Proteome Analysis of Whole-Body Responses in Medaka Experimentally Exposed to Fish-Killing Dinoflagellate Karenia mikimotoi

Abstract

Karenia mikimotoi is a well-known harmful algal bloom species. Blooms of this dinoflagellate have become a serious threat to marine life, including fish, shellfish, and zooplanktons and are usually associated with massive fish death. Despite the discovery of several toxins such as gymnocins and gymnodimines in K. mikimotoi, the mechanisms underlying the ichthyotoxicity of this species remain unclear, and molecular studies on this topic have never been reported. The present study investigates the fish-killing mechanisms of K. mikimotoi through comparative proteomic analysis. Marine medaka, a model fish organism, was exposed to K. mikimotoi for a three-part time period (LT25, LT50 and LT90). Proteins extracted from the whole fish were separated by using two-dimensional gel electrophoresis, and differentially expressed proteins were identified with reference to an untreated control. The change in fish proteomes over the time-course of exposure were analyzed. A total of 35 differential protein spots covering 19 different proteins were identified, of which most began to show significant change in expression levels at the earliest stage of intoxication. Among the 19 identified proteins, some are closely related to the oxidative stress responses, energy metabolism, and muscle contraction. We propose that oxidative stress-mediated muscle damage might explain the symptoms developed during the ichthyotoxicity test, such as gasping for breath, loss of balance, and body twitching. Our findings lay the foundations for more in-depth studies of the mechanisms of K. mikimotoi’s ichthyotoxicity.

Review of Harmful Algal Blooms (HABs) Causing Marine Fish Kills: Toxicity and Mitigation

“…Large-scale fish kills have been documented along the coastal seas of Europe and Asia, resulting in significant economic losses [50,52]. Three main mechanisms by which K. mikimotoi may cause fish deaths include [53]: (1) the production of reactive oxygen species (ROS) that damage fish respiratory systems and destabilize their antioxidant defenses; (2) fish suffocation due to lack of dissolved oxygen; and (3) the release of cytotoxic toxins like gymnocins, hemolysins, and specific polyunsaturated fatty acids. Another evolving neurotoxin known as gymnodimines has also been reported in fish kills and is produced by Karenia selliformis [54].…”

“…To identify differentially expressed proteins, entire proteomes of medaka (the Japanese rice fish) were studied using 2-DE (two-dimensional electrophoresis) by varying the toxin dosage of K. mikimotoi (LT25, LT50, and LT90) [53]. A total of 35 differential protein locations with at least two-fold variations were confirmed with mass spectrometric analysis.…”

“…Several unfavorable symptoms that emerged during the exposure time, such as asphyxiation, losing balance, and body jerking, which are all symptoms of muscle injury, were observed. Kwok et al [53] reported a novel approach employing molecular analysis to study ichthyotoxicity processes; the exact toxicity pathways of fish kills are still at the infant stage, as inferred by these authors.…”