Species-specific detection windows for human STR profiling from blood-fed mosquitoes and sand flies

Hematophagous insects found at crime scenes can be forensically useful, as human DNA from their blood meals may help identify suspects or victims. This study evaluates the use of two commercial STR kits, IG_17Plex (17-loci) and IG_GrandPlex (28-loci), to obtain human short tandem repeat (STR) profiles from blood-fed Culex pipiens , Anopheles stephensi , and Phlebotomus papatasi . Both kits were tested for amplification reliability and compared for performance. The effect of post-feeding time was assessed by euthanizing insects at 12-h intervals up to 72 h. We also tested mixed blood meals in both An. stephensi and Cx. pipiens , finding comparable results between the two species. Additionally, we compared four preservation methods, ethanol immersion (with or without cuticle) and freezing (with or without cuticle), on DNA integrity in blood-fed An. stephensi . STR kits consistently amplified complete profiles from blood-fed mosquitoes, but sand flies yielded only partial profiles. Complete DNA profiles were obtainable up to 60 h post-blood meal (PBM) from Cx. pipiens and up to 48 h PBM from An. stephensi , and this temporal window remained consistent regardless of whether the blood meal was single or mixed. All preservation methods performed similarly, though freezing without dissection was preferable. Overall, blood from Cx. pipiens and An. stephensi mosquitoes serves as a reliable forensic resource for human identification up to 48–60 h PBM, including in cases of mixed-feeding scenarios.