Metabolomic analysis of larval stages of Onchocerca japonica (Spirurida: Onchocercidae), raised in black fly (Diptera: Simuliidae) vectors, by gas chromatography-tandem mass spectrometry

To monitor and prevent the spread of zoonotic onchocerciasis, identification of the natural vectors (blood-sucking insects) of its causative agents, Onchocerca species, is crucial. To date, vector identification depends on the detection of infective third-stage larvae in insects by traditional dissection. We aimed to develop a novel, more efficient method for the discrimination of the four larval stages, i.e. microfilariae (Mf), first-stage larvae (L1), second-stage larvae (L2), and third-stage larvae (L3), of O. japonica by metabolomic analysis. Microfilariae of O. japonica, the causative agent of zoonotic onchocerciasis in Japan, were obtained from skin snips of wild boars and injected into newly-emerged black flies (Diptera: Simuliidae) to enable further larval development. Metabolites obtained from Mf, L1, L2, and L3 were analyzed using a gas chromatography-tandem mass spectrometer. Multivariate analysis of the data of metabolites showed the complete separation of the four larval stages. The highest level of acetoacetic acid and hydroxylamine was found in Mf and L3, respectively. Consequently, we propose that hydroxylamine is a potential marker to detect infective larvae of O. japonica in natural infections and could be a valuable tool in vector surveys, transmission studies and epidemiological surveys.