Transcriptomic and proteomic analyses reveal the first occurrence of diverse toxin groups in Millepora alcicornis.

Millepora alcicornis is a reef-forming cnidarian widely distributed in the Mexican Caribbean. Millepora species or "fire corals" inflict a painful stinging reaction in humans when touched. Even though hundreds of organic and polypeptide toxins have been characterized from sea anemones and jellyfish, there are few reports regarding the diversity of toxins synthesized by fire corals. Here, based on transcriptomic analysis of M. alcicornis, several predicted proteins that show amino acid sequence similarity to toxins were identified, including neurotoxins, metalloproteases, hemostasis-impairing toxins, serin proteases, cysteine-rich venom proteins, phospholipases, complement system-impairing toxins, phosphodiesterases, pore-forming toxins, and L-aminoacid oxidases. The soluble nematocyst proteome of this organism was shown to induce hemolytic, proteolytic, and phospholipase A2 effects by gel zymography. Protein bands or spots on 1D- and 2D-PAGE gels corresponding to zones of hemolytic and enzymatic activities were excised, subjected to in-gel digestion with trypsin, and analyzed by mass spectrometry. These proteins exhibited sequence homology to PLA2s, metalloproteinases, pore-forming toxins, and neurotoxins, such as actitoxins and CrTX-A. The complex array of venom-related transcripts that were identified in M. alcicornis, some of which are first reported in "fire corals", provide novel insight into the structural richness of Cnidarian toxins and their distribution among species. SIGNIFICANCE: Marine organisms are a promising source of bioactive compounds with valuable contributions in diverse fields such as human health, pharmaceuticals, and industrial application. Currently, not much attention has been paid to the study of fire corals, which possess a variety of molecules that exhibit diverse toxic effects and therefore have great pharmaceutical and biotechnological potential. The isolation and identification of novel marine-derived toxins by classical approaches are time-consuming and have low yields. Thus, next-generation strategies, like base-'omics technologies, are essential for the high-throughput characterization of venom compounds such as those synthesized by fire corals. This study moves the field forward because it provides new insights regarding the first occurrence of diverse toxin groups in Millepora alcicornis. The findings presented here will contribute to the current understanding of the mechanisms of action of Millepora toxins. This research also reveals important information related to the potential role of toxins in the defense and capture of prey mechanisms and for designing appropriate treatments for fire coral envenomation. Moreover, due to the lack of information on the taxonomic identification of Millepora, the insights presented here can advise the taxonomic classification of the species of this genus.

Transcriptomic differences between bleached and unbleached hydrozoan Millepora complanata following the 2015-2016 ENSO in the Mexican Caribbean.

The 2015-2016 El Niño-southern oscillation or "ENSO" caused many M. complanata colonies that live in the Mexican Caribbean to experience extensive bleaching. The purpose of this work was to analyze the effect of bleaching on the cellular response of M. complanata, employing a transcriptomic approach with RNA-seq. As expected, bleached specimens contained a significantly lower chlorophyll content than unbleached hydrocorals. The presence of algae of the genera Durusdinium and Cladocopium was only found in tissues of unbleached M. complanata, which could be associated to the greater resistance that these colonies exhibited during bleaching. We found that 299 genes were differentially expressed in M. complanata bleached colonies following the 2015-2016 ENSO in the Mexican Caribbean. The differential expression analysis of bleached M. complanata specimens evidenced enriched terms for functional categories, such as ribosome, RNA polymerase and basal transcription factors, chaperone, oxidoreductase, among others. Our results suggest that the heat-shock response mechanisms displayed by M. complanata include: an up-regulation of endogenous antioxidant defenses; a higher expression of heat stress response genes; up-regulation of transcription-related genes, higher expression of genes associated to transport processes, inter alia. This study constitutes the first differential gene expression analysis of the molecular response of a reef-forming hydrozoan during bleaching.

Living on fire: Deactivating fire coral polyps for larval settlement and symbiosis in the fire coral-associated barnacle Wanella milleporae (Thoracicalcarea: Wanellinae).

