Organ-Wide qPCR Profiling Reveals High Trypanosome Intensity in Oral Mucosa of Cultured Larimichthys crocea

Trypanosomiasis has emerged as a significant health concern in cultured large yellow croaker (Larimichthys crocea), yet the organ-level distribution of the causative trypanosome within this host remains poorly characterized. The trypanosome was confirmed as Trypanosoma sp. Lc by 18S rRNA gene sequencing (99.9% identity to two isolates from L. crocea in Fujian); its precise taxonomic placement remains under discussion. We quantified trypanosome distribution across 12 organ types of naturally microscopy-confirmed infected L. crocea (n = 6; collected from Zhujiajian Island, Zhoushan, China) by RT-qPCR targeting the trypanosome 18S rRNA gene, with conspecific fish confirmed uninfected by blood smear microscopy (n = 3) included as biological negative controls. Trypanosome 18S rRNA signal was detected in all 12 organ types from infected L. crocea but not in any biological negative controls. Among organ types, the oral mucosa exhibited the highest trypanosome mean intensity (554.1 copies/μg RNA) significantly higher than that of mesentery (0.5 copies/μg RNA), muscle (0.8 copies/μg RNA) and gonad (1.5 copies/μg RNA) (p < 0.05 in all cases), followed by comparatively high levels in the heart (219.3 copies/μg RNA), gill (87.4 copies/μg RNA) and kidney (39.9 copies/μg RNA). In parallel, trypanosome morphology and subcellular structures were examined using scanning and transmission electron microscopy, supplemented by fluorescence staining. Ultrastructural observations identified typical trypomastigote features, including an elongated cell body with an undulating membrane and flagellum, as well as the kinetoplast, nucleus and paraflagellar rod. Together, these findings provide an organ-level distribution profile of Trypanosoma sp. Lc in large yellow croaker and define key ultrastructural characteristics that complement molecular identification. The high trypanosome 18S rRNA mean intensity observed in oral mucosa and heart may help inform sample collecting for molecular detection and field surveillance in large yellow croaker aquaculture.