Multiplex PCR for sexing Schistosoma japonicum cercariae and its utility

Accurate discrimination of the Schistosoma japonicum cercariae gender is very important for establishing monosexual infection animal models and for standardizing the real intensity of infection. In this study, a multiplex PCR technique consisting of two pairs of primers, of which one amplifies a 185-bp band specific for the W chromosome and the other amplifies a 420-bp band for the Z chromosome, was established to sex the S. japonicum cercariae. For male cercariae (ZZ), a single 420-bp band is expected, and for female cercariea (ZW), two distinct 185-bp and 420-bp bands can be observed. There was no cross-reaction with S. mansoni, S. haematobium, Clonorchis sinensis, Paragonimus westermani, and Trichinella spiralis. After sexing the cercariae escaped from a single snail, mice in group A were infected with 60 male cercariae and mice of group B were infected with 40 female cercariae. Meanwhile, mice in group C were infected with 10 male and 10 female cercariae that were sexed by multiplex PCR. At 45 days postinfection, male and female adult worms were recovered to verify the accuracy of multiplex PCR for sexing S. japonicum cercariae and to calculate the male and female survival rate and paired worm ratio. Our results showed that the multiplex PCR technique could distinguish male cercariae with 100% accuracy. However, sometimes the discrimination results of multiplex PCR mis-scored mixed sexual cercariae as female cercariae. The mean male adult worm burden in mice of group C was 10.7 ± 2.4, and the mean female adult worm burden was 7.7 ± 2.5. There was a significant difference between the male worm burden and female worm burden in group C. The P value was 0.013. The real paired worm ratio of group C was 74.2% (95%CI 56.6~91.8%). These results demonstrated a male-biased sex ratio in the mice model with equilibrated sex ratio cercariae infection, as predicted by our multiplex PCR technique. In conclusion, our multiplex PCR technique is an effective tool for sexing S. japonicum cercariae, especially for distinguishing male cercariae, which is of great value for establishing monosexual cercariae infection mice models to harvest male adult worms for anti-schistosomal drug screening.