Species Diversity of the Ichthyotoxic Dinoflagellates Kareniaceae (Gymnodiniales) in Malaysia Waters Based on Morphology and Molecular Analysis, and Their Toxicologic Effect towards the Brine Shrimp (Artemia sp.) and Fish Fingerling (Lates calcarifer)

Sheryl Uncha, anak Andrew Chiba (2024) Species Diversity of the Ichthyotoxic Dinoflagellates Kareniaceae (Gymnodiniales) in Malaysia Waters Based on Morphology and Molecular Analysis, and Their Toxicologic Effect towards the Brine Shrimp (Artemia sp.) and Fish Fingerling (Lates calcarifer). Masters thesis, Universiti Malaysia Sarawak.

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Abstract

Kareniaceae is a known family of naked dinoflagellates associated with harmful algal blooms (HABs) worldwide is initially made up of three genera namely Karenia,Karlodinium and Takayama, and four recently established genera Asterodinium, Brachidinium, Gertia, and Shimiella. In Malaysia, Karlodinium australe had reported as the culprit in mass fish mortality in West Johor Straits in 2014 and the recurrence a year later had caused greater loss of mariculture. The aims of this study were to investigate the species diversity of Kareniaceae in Malaysia waters by morphological and molecular means, to investigate the survival of fish exposed to different cell densities and cell conditions of Karlodinium australe, and to examine the toxigenic effect of family Kareniaceae towards the brine shrimp Artemia sp. and fish. A total of 59 strains of Kareniaceae were established from the waters of Peninsular Malaysia and Malaysian Borneo. Ten Kareniaceae species, Karenia mikimotoi, Karlodinium armiger, K. australe, K. azanzae, K. ballantinum, K. decipiens, K. gentienii, K. veneficum, K. zhouanum and Takayama acrotrocha, were confirmed morphologically by light and scanning electron microscopy, and identified by key features such as cell shape, cell size, apical grove, ventral pore, and nucleus placement with the well-described species. Molecular phylogeny of internal transcribed spacer (ITS1─5.8S─ITS2) and large-subunit (LSU) ribosomal DNA were further confirmed the identity of the ten species above revealing polyphyletic clade of Karenia, Takayama and Karlodinium species. ITS2 secondary-structure transcript was applied to further support the species delineation of the Karlodinium species with presence of the Compensatory Base Changes (CBCs) and Hemi-Compensatory Base Changes (hCBCs) between the closely related species. Bioassay was conducted by exposing brine shrimp Artemia sp. to each Kareniaceae species in this study. Naupliar mortality was observed at initial 6-h of exposure and mortality rates increased to 50% after 24-h exposure. The 100% mortality of naupliar was observed after 48 hours in K. australe (cell density: 2.3 x 103 cells mL-1), K. azanzae (2.3 x 103 cells mL-1), K. veneficum (2.8 x 103 cells mL-1), K. armiger (2.3 x 103 cells mL-1), K. gentienii (2.7 x 103 cells mL-1), and T. acrotrocha (3.0 x 103 cells mL-1). In addition, further bioassay was conducted by exposing 1-inch fingerling seabass Lates calcarifer to K. australe in different cell densities and conditions. Fish mortality was observed at the first 10 min exposure at cell densities of 2.4 x 103 cells mL-1, 2.1 x 103 cells mL-1, and 1.8 x 103 cells mL-1, and 100% mortality occurred within 24-h at cell densities of 2.4 x 103 cells mL-1, 2.1 x 103 cells mL-1, 1.8 x 103 cells mL-1, 1.7 x 103 cells mL-1, and 1.5 x 103 cells mL-1. The result in this study showed K. mikimotoi, K. armiger, K. australe, K. azanzae, K. ballantinum, K. decipiens, K. veneficum, and T. acrotrocha were lethal to brine shrimp, and K. australe is toxic and harmful to fish. The presence of ten Kareniceae species in this study is the first record on high diversity family Kareniacea in Malaysia waters, where two species were recorded in Johor and Perak waters, five species recorded in Sarawak waters and seven species recorded in Sabah waters.

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