Epigenetic regulation of gene expression by covalent modification of histones is 
important for germ line cell development. In mammals, histone H3 lysine 9 
(H3K9)-specific histone methyltransferases (HMTases), such as G9a, SETDB1, and 
SUV39H, play critical roles, but the contribution of H3K9-specific HMTases in 
Drosophila remains to be clarified, especially in male sperm. Here, we performed 
immunocytochemical analyses with a specific antibody to dG9a, Drosophila G9a 
ortholog, and demonstrated localization in the cytoplasm from the growth to 
elongation stages of spermatogenesis. In the subsequent early canoe stage, 
strong dG9a signals were detected exclusively in nuclei, suggesting a regulatory 
role. However, mono-, di-, and trimethylated H3K9 signals were not extensively 
decreased in a homozygous dG9a null mutant throughout these stages. In contrast, 
mono- and trimethylated H3K9 signals were extensively decreased in a 
heterozygous DmSetdb1 mutant during spermatogenesis, and similar reduction in 
monomethylated H3K9 signals was observed in a homozygous Su(var)3-9 mutant. 
Therefore, DmSETDB1 is likely to be mainly responsible for mono- and 
trimethylation of H3K9 and SU(VAR)3-9 for monomethylation of H3K9 during 
spermatogenesis. However, the reduced methylation of H3K9 in premeiotic 
spermatocytes did not influence X-Y chromosome disjunction in male meiosis, 
suggesting that it may not be critical for spermatogenesis in Drosophila.