invasiveness. We also took advantage of the asymmetric nature of the assay in order to show that fibronectin enhances the invasiveness of breast cancer cells more potently than laminin. The asymmetric properties of the assay were also used to demonstrate that soluble factors derived from fibroblasts can preferentially attract invading breast cancer cells. CONCLUSION: Our assay displays several advantages over previous invasion assays as it is allows the quantitative analysis of directional invasive behavior of tumor cells in a 3D environment mimicking the tumor microenvironment. It should be particularly useful for the study of the effects of components of the tumor microenvironment on tumor cell invasiveness.

Functional and proteomic analysis of Ceratonova shasta (Cnidaria: Myxozoa) polar capsules reveals adaptations to parasitism.

Myxozoa is a diverse, speciose group of microscopic parasites, recently placed within the phylum Cnidaria. Myxozoans are highly reduced in size and complexity relative to free-living cnidarians, yet they have retained specialized organelles known as polar capsules, akin to the nematocyst stinging capsules of free-living species. Whereas in free-living cnidarians the stinging capsules are used for prey capture or defense, in myxozoans they have the essential function of initiating the host infection process. To explore the evolutionary adaptation of polar capsules to parasitism, we used as a model organism Ceratonova shasta, which causes lethal disease in salmonids. Here, we report the first isolation of C. shasta myxospore polar capsules using a tailored dielectrophoresis-based microfluidic chip. Using electron microscopy and functional analysis we demonstrated that C. shasta tubules have no openings and are likely used to anchor the spore to the host. Proteomic analysis of C. shasta polar capsules suggested that they have retained typical structural and housekeeping proteins found in nematocysts of jellyfish, sea anemones and Hydra, but have lost the most important functional group in nematocysts, namely toxins. Our findings support the hypothesis that polar capsules and nematocysts are homologous organelles, which have adapted to their distinct functions.