COX4-like, a Nuclear-Encoded Mitochondrial Gene Duplicate, Is Essential for Male Fertility in Drosophila melanogaster

Genes (Basel). 2022 Feb 25;13(3):424. doi: 10.3390/genes13030424.


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.

Keywords: COX4L; CRISPR knockout; Drosophila melanogaster; gene duplication; nuclear-encoded mitochondrial gene; spermatogenesis.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Drosophila / genetics
  • Drosophila Proteins* / genetics
  • Drosophila Proteins* / metabolism
  • Drosophila melanogaster / genetics
  • Drosophila melanogaster / metabolism
  • Electron Transport Complex IV / genetics
  • Fertility / genetics
  • Genes, Mitochondrial
  • Humans
  • Infertility, Male* / genetics
  • Male


  • Drosophila Proteins
  • Electron Transport Complex IV