Introduction From 2023 to 2025, Trypanosoma spp. have emerged as a significant threat to the sustainable aquaculture of Larimichthys crocea, causing substantial economic losses. Current strategies for controlling andpreventing trypanosomiasis in thisspecies remain limited. Early diagnosis is crucial for disease prevention and control, but existing detection methods have limitations such as low sensitivity and cumbersome operation.

Objective This study aims to establish a rapid and accurate detection technology for Trypanosoma sp. in L. crocea and explore its tissue distribution characteristics, providing support for early disease diagnosis and epidemiological investigation.

Methods Specific primers and TaqMan probes were designed based on the conserved sequence of Trypanosoma spp. 18S rDNA in GenBank, and a fluorescence quantitative PCR (qPCR) method was constructed; a standard curve was established through gradient dilution of recombinant plasmids to verify the specificity, sensitivity, and repeatability of the method; samples of L. crocea from Sandu Island, Xiapu, and Lianjiang breeding areas were collected to detect infection rates and analyze the differences in Trypanosoma spp. content in 9 tissues.

Results The standard curve showed a good linear relationship (R²=0.997), with a detection limit of 25 copies/µL; the inter-group and intra-group cycle threshold variation coefficients were respectively <2.0% and <1.5%, and the repeatability was stable; there was no cross-reaction with common aquatic pathogens such as ciliates, Vibrio parahaemolyticus, and neurotropic virus, and the specificity was significant. The overall infection rate of 60 samples was 93.33%, with the highest infection rate in Sandu Island (100%); the tissue distribution showed that the content of Trypanosoma spp. in blood, heart, and spleen was significantly higher than that in gills, liver, and kidneys, etc. (P<0.01). In addition, only a small number of Mugil cephalus and Pagrus major were infected, with extremely low levels of pathogens. Among non-fish samples, Undaria pinnatifida (Wakame) had the highest detection rate (93.33%), while a small amount of pathogens were detected in netting and oysters, and no pathogens were detected in the remaining samples.

Conclusion The fluorescence quantitative PCR method established in the study has high specificity, sensitivity, and repeatability, and can be used for early diagnosis of Trypanosoma spp. disease; the tendency of Trypanosoma spp. enrichment in tissues such as blood, heart, and spleen provides a scientific basis for analyzing its pathogenic mechanism and formulating targeted control strategies, and is of great significance for the healthy development of the L. crocea aquaculture industry.