Symbiosis is increasingly recognized as being an important component in marine systems, and many such relationships are initiated when free-swimming larvae of one partner settle and become sedentary on a host partner. Therefore, several crucial questions emerge such as the larva's mechanism of locating a host, selection of substratum and finally settlement on the surface of its future partner. Here, we investigated these mechanisms by studying how larvae of the fire coral-associated barnacle Wanella milleporae move, settle and establish symbiosis with their host, Millepora tenera. Cyprids of W. milleporae possess a pair of specialized antennules with bell-shaped attachment discs that enable them to explore and settle superficially on the hostile surface of the fire coral. Intriguingly, the stinging polyps of the fire coral remain in their respective pores when the cyprids explore the fire coral surface. Even when cyprids come into contact with the nematocysts on the extended stinging polyps during the exploratory phase, no immobilization effects against the cyprids were observed. The exploratory phase of Wanella cyprids can be divided into a sequence of wide searching (large step length and high walking speed), close searching (small step length and low speed) and inspection behavior, eventually resulting in permanent settlement and metamorphosis. After settlement, xenogeneic interactions occur between the fire coral and the newly metamorphosed juvenile barnacle. This involved tissue necrosis and regeneration in the fire coral host, leading to a callus ring structure around the juvenile barnacle, enhancing survival rate after settlement. The complex exploratory and settlement patterns and interactions documented here represent a breakthrough in coral reef symbiosis studies to show how invertebrates start symbiosis with fire corals.

Ghost fishing impacts on hydrocorals and associated reef fish assemblages.

Ghost fishing is a threat to many marine environments, as lost or discarded fishing gear (e.g., fishing lines, nets) continues to fish by entangling, damaging or killing various organisms. Among the benthic organisms that live on tropical reefs, the group probably most affected, due to their shape, are the branching corals. These corals provide refuge, foraging and breeding sites, especially for fishes and therefore impacts on coral structure could compromise the ecology of associated species. We tested if fishing lines entangled on the branching coral Millepora alcicornis would result in an increase in colony mortality, decrease in abundance and richness of fishes and changes in the behavior of associated reef fish. In the field, we estimated the volume of M. alcicornis colonies and its mortality percentages, and videos were recorded to evaluate abundance and richness of fish assemblages and fish behavior. Our results showed that coral mortality increased with increasing amounts of entangled fishing lines. Fish assemblages were similar in M. alcicornis colonies with or without entangled fishing lines. Nevertheless, we observed a significant decrease in the frequency of feeding attempts in two herbivore fish species (Acanthurus bahianus and Ophioblennius trinitatis) that play an important role in coral-reef dynamics, controlling algae abundances. Therefore, ghost fishing has negative impacts on shallow reef ecosystems, directly affecting branching corals and important coral-fish interactions. Management of tropical shallow reef environments should consider regulation and monitoring of coastal fisheries to ensure reef integrity.

Impact of El Niño-Southern Oscillation 2015-2016 on the soluble proteomic profile and cytolytic activity of Millepora alcicornis ("fire coral") from the Mexican Caribbean.

Reef-forming cnidarians are extremely susceptible to the "bleaching" phenomenon caused by global warming. The effect of elevated seawater temperature has been extensively studied on Anthozoans; however, to date the impact of thermal stress on the expression of genes and proteins in Hydrozoan species has not been investigated. The present study aimed to determine the differential proteomic profile of Millepora alcicornis, which inhabits the Mexican Caribbean, in response to the El Niño-Southern Oscillation 2015-2016. Additionally, the cytolytic activity of the soluble proteomes obtained from normal and bleached M. alcicornis was assessed. Bleached specimens showed decreased symbiont's density and chlorophyll a and c2 levels. After bleaching, we observed a differential expression of 17 key proteins, tentatively identified as related to exocytosis, calcium homeostasis, cytoskeletal organization, and potential toxins, including a metalloprotease, a phospholipase A2 (PLA2), and an actitoxin. Although, some of the differentially expressed proteins included potential toxins, the hemolytic, PLA2, and proteolytic activities elicited by the soluble proteomes from bleached and normal specimens were not significantly different. The present study provides heretofore-unknown evidence that thermal stress produces a differential expression of proteins involved in essential cellular processes of Hydrozoan species. Even though our results showed an over-expression of some potential toxin-related proteins, the cytolytic effect (as assessed by hemolytic, PLA2, and caseinolytic activities) was not increased in bleached M. alcicornis, which suggests that the cytolysis is mainly produced by toxins whose expression was not affected by temperature stress. These findings allow hypothesizing that this hydrocoral is able to prey heterotrophically when suffering from moderate bleaching, giving it a better chance to withstand the effects of high temperature.

Millepora species (Fire Coral) Sting: A Case Report and Review of Recommended Management.

Fire corals (Millepora spp) are the second most common reef-forming organisms and are frequently found in tropical and subtropical waters. Fire corals are not true corals but rather hydrozoans more closely related to jellyfish and sea nettles. Rigidly affixed to the reef and with a branching structure, each fire coral is a colony of numerous individual hydrozoans forming a collective symbiotic organism. It is common for divers to accidentally make contact with fire corals. Fire coral contact is characterized by the immediate onset of burning pain caused by venom discharge from numerous tiny nematocysts located externally on the creature. Treatment consists of saltwater irrigation of the wound, nematocyst removal, and supportive care of the associated symptoms of pain, dermatitis, and pruritus. Rarely, fire coral can cause systemic toxicity. We present a case report of a 30-y-old recreational diver who experienced a fire coral sting of her left anterior thigh and review the recommended prevention and management of fire coral stings.

