Homologues of Piwi control transposable elements and development of male germline in Penaeus monodon

Abstract

PIWI belongs to the Argonaute protein family, which is a major protein component in RNA silencing pathway. Piwi proteins play roles in the control of transposons and germline development. They have been widely studied in vertebrates and flies, while very little is known in crustacean so far. We have previously identified and characterized a cDNA encoding Piwi protein (PmPiwi1) in the black tiger shrimp Penaeus monodon. In this study, a cDNA encoding another Piwi protein namely PmPiwi2 was identified by rapid amplification of cDNA ends (RACEs). PmPiwi2 was expressed solely in shrimp testis and ovary, indicating its potential role in germ cell development. Similar to PmPiwi1, PmPiwi2 also plays a part in the control of transposons as PmPiwi2-knockdown shrimp showed a significant increase in the expression of gypsy2 retrotransposon and mariner element in the testis. In addition, a reduction of sperm numbers in the spermatophore of PmPiwi2-knockdown shrimp suggests that PmPiwi2 is required for spermatogenesis similar to PmPiwi1. This study further demonstrated that apoptotic cell death was strongly detected in spermatogonia and spermatocyte cells of both PmPiwi-knockdown shrimp and thus, could be the cause of reduced sperm count. Investigation of sperm morphology showed a remarkably high proportion of abnormal sperms in the spermatophore of the PmPiwi1-knockdown shrimp, while PmPiwi2-knockdown shrimp had comparable percentage of abnormal sperms to the control shrimp. Consistently, the expression of KIFC1, a gene that is necessary for spermiogenesis was significantly reduced upon PmPiwi1 silencing, but not in the PmPiwi2-knockdown shrimp. Our results suggested that while both PmPiwis are required for the development of spermatid, only PmPiwi1 is possibly involved in the final stage of sperm maturation.