Most of the genes encoding proteins that function in the mitochondria are located in the nucleus and are called nuclear-encoded mitochondrial genes, or N-mt genes. In Drosophila melanogaster, about 23% of N-mt genes fall into gene families, and all duplicates with tissue-biased expression (76%) are testis biased. These genes are enriched for energy-related functions and tend to be older than other duplicated genes in the genome. These patterns reveal strong selection for the retention of new genes for male germline mitochondrial functions. The two main forces that are likely to drive changes in mitochondrial functions are maternal inheritance of mitochondria and male–male competition for fertilization. Both are common among animals, suggesting similar N-mt gene duplication patterns in different species. To test this, we analyzed N-mt genes in the human genome. We find that about 18% of human N-mt genes fall into gene families, but unlike in Drosophila, only 28% of the N-mt duplicates have tissue-biased expression and only 36% of these have testis-biased expression. In addition, human testis-biased duplicated genes are younger than other duplicated genes in the genome and have diverse functions. These contrasting patterns between species might reflect either differences in selective pressures for germline energy-related or other mitochondrial functions during spermatogenesis and fertilization, or differences in the response to similar pressures.

Recent studies on nuclear-encoded mitochondrial genes (N-mt genes) in Drosophila melanogaster 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., Cytochrome c oxidase 4-like (COX4L)—in order to understand its function and, consequently, the reason behind its retention in the D. melanogaster genome. The COX4L gene is a duplicate of the Cytochrome c oxidase 4 (COX4) gene of OXPHOS complex IV. While the parental COX4 gene has been found in all eukaryotes, including single-cell eukaryotes such as yeast, we show that COX4L is only present in the Brachycera suborder of Diptera; thus, both genes are present in all Drosophila species, but have significantly different patterns of expression: COX4 is highly expressed in all tissues, while COX4L has a testis-specific expression. To understand the function of this new gene, we first knocked down its expression in the D. melanogaster germline using two different RNAi lines driven by the bam-Gal4 driver; second, we created a knockout strain for this gene using CRISPR-Cas9 technology. Our results showed that knockdown and knockout lines of COX4L 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 COX4L-HA in the germline, but not when driving COX4-HA. In addition, ectopic expression of COX4L in the soma caused embryonic lethality, while overexpression in the germline led to a reduction in male fertility. COX4L-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 Drosophila sperm proteome, suggests that COX4L is a paralogous, specialized gene that is assembled in OXPHOS complex IV of male germline cells and/or sperm mitochondria.

Background The nuclear transport machinery is involved in a well-known male meiotic drive system in Drosophila. Fast gene evolution and gene duplications have been major underlying mechanisms in the evolution of meiotic drive systems, and this might include some nuclear transport genes in Drosophila. So, using a comprehensive, detailed phylogenomic study, we examined 51 insect genomes for the duplication of the same nuclear transport genes. Results We find that most of the nuclear transport duplications in Drosophila are of a few classes of nuclear transport genes, RNA mediated and fast evolving. We also retrieve many pseudogenes for the Ran gene. Some of the duplicates are relatively young and likely contributing to the turnover expected for genes under strong but changing selective pressures. These duplications are potentially revealing what features of nuclear transport are under selection. Unlike in flies, we find only a few duplications when we study the Drosophila duplicated nuclear transport genes in dipteran species outside of Drosophila, and none in other insects. Conclusions These findings strengthen the hypothesis that nuclear transport gene duplicates in Drosophila evolve either as drivers or suppressors of meiotic drive systems or as other male-specific adaptations circumscribed to flies and involving a handful of nuclear transport functions.