Cytolytic and systemic toxic effects induced by the aqueous extract of the fire coral Millepora alcicornis collected in the Mexican Caribbean and detection of two types of cytolisins.

BACKGROUND: Millepora alcicornis is a branching hydrocoral common throughout the Caribbean Sea. Like other members of this genus, this species is capable of inducing skin eruptions and blisters with severe pain after contact. In the present study, we investigated the toxicity of the M. alcicornis aqueous extract on several animal models. Considering that some cnidarian hemolysins have been associated to local tissue damage, since they also induce lysis of other cell types, we also made a partial characterization of the hemolytic activity of M. alcicornis aqueous extract. This information is important for understanding the defense mechanisms of the "fire corals". METHODS: The effects of pH, temperature, and some divalent cations on the hemolytic activity of the extract were assayed, followed by a zymogram analysis to detect the cytolysins and determine their approximate molecular weight. The toxicity of the aqueous extract was assayed in mice, by intravenous administration, and histopathological changes on several tissues were analyzed by light microscopy. The toxicity of the extract was also tested in Artemia salina nauplii, and the damages caused on the crustaceans were analyzed by transmission and scanning electron microscopy. RESULTS: The hemolytic activity of the hydrocoral extract was enhanced in the presence of Ca(2+) (≥2 mM), Mg(2+) (≥6 mM), and Ba(2+) (≥0.1 mM); however, it was reduced in the presence of Cu(2+) (≥0.1 mM), Zn(2+) (≥6 mM), and EDTA (≥0.34 mM). Differences in the pH did not affect the hemolytic activity, but it was temperature-sensitive, since preincubation at ≥ 50 °C sharply reduced hemolysis. The zymogram showed the presence of two types of hemolysins: ~ 28-30 kDa proteins with phospholipase A2 activity and ~ 200 kDa proteins that do not elicit enzymatic activity. The aqueous extract of this cnidarian was lethal to mice (LD50 = 17 µg protein/g), and induced kidney, liver, and lung damages. Under denaturing conditions, the aqueous extract completely lost its toxic and hemolytic activities. CONCLUSIONS: The results showed that the M. alcicornis aqueous extract contains two types of thermolabile hemolysins: proteins of approximately 28-30 kDa with PLA2 activity, while the others are larger proteins of approximately 200 kDa, which do not possess PLA2 activity. Those thermolabile cytolysins, which are stable to pH changes and whose activity is calcium dependent, are capable of inducing damage in lung, kidney and liver tissues, resulting in a slow death of mice. M. alcicornis cytolysins also provoke tissue dissociation in Artemia salina nauplii that might be attributed to pore forming mechanisms.

Cytolytic and systemic toxic effects induced by the aqueous extract of the fire coral Millepora alcicornis collected in the Mexican Caribbean and detection of two types of cytolisins

Background Millepora alcicornis is a branching hydrocoral common throughout the Caribbean Sea. Like other members of this genus, this species is capable of inducing skin eruptions and blisters with severe pain after contact. In the present study, we investigated the toxicity of theM. alcicornis aqueous extract on several animal models. Considering that some cnidarian hemolysins have been associated to local tissue damage, since they also induce lysis of other cell types, we also made a partial characterization of the hemolytic activity of M. alcicornis aqueous extract. This information is important for understanding the defense mechanisms of the fire corals.Methods The effects of pH, temperature, and some divalent cations on the hemolytic activity of the extract were assayed, followed by a zymogram analysis to detect the cytolysins and determine their approximate molecular weight. The toxicity of the aqueous extract was assayed in mice, by intravenous administration, and histopathological changes on several tissues were analyzed by light microscopy. The toxicity of the extract was also tested inArtemia salina nauplii, and the damages caused on the crustaceans were analyzed by transmission and scanning electron microscopy.Results The hemolytic activity of the hydrocoral extract was enhanced in the presence of Ca 2+ (2 mM), Mg 2+ (6 mM), and Ba2+ (0.1 mM); however, it was reduced in the presence of Cu2+(0.1 mM), Zn 2+ (6 mM), and EDTA (0.34 mM). Differences in the pH did not affect the hemolytic activity, but it was temperature-sensitive, since preincubation at 50 °C sharply reduced hemolysis. The zymogram showed the presence of two types of hemolysins: ~ 2830 kDa proteins with phospholipase A 2 activity and ~ 200 kDa proteins that do not elicit enzymatic activity. The aqueous extract of this cnidarian was lethal to mice (LD 50 = 17 g protein/g), and induced kidney, liver, and lung damages. Under denaturing conditions, the aqueous extract completely lost its toxic and hemolytic activities.Conclusions The results showed that the M. alcicornis aqueous extract contains two types of thermolabile hemolysins: proteins of approximately 2830 kDa with PLA 2 activity, while the others are larger proteins of approximately 200 kDa, which do not possess PLA 2activity. Those thermolabile cytolysins, which are stable to pH changes and whose activity is calcium dependent, are capable of inducing damage in lung, kidney and liver tissues, resulting in a slow death of mice. M. alcicorniscytolysins also provoke tissue dissociation inArtemia salina nauplii that might be attributed to pore forming mechanisms.

