We investigated gene expression patterns in
Lutzomyia
and
Phlebotomus
sand fly vectors of leishmaniases. Using quantitative PCR, we assessed the expression stability of potential endogenous control genes commonly used in dipterans. We analyzed
Lutzomyia longipalpis
and
Phlebotomus papatasi
samples from L3 and L4 larval stages, adult sand flies of different sexes, diets, dsRNA injection, and
Leishmania
infection. Six genes were evaluated: actin, α-tubulin, GAPDH, 60 S ribosomal proteins L8 and L32 (RiboL8 and RiboL32), and elongation factor 1-α (EF1-α). EF1-α was among the most stably expressed along with RiboL8 in
L. longipalpis
larvae and RiboL32 in adults. In
P. papatasi
, EF1-α and RiboL32 were the top in larvae, while EF1-α and actin were the most stable in adults. RiboL8 and actin were the most stable genes in dissected tissues and infected guts. Additionally, five primer pairs designed for
L. longipalpis
or
P. papatasi
were effective in PCR with
Lutzomyia migonei
,
Phlebotomus duboscqi
,
Phlebotomus perniciosus
, and
Sergentomyia schwetzi
cDNA. Furthermore,
L. longipalpis
RiboL32 and
P. papatasi
α-tubulin primers were suitable for qPCR with cDNA from the other four species. Our research provides tools to enhance relative gene expression studies in sand flies, facilitating the selection of endogenous control for qPCR.
Supplementary Information
The online version contains supplementary material available at 10.1038/s41598-024-74776-9.