Abstract
The hatching enzymes or choriolysins are key proteases in fish life cycle controlling the release of larvae to surrounding environment that have been suggested as target for novel biotechnological uses. Due to the large amounts of eggs released by the flatfish Solea senegalensis, during the spawning season, the hatching liquid properties and choriolysin-encoding genes were investigated in this species. A genomic analysis identified four putative genes referred to as SseHCEa, SseHCEb, SseLCE and SseHE. The phylogenetic analysis classified these paralogs into two clades, the clade I containing SseHCE paralogs and the clade II containing two well-supported subclades named as HE and LCE. The two SseHCE paralogs were intron-less and both genes were tandemly arrayed very close in the genome. The synteny and gene rearrangement identified in the flatfish lineage indicated that the duplication of these two paralogs occurred recently and they are under divergent evolution. The genes SseHE and SseLCE were structured in 8 exons and 7 introns and the synteny was conserved in teleosts. Expression studies confirmed that the four genes were expressed in the hatching gland cells and they migrate co-ordinately from the head to around the yolk sac close to the hatch with specific temporal and intensity expression profiles. Although the mRNA levels of the four genes peaked in the hours previous to larval hatching, the SseHCE and SseLCE paralogs kept a longer expression than SseHE after hatching. These expression patterns were consistent even when larvae were incubated at different temperatures that modified hatching times. The analysis of hatching-liquid using SDS-PAGE and zymography analyses of hatching liquid identified a major band of expected choriolysin size. The optimal pH for protease activity was 8.5 and inhibition assays using EDTA demonstrated that most of the activity in the hatching liquid was due to metalloproteases with Ca2+ ions acting as the most effective metal to restore the activity. All these data provide new clues about the choriolysin evolution and function in flatfish with impact in the aquaculture and the blue cosmetic industry.
Highlights
The hatching enzymes, known as choriolysins, are essential proteases for reproduction success and larval survival
This is of particular interest in those fish produced in aquaculture such as the Senegalese sole (Solea senegalensis) in which the enzymes play a key role in the quality and survival of hatched larvae and an excess volume of fertilized eggs are produced during the spawning season that can be used for biotechnological purposes
All the four predicted matured enzymes had the consensus active site sequence of the astacin family protease, HExxHxxGFxHExxRxDR, the conserved sequence SXMHY and six conserved cysteine residues specific for fish hatching enzymes (S1 Fig)
Summary
The hatching enzymes, known as choriolysins, are essential proteases for reproduction success and larval survival. These enzymes accumulate in the hatching gland cells as proenzymes and they are secreted and activated at the onset of larval hatching Both enzyme classes act cooperatively to disintegrate the egg envelope in a stepwise manner with HCE swelling the chorion and LCE fully solubilizing of chorionic proteins [4, 6]. These two choriolysin classes are conserved in most teleosts, the number of genes encoding them is highly variable across species due to the teleost-specific genome duplication (3R) and retrocopy mechanisms [5,6,7,8]. This is of particular interest in those fish produced in aquaculture such as the Senegalese sole (Solea senegalensis) in which the enzymes play a key role in the quality and survival of hatched larvae and an excess volume of fertilized eggs are produced during the spawning season that can be used for biotechnological purposes
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