Cytolytic and systemic toxic effects induced by the aqueous extract of the fire coral Millepora alcicornis collected in the Mexican Caribbean and detection of two types of cytolisins

Background Millepora alcicornis is a branching hydrocoral common throughout the Caribbean Sea. Like other members of this genus, this species is capable of inducing skin eruptions and blisters with severe pain after contact. In the present study, we investigated the toxicity of theM. alcicornis aqueous extract on several animal models. Considering that some cnidarian hemolysins have been associated to local tissue damage, since they also induce lysis of other cell types, we also made a partial characterization of the hemolytic activity of M. alcicornis aqueous extract. This information is important for understanding the defense mechanisms of the "fire corals".Methods The effects of pH, temperature, and some divalent cations on the hemolytic activity of the extract were assayed, followed by a zymogram analysis to detect the cytolysins and determine their approximate molecular weight. The toxicity of the aqueous extract was assayed in mice, by intravenous administration, and histopathological changes on several tissues were analyzed by light microscopy. The toxicity of the extract was also tested inArtemia salina nauplii, and the damages caused on the crustaceans were analyzed by transmission and scanning electron microscopy.Results The hemolytic activity of the hydrocoral extract was enhanced in the presence of Ca 2+ (≥2 mM), Mg 2+ (≥6 mM), and Ba2+ (≥0.1 mM); however, it was reduced in the presence of Cu2+(≥0.1 mM), Zn 2+ (≥6 mM), and EDTA (≥0.34 mM). Differences in the pH did not affect the hemolytic activity, but it was temperature-sensitive, since preincubation at ≥ 50 °C sharply reduced hemolysis. The zymogram showed the presence of two types of hemolysins: ~ 28-30 kDa proteins with phospholipase A 2 activity and ~ 200 kDa proteins that do not elicit enzymatic activity. The aqueous extract of this cnidarian was lethal to mice (LD 50 = 17 μg protein/g), and induced kidney, liver, and lung damages. Under denaturing conditions, the aqueous extract completely lost its toxic and hemolytic activities.Conclusions The results showed that the M. alcicornis aqueous extract contains two types of thermolabile hemolysins: proteins of approximately 28-30 kDa with PLA 2 activity, while the others are larger proteins of approximately 200 kDa, which do not possess PLA 2activity. Those thermolabile cytolysins, which are stable to pH changes and whose activity is calcium dependent, are capable of inducing damage in lung, kidney and liver tissues, resulting in a slow death of mice. M. alcicorniscytolysins also provoke tissue dissociation inArtemia salina nauplii that might be attributed to pore forming mechanisms.(AU)

Characteristics of hemolytic activity induced by the aqueous extract of the Mexican fire coral Millepora complanata.

