Bulk identification of Anophelinae larvae from fishponds using DNA metabarcoding

Aquaculture can contribute to the increased abundance of malaria vectors in endemic settings across the Amazon Basin. Accurate identification of anopheline larvae breeding in fish-farming ponds is essential for the effectiveness of mosquito control interventions through entomological surveillance. A protocol was developed for bulk molecular identification, at the species level, of anopheline larvae present in fishponds before, during, and after an intervention with biolarvicides. DNA was extracted from total 14,994 third (L3) and fourth (L4) larval instar stages, grouped into 161 pools. The D2 region of the 28S RNA gene was amplified in each larvae pool. Taxonomic assignment was performed for 1,357 Amplicon Sequence Variants (ASVs). The following species were assigned to 1,315 ASVs: Nyssorhynchus albitarsis H / Nyssorhynchus marajoara (7.60%), Nyssorhynchus braziliensis (0.53%), Nyssorhynchus goeldii / Nyssorhynchus dunhami (0.53%), Anopheles costai G1 (0.08%), Anopheles near malefactor (0.46%), Nyssorhynchus tadei (0.46%), Anopheles peryassui (34.07%), Nyssorhynchus rangeli (11.79%), Nyssorhynchus darlingi (15.36%), and Nyssorhynchus triannulatus (29.12%). Identification to species level was not possible for 42 ASVs. These ASVs were designated as Nyssorhynchus sp. and Anopheles sp. Interspecific genetic distances of the D2 region were calculated using the Kimura 2-parameter distance and ranged from 0% to 24.93%. All mosquito species mentioned above were found in fishponds prior to the application of biolarvicide. During treatment, Nyssorhynchus sp., Ny. goeldii / Ny. dunhami, Ny. braziliensis, An. costai G1, and An. near malefactor were not collected. In the post-treatment period, only Ny. albitarsis H / Ny. marajoara, Ny. triannulatus, and Ny. darlingi were found. Here a protocol for bulk molecular identification of anopheline larvae is described and it can be promising to entomological surveillance during larval control.