Harmful algal blooms (HABs) involving small unarmored dinoflagellates are a growing ecological and economic concern in the Philippines, particularly in aquaculture-dominated coastal areas where recurring fish kills are frequently reported. Among the most problematic taxa are members of the family Kareniaceae, such as Takayama, Karlodinium, and Karenia, which have been associated with massive fish mortalities and significant ecosystem disruption. Traditional microscopic methods often fail to provide reliable and timely species-level identification due to the morphological similarity of these taxa. Because Takayama acrotrocha has been directly associated with recurring fish kill events in Philippine waters, we developed and optimized a species-specific quantitative PCR (qPCR) assay targeting the internal transcribed spacer (ITS) region for this species. Primer sets and synthetic gene fragments were designed, synthesized, and validated in silicothrough IDT, Primer-BLAST, and NCBI BLAST, and further optimized using DNA extracts from cultured isolates using conventional PCR and qPCR. Robust amplification with high specificity was achieved at amplification efficiency (AE) of 91.3% at 250nM concentration of the cycle threshold (C_T) with an R² of 0.9975% and a slope of -3.550. Limit of detection (LOD) was established at 10² gene copies while the limit of quantification (LOQ) of this study had 10³ gene copies. Field validation of the assay was conducted in Central Luzon, particularly in Obando, Bulacan, where seasonal sampling revealed recurring blooms of T. acrotrocha in association with small-scale fish kill events. As an additional line of evidence, metabarcoding was applied to the water samples collected during the bloom (April 2025) and non-bloom period (June 2025) to characterize the broader phytoplankton community and screen for other potentially harmful Kareniaceae co-occurring with T. acrotrocha. This survey confirmed a dinoflagellate-dominated community in which Kareniaceae accounted for the majority of amplicon reads, with Karlodinium zhouanum and Takayama acrotrocha as the most abundant taxa and Karlodinium veneficum detected at low abundance, indicating that additional toxin-producing species co-occur with T. acrotrocha in these waters. Together, the targeted qPCR assay and the metabarcoding-based community survey provide a complementary framework combining rapid, sensitive, species-specific detection of the fish-kill-associated species with a broader assessment of co-occurring harmful Kareniaceae. This approach strengthens routine HAB surveillance and contributes essential baseline data for ecological assessments, aquaculture protection, and coastal management strategies in Philippine waters.

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