Comparative Analysis of the Soluble Proteome and the Cytolytic Activity of Unbleached and Bleached Millepora complanata ("Fire Coral") from the Mexican Caribbean

Abstract

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.

Keywords: Millepora complanata; coral bleaching; hydrozoa; proteomics; toxins.

Figure 1

(A) Photographs of representative fragments of unbleached (UMc) and bleached (BMc) M. complanata collected in 2016 in the Mexican Caribbean. (B) Symbiont density quantified (n = 3) and expressed per cm² for UMc and BMc. Data are mean ± SEM. (*) Indicate significant difference (p < 0.05) in symbiont density between UMc and BMc. Photographs from UMc and BMc specimens were taken by Víctor Hugo Hernández-Elizárraga and Norma Olguín-Lopez.

Figure 2

Representative electrophoresis gel showing the protein profiles of the soluble proteomes from unbleached [UMc (1)] and bleached [BMc (2)] M. complanata specimens. Samples (80 μg of protein) were separated by one dimensional SDS-PAGE using 12% (w/v) polyacrylamide under non-reducing conditions. (MW) Molecular weight marker. Protein bands were stained using Coomassie Blue R-250. MW and samples 1 and 2 were resolved in the same gel, lanes with lower protein concentrations were removed.

Figure 3

Isolectric point (pI) and molecular weight distribution of proteins found in the soluble proteomes from unbleached (UMc) (n = 3) and bleached (n = 3) specimens (BMc) of M. complanata. (A) Distribution graph of molecular weights. (B) Distribution graph of isoelectric points.

Figure 4

Representative 2DE “master” gels of M. complanata-soluble proteomes. This master gel displays combined proteomes from both bleached (n = 3) and unbleached conditions (n = 3). The marks and numbers on the 2DE gels show the differentially expressed proteins (fold change ≥ 2). Significant changes in the levels of 35 proteins were observed. Protein spots in M. complanata-soluble proteome which exhibited higher levels in bleached samples are indicated as green marks. Protein spots whose levels were lower in bleached hydrocorals are indicated as red marks.

Figure 5

(A) Concentration-response curves showing the hemolytic activity elicited on rat erythrocytes by the soluble proteomes of unbleached (UMc) and bleached (BMc) specimens of M. complanata. (B) Progress curve for the PLA2 activity induced by UMC- and BMc- soluble proteomes at 414 nm. Positive control: PLA2 from Apis mellifera. Each point of the curves represents the mean of three replicates.