, a Nuclear-Encoded Mitochondrial Gene Duplicate, Is Essential for Male Fertility in

Recent studies on nuclear-encoded mitochondrial genes (N-mt genes) in have shown a unique pattern of expression for newly duplicated N-mt genes, with many duplicates having a testis-biased expression and playing an essential role in spermatogenesis. In this study, we investigated a newly duplicated N-mt gene-i.e., ()-in order to understand its function and, consequently, the reason behind its retention in the genome. The gene is a duplicate of the () gene of OXPHOS complex IV. While the parental gene has been found in all eukaryotes, including single-cell eukaryotes such as yeast, we show that is only present in the Brachycera suborder of Diptera; thus, both genes are present in all species, but have significantly different patterns of expression: is highly expressed in all tissues, while has a testis-specific expression. To understand the function of this new gene, we first knocked down its expression in the germline using two different RNAi lines driven by the driver; second, we created a knockout strain for this gene using CRISPR-Cas9 technology. Our results showed that knockdown and knockout lines of produce partial sterility and complete sterility in males, respectively, where a lack of sperm individualization was observed in both cases. Male infertility was prevented by driving -HA in the germline, but not when driving -HA. In addition, ectopic expression of in the soma caused embryonic lethality, while overexpression in the germline led to a reduction in male fertility. -KO mitochondria show reduced membrane potential, providing a plausible explanation for the male sterility observed in these flies. This prominent loss-of-function phenotype, along with its testis-biased expression and its presence in the sperm proteome, suggests that is a paralogous, specialized gene that is assembled in OXPHOS complex IV of male germline cells and/or sperm mitochondria.