BACKGROUND: Millepora complanata is a plate-like fire coral common throughout the Caribbean. Contact with this species usually provokes burning pain, erythema and urticariform lesions. Our previous study suggested that the aqueous extract of M. complanata contains non-protein hemolysins that are soluble in water and ethanol. In general, the local damage induced by cnidarian venoms has been associated with hemolysins. The characterization of the effects of these components is important for the understanding of the defense mechanisms of fire corals. In addition, this information could lead to better care for victims of envenomation accidents. METHODS: An ethanolic extract from the lyophilized aqueous extract was prepared and its hemolytic activity was compared with the hemolysis induced by the denatured aqueous extract. Based on the finding that ethanol failed to induce nematocyst discharge, ethanolic extracts were prepared from artificially bleached and normal M. complanata fragments and their hemolytic activity was tested in order to obtain information about the source of the heat-stable hemolysins. RESULTS: Rodent erythrocytes were more susceptible to the aqueous extract than chicken and human erythrocytes. Hemolytic activity started at ten minutes of incubation and was relatively stable within the range of 28-50°C. When the aqueous extract was preincubated at temperatures over 60°C, hemolytic activity was significantly reduced. The denatured extract induced a slow hemolytic activity (HU50 = 1,050.00 ± 45.85 µg/mL), detectable four hours after incubation, which was similar to that induced by the ethanolic extract prepared from the aqueous extract (HU50 = 1,167.00 ± 54.95 µg/mL). No significant differences were observed between hemolysis induced by ethanolic extracts from bleached and normal fragments, although both activities were more potent than hemolysis induced by the denatured extract. CONCLUSIONS: The results showed that the aqueous extract of M. complanata possesses one or more powerful heat-labile hemolytic proteins that are slightly more resistant to temperature than jellyfish venoms. This extract also contains slow thermostable hemolysins highly soluble in ethanol that are probably derived from the body tissues of the hydrozoan.

Characteristics of hemolytic activity induced by the aqueous extract of the Mexican fire coral Millepora complanata

Background Millepora complanata is a plate-like fire coral common throughout the Caribbean. Contact with this species usually provokes burning pain, erythema and urticariform lesions. Our previous study suggested that the aqueous extract of M. complanata contains non-protein hemolysins that are soluble in water and ethanol. In general, the local damage induced by cnidarian venoms has been associated with hemolysins. The characterization of the effects of these components is important for the understanding of the defense mechanisms of fire corals. In addition, this information could lead to better care for victims of envenomation accidents.Methods An ethanolic extract from the lyophilized aqueous extract was prepared and its hemolytic activity was compared with the hemolysis induced by the denatured aqueous extract. Based on the finding that ethanol failed to induce nematocyst discharge, ethanolic extracts were prepared from artificially bleached and normal M. complanata fragments and their hemolytic activity was tested in order to obtain information about the source of the heat-stable hemolysins.Results Rodent erythrocytes were more susceptible to the aqueous extract than chicken and human erythrocytes. Hemolytic activity started at ten minutes of incubation and was relatively stable within the range of 28-50°C. When the aqueous extract was preincubated at temperatures over 60°C, hemolytic activity was significantly reduced. The denatured extract induced a slow hemolytic activity (HU50= 1,050.00 ± 45.85 g/mL), detectable four hours after incubation, which was similar to that induced by the ethanolic extract prepared from the aqueous extract (HU50= 1,167.00 ± 54.95 g/mL). No significant differences were observed between hemolysis induced by ethanolic extracts from bleached and normal fragments, although both activities were more potent than hemolysis induced by the denatured extract...

Characteristics of hemolytic activity induced by the aqueous extract of the Mexican fire coral Millepora complanata

Background Millepora complanata is a plate-like fire coral common throughout the Caribbean. Contact with this species usually provokes burning pain, erythema and urticariform lesions. Our previous study suggested that the aqueous extract of M. complanata contains non-protein hemolysins that are soluble in water and ethanol. In general, the local damage induced by cnidarian venoms has been associated with hemolysins. The characterization of the effects of these components is important for the understanding of the defense mechanisms of fire corals. In addition, this information could lead to better care for victims of envenomation accidents.Methods An ethanolic extract from the lyophilized aqueous extract was prepared and its hemolytic activity was compared with the hemolysis induced by the denatured aqueous extract. Based on the finding that ethanol failed to induce nematocyst discharge, ethanolic extracts were prepared from artificially bleached and normal M. complanata fragments and their hemolytic activity was tested in order to obtain information about the source of the heat-stable hemolysins.Results Rodent erythrocytes were more susceptible to the aqueous extract than chicken and human erythrocytes. Hemolytic activity started at ten minutes of incubation and was relatively stable within the range of 28-50°C. When the aqueous extract was preincubated at temperatures over 60°C, hemolytic activity was significantly reduced. The denatured extract induced a slow hemolytic activity (HU50= 1,050.00 ± 45.85 μg/mL), detectable four hours after incubation, which was similar to that induced by the ethanolic extract prepared from the aqueous extract (HU50= 1,167.00 ± 54.95 μg/mL). No significant differences were observed between hemolysis induced by ethanolic extracts from bleached and normal fragments, although both activities were more potent than hemolysis induced by the denatured extract.Conclusions The results showed that the aqueous extract of M. complanata possesses one or more powerful heat-labile hemolytic proteins that are slightly more resistant to temperature than jellyfish venoms. This extract also contains slow thermostable hemolysins highly soluble in ethanol that are probably derived from the body tissues of the hydrozoan.(AU)