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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SUMMARYOrganic matter dynamics and nutrient availability in saline agricultural soils of the State of Guanajuato might provide information for remediation strategies. 14 C labeled glucose with or without 200 mg kg -1 of NH 4 + -N soil was added to two clayey agricultural soils with different electrolytic conductivity (EC), i.e. 0.94 dS m -1 (low EC; LEC) and 6.72 dS m -1 (high EC; HEC), to investigate the effect of N availability and salt content on organic material decomposition. Inorganic N dynamics and production of CO 2 and 14 CO 2 were monitored. Approximately 60 % of the glucose-14 C added to LEC soil evolved as 14 CO 2 , but only 20 % in HEC soil after the incubation period of 21 days. After one day, < 200 mg 14 C was extractable from LEC soil, but > 500 mg 14 C from HEC soil. No N mineralization occurred in the LEC and HEC soils and glucose addition reduced the concentrations of inorganic N in unamended soil and soil amended with NH 4 + -N. The NO 2 -and NO 3 -concentrations were on average higher in LEC than in HEC soil, with exception of NO 2 -in HEC amended with NH 4 + -N. It was concluded that increases in soil EC reduced mineralization of the easily decomposable C substrate and resulted in Ndepleted soil.Index terms: dynamics of inorganic N, emission of 14 CO 2 and 12 CO 2 , saline soils. Os conhecimentos sobre a dinâmica da matéria orgânica e disponibilidade de nutrientes em solos salinos podem ser úteis para nortear a adoção de estratégias de recuperação e manejo. O objetivo deste trabalho foi avaliar o efeito da condutividade elétrica (EC) e presença de N inorgânico na decomposição da matéria orgânica a dois solos salinos localizados em Guanajuato (México). Nesse sentido, glicose marcada com 14 C com ou sem amônio na dose de 200 mg kg -1 de N foi adicionada em dois tipos de solo com valores de condutividade elétrica de 0,94 dS m -1 (baixo EC; LEC) e 6,72 dS m -1 (alta EC; HEC). Amostras de solos foram incubadas durante 21 dias e, ao longo desse tempo, foram avaliadas as concentrações de N inorgânico e a produção de 12 CO 2 e 14 CO 2 . Após o período de incubação, aproximadamente 60 % da glicose marcada com 14 C adicionado ao solo LEC evoluiu como 14 CO 2 , mas somente 20 % no solo HEC a evolução de 14 CO 2 foi de apenas 20 %. Decorrido um dia de incubação, menos de 200 mg de 14 C foram extraídos do solo LEC, ao passo que no solo HEC foram extraídos mais de 500 mg de 14 C. Não ocorreu mineralização de N nos dois solos e a adição de glicose reduziu as concentrações de N inorgânico no solo controle e no solo que recebeu N-NH 4 + . As concentrações de NO 2 -e NO 3 -foram, em média, maiores no solo LEC do que no solo HEC, com exceção do teor de NO 2 -, que foi maior no HEC tratado com NH 4 + . O aumento da condutividade elétrica promoveu a redução da mineralização de substrato facilmente decomponível e a diminuição do teor de N no solo.Termos de indexação: dinâmica de nitrogênio inorgânico, emissão de 12 CO 2 e 14 CO 2 , solos salinos.

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.

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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.