Here, the main goal is to assess natural infections of Plasmodium spp. in anophelines in a forest reserve from the same region where we previously found a surprisingly high rate (5.2%) of plasmodia infections (n = 25) in Kerteszia mosquitoes (N = 480) on the slopes of Serra do Mar, Atlantic Forest, Brazil. The mosquito collection sampling was carried out at the Legado das Águas Forest Reserve using CDC light traps and Shannon traps at night (5–10 pm) in 3-day collections in November 2021 and March, April, May, and November 2022. The captured specimens were morphologically identified at the species level and had their genomic DNA extracted in pools of up to 10 mosquitoes/pool. Each pool was tested using 18S qPCR and cytb nested PCR plus sequencing. A total of 5301 mosquitoes, mostly belonging to the genus Kerteszia (99.7%), were sampled and sorted into 773 pools. Eight pools positive for Plasmodium spp. were identified: four for Plasmodium spp., one for P. vivax or P. simium, one for P. malariae or P. brasilianum, and two for the P. falciparum-like parasite. After Sanger sequencing, two results were further confirmed: P. vivax or P. simium and P. malariae or P. brasilianum. The minimum infection rate for Kerteszia mosquitoes was 0.15% (eight positive pools/5285 Kerteszia mosquitoes). The study reveals a lower-than-expected natural infection rate (expected = 5.2% vs. observed = 0.15%). This low rate relates to the absence of Alouatta monkeys as the main simian malaria reservoir in the studied region. Their absence was due to a significant population decline following the reemergence of yellow fever virus outbreaks in the Atlantic Forest from 2016 to 2019. However, this also indicates the existence of alternative reservoirs to infect Kerteszia mosquitoes. The found zoonotic species of Plasmodium, including the P. falciparum-like parasite, may represent a simian malaria risk and thus a challenge for malaria elimination in Brazil.
