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Species of the Karenia genus are widely distributed in global waters and frequently cause harmful algal blooms (HABs), posing significant threats to coastal ecosystems, aquaculture, and human safety. Among them, Karenia brevis is one of the most extensively studied species due to its production of brevetoxins, which are highly toxic to marine life and can adversely impact public health. In this study, we developed a rapid detection method that combines Recombinase-aided amplification (RAA) with CRISPR/LbCas12a, aimed at the precise identification of K. brevis. Specific RAA primers and crRNAs were designed based on the highly variable regions of the internal transcribed spacer (ITS) sequence of K. brevis. After screening multiple RAA primer pairs and crRNAs, the optimal combination was identified, ensuring both high efficiency and specificity of the detection system. For field application, two detection modes were employed: fluorescence-based (FQ) and lateral flow dipstick (LFD), along with a simple and rapid DNA extraction method. Sensitivity tests demonstrated that the detection limit of this method was 5.9 × 10³ copies/µL for plasmid DNA and 1 cell/mL for live cells. Environmental water samples collected during a HAB event in Lianjiang, Fujian Province, in May 2024, tested negative for K. brevis using the RAA-CRISPR/LbCas12a system. However, two other Karenia species—Karenia mikimotoi and Karenia longicanalis—were identified through ITS fragment amplification, cloning sequencing and phylogenetic analysis. In spiked experiments where K. brevis was introduced into natural water samples, the RAA-CRISPR/LbCas12a system accurately detected its presence. Overall, the RAA-CRISPR/LbCas12a detection system demonstrated excellent sensitivity, specificity and operational simplicity, making it a promising tool for rapid field detection of K. brevis and potentially suitable for broader applications in HAB monitoring.
Species of the Karenia genus are widely distributed in global waters and frequently cause harmful algal blooms (HABs), posing significant threats to coastal ecosystems, aquaculture, and human safety. Among them, Karenia brevis is one of the most extensively studied species due to its production of brevetoxins, which are highly toxic to marine life and can adversely impact public health. In this study, we developed a rapid detection method that combines Recombinase-aided amplification (RAA) with CRISPR/LbCas12a, aimed at the precise identification of K. brevis. Specific RAA primers and crRNAs were designed based on the highly variable regions of the internal transcribed spacer (ITS) sequence of K. brevis. After screening multiple RAA primer pairs and crRNAs, the optimal combination was identified, ensuring both high efficiency and specificity of the detection system. For field application, two detection modes were employed: fluorescence-based (FQ) and lateral flow dipstick (LFD), along with a simple and rapid DNA extraction method. Sensitivity tests demonstrated that the detection limit of this method was 5.9 × 10³ copies/µL for plasmid DNA and 1 cell/mL for live cells. Environmental water samples collected during a HAB event in Lianjiang, Fujian Province, in May 2024, tested negative for K. brevis using the RAA-CRISPR/LbCas12a system. However, two other Karenia species—Karenia mikimotoi and Karenia longicanalis—were identified through ITS fragment amplification, cloning sequencing and phylogenetic analysis. In spiked experiments where K. brevis was introduced into natural water samples, the RAA-CRISPR/LbCas12a system accurately detected its presence. Overall, the RAA-CRISPR/LbCas12a detection system demonstrated excellent sensitivity, specificity and operational simplicity, making it a promising tool for rapid field detection of K. brevis and potentially suitable for broader applications in HAB monitoring.
The dinoflagellate genus Karlodinium J. Larsen is well known to form harmful algal blooms (HABs), some of which can produce karlotoxins or other ichthyotoxins and thus cause fish-killing events. Among the 16 currently accepted species of Karlodinium (about half of which are reported to be toxic), six species (K. australe, K. decipiens, K. digitatum, K. elegans, K. veneficum, and K. zhouanum) have been reported or described in the coastal waters of China. However, a fine morphological and molecular characterization of the seldom-observed species K. decipiens has not been conducted; moreover, the negative effects of this species on aquatic animals have not been investigated. This work reports the morphological and phylogenetic characterization of a strain of K. decipiens isolated from Jiaozhou Bay, China, in 2019. The characterization of the strain was conducted using light and scanning electron microscopy, LSU, SSU rDNA, and ITS sequences-based systematic analyses, pigment analysis, and a detailed investigation of its potential toxic/harmful activity on aquatic animals. We observed the typical diagnostic features of K. decipiens, including its relatively large size, ellipsoidal or ovoid cell shape, ventral pore, ventral ridge connecting the two displaced ends of the cingulum, cingulum with a displacement of about one-third of the cell length, numerous polyhedral or slightly elongated chloroplasts distributed peripherally, and large nucleus located centrally. However, we also observed a large amphiesmal vesicle at the dorsal end of the ASC at the dorsal epicone, which is a novel feature that has never been reported from any species of the genus. Based on the results of this study, it is not clear whether this feature is a specific structure of the species or a common characteristic of the genus; therefore, this novel feature is worthy of further examination. Fucoxanthin was the most abundant pigment among all the carotenoids detected. The phylogenies inferred using Bayesian inference (BI) and maximum likelihood (ML) techniques confirmed the conspecificity of our isolate with the holotype K. decipiens (accession no. EF469236). In molecular trees, K. decipiens and K. antarcticum form a separate clade from other species of Karlodinium, and it should be examined whether a large amphiesma vesicle may be a characteristic of this clade. The exposure bioassays using brine shrimp (Artemia salina) indicated that K. decipiens exhibited toxicity to zooplankton, with 100% and 68% mortality observed in brine shrimp using live cell cultures and cell culture lysates over 120 h, respectively. Our work provides a detailed morphological and molecular characterization of K. decipiens from China. The results of this study broaden the known geographical distribution of this species and demonstrate it to be a harmful dinoflagellate.
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