Bulk identification of Anophelinae larvae from fishponds using DNA metabarcoding

Area of Science:

• Medical Entomology
• Molecular Biology
• Public Health

Background:

• Aquaculture practices in the Amazon Basin can increase the abundance of malaria vectors.
• Effective mosquito control relies on accurate identification of anopheline larvae breeding sites, such as fish-farming ponds.
• Entomological surveillance is crucial for targeted interventions against malaria vectors.

Purpose of the Study:

• To develop and validate a protocol for the bulk molecular identification of anopheline larvae species in fishponds.
• To assess the effectiveness of biolarvicide interventions on anopheline larval populations.
• To provide a tool for improved entomological surveillance in aquaculture-associated malaria vector habitats.

Main Methods:

• DNA extraction from 14,994 third (L3) and fourth (L4) instar anopheline larvae, pooled into 161 samples.
• Amplification of the D2 region of the 28S RNA gene for each larval pool.
• Taxonomic assignment of Amplicon Sequence Variants (ASVs) using molecular identification.

Main Results:

• The protocol successfully identified multiple Anopheles and Nyssorhynchus species, including Nyssorhynchus albitarsis H / Nyssorhynchus marajoara, Nyssorhynchus darlingi, and Nyssorhynchus triannulatus.
• All identified mosquito species were present before biolarvicide application.
• Post-intervention, only Nyssorhynchus albitarsis H / Nyssorhynchus marajoara, Nyssorhynchus triannulatus, and Nyssorhynchus darlingi were detected, indicating a reduction in species diversity.

Conclusions:

• A bulk molecular identification protocol using the 28S RNA gene is effective for species-level identification of anopheline larvae in fishponds.
• This method is a promising tool for entomological surveillance during larval control interventions.
• Understanding larval populations in aquaculture settings is key to managing malaria vector abundance.