A practical limitation to many metabarcoding initiatives is that sampling methods tend to collect many non-target taxa, which become “amplicon noise” that can saturate Next Generation Sequencing results and lead to both financial and resource inefficiencies. An available molecular tool that can significantly decrease these non-target amplicons and decrease the need for pre-DNA-extraction sorting of bycatch is the design of PCR primers tailored to the taxa under investigation. We assessed whether the D2 extension segment of the 28S ribosomal operon can limit this shortcoming within the context of mosquito (Culicidae) monitoring. We designed PCR primers that are fully conserved across mosquitos and exclude from amplification most other taxa likely to be collected with current sampling apparatuses. We show that, given enough sequencing depth, D2 is an effective marker for the detection of mosquito sequences within mock genomic DNA pools. As few as 3,050 quality-filtered Illumina reads were able to recover all 17 species in a bulk pool containing as little as 0.2% of constituent DNA from single taxa. We also mixed these mosquito DNA pools with high concentrations of non-Culicidae bycatch DNA and show that the component mosquito species are generally still recoverable and faithful to their original relative frequencies. Finally, we show that there is little loss of fidelity in abundance parameters when pools from degraded DNA samples were sequenced using the D2 primers.

The rapid and economical monitoring of mosquitos is imperative to understanding the dynamics of both disease vectors and nuisance species. In light of technological advances in mosquito sampling and DNA sequencing, health agencies can now utilize the full potential of metabarcoding pipelines for rapid and standardizable surveillance. Here, we describe mosquito spatial and temporal variation, with particular focus on Mansonia Blanchard species, in the Madeira (Rondônia State) and the Ribeira (São Paulo) watersheds, Brazil using metabarcoding of the D2 rDNA marker. Sampling and molecular pipelines were used to evaluate the taxonomic contribution of mosquitos in pools of culicids collected en masse from macrophyte-roots (immatures) and from Mosquito Magnet traps and protected human landings (adults). Results for adult captures are comparable to morphological diagnoses and clarify previously unknown temporal and spatial species turnover. Metabarcoding of immature stages also confirmed the extent of the geographical distribution of some species and each taxon’s association with macrophyte species. Given the benefits of metabarcoding, such as taxonomic acuity, high throughput processing, and objectivity, we suggest such techniques should be more fully incorporated into culicid monitoring schemes. The metabarcoding protocol described herein paired with standardized field sampling schemes, when used by mosquito monitoring professionals, offers substantial improvements in terms of practicality, speed and cost.

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High abundance of hematophagous mosquitoes of the genus Mansonia Blanchard, 1901 (Diptera: Culicidae) threatens human and domestic animal health and well-being. Knowledge of the biology of nuisance mosquito species is necessary to understand specific ecological and biological factors to enable rapid and effective monitoring measures for sustainable control programs. The establishment and dispersion of Mansonia species are associated with the occurrence of aquatic macrophytes species, which are indispensable for the development of larvae and pupae. To increase knowledge of the host plants for Mansonia immature stages in Porto Velho, Rondonia State, Brazil, specimens of four plant species, which occur across the tributaries of the Madeira River were sampled and inspected for the presence of egg batches, larvae, and pupae. A total of 1,386 larvae and pupae of Mansonia spp. were collected attached to the roots of Eichhornia crassipes (Mart.) Solms (Commelinales: Pontederiaceae), Pistia stratiotes L. (Alismatales: Araceae), and Limnobium laevigatum (Humb. and Bonpl. Ex Willd.) Heine (Alismatales: Hydrocharitaceae). The novel association of Mansonia species with L. laevigatum is presented. Egg batches of Mansonia spp. were found only on Salvinia molesta D.S. Mitch. (Salviniales: Salviniaceae). Possible differences in the roles played by E. crassipes and S. molesta in the reproductive cycle of Mansonia spp. in the surveyed area are discussed. All species of host plants including E. crassipes, P. stratiotes, S. molesta, and L. laevigatum should be considered when planning macrophyte management for the control of Mansonia species.