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.

Coral bleaching caused by global warming has resulted in massive damage to coral reefs worldwide. Studies addressing the consequences of elevated temperature have focused on organisms of the class Anthozoa, and up to now, there is little information regarding the mechanisms by which reef forming Hydrozoans face thermal stress. In this study, we carried out a comparative analysis of the soluble proteome and the cytolytic activity of unbleached and bleached Millepora complanata (“fire coral”) that inhabited reef colonies exposed to the 2015–2016 El Niño-Southern Oscillation in the Mexican Caribbean. A differential proteomic response involving proteins implicated in key cellular processes, such as glycolysis, DNA repair, stress response, calcium homeostasis, exocytosis, and cytoskeleton organization was found in bleached hydrocorals. Four of the proteins, whose levels increased in bleached specimens, displayed sequence similarity to a phospholipase A2, an astacin-like metalloprotease, and two pore forming toxins. However, a protein, which displayed sequence similarity to a calcium-independent phospholipase A2, showed lower levels in bleached cnidarians. Accordingly, the hemolytic effect of the soluble proteome of bleached hydrocorals was significantly higher, whereas the phospholipase A2 activity was significantly reduced. Our results suggest that bleached M. complanata is capable of increasing its toxins production in order to balance the lack of nutrients supplied by its symbionts.

BackgroundMillepora 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”.MethodsThe 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.ResultsThe hemolytic activity of the hydrocoral extract was enhanced in the presence of Ca2+ (≥2 mM), Mg2+ (≥6 mM), and Ba2+ (≥0.1 mM); however, it was reduced in the presence of Cu2+ (≥0.1 mM), Zn2+ (≥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.ConclusionsThe 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.

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To date, few studies have been carried out aimed at characterizing the toxins synthesized by hydrocorals of the genus Millepora. The purpose of this study was to explore the toxin diversity and antibacterial activity of the “fire coral” M. complanata using a transcriptomic data mining approach. In addition, the cytolytic and antibacterial activities of the M. complanata nematocyst proteome were experimentally confirmed. Cytolysins were predicted from the transcriptome by comparing against the Animal Toxin Annotation Project database, resulting in 190 putative toxins, including metalloproteases, hemostasis-impairing toxins, phospholipases, among others. The M. complanata nematocyst proteome was analyzed by 1D and 2D electrophoresis and zymography. The zymograms showed different zones of cytolytic activity: two zones of hemolysis at ~25 and ~205 kDa, two regions corresponding to phospholipase A2 (PLA2) activity around 6 and 25 kDa, and a proteolytic zone was observed between 50 and 205 kDa. The hemolytic activity of the proteome was inhibited in the presence of PLA2 and proteases inhibitors, suggesting that PLA2s, trypsin, chymotrypsin, serine-proteases, and matrix metalloproteases are responsible for the hemolysis. On the other hand, antimicrobial peptide sequences were retrieved from their transcripts with the amPEPpy software. This analysis revealed the presence of homologs to SK84, cgUbiquitin, Ubiquicidin, TroTbeta4, SPINK9-v1, and Histone-related antimicrobials in the transcriptome of this cnidarian. Finally, by employing disk diffusion and microdilution assays, we found that the nematocyst peptidome of M. complanata showed inhibitory activity against both Gram-positive and Gram-negative bacteria including S. enteritidis, P. perfectomarina, E. coli, and C. xerosis, among others. This is the first transcriptomic data mining analysis to explore the diversity of the toxins synthesized by an organism of the genus Millepora. Undoubtedly, this work provides information that will broaden our general understanding of the structural richness of cnidarian toxins.