Efficient Monitoring of Adult and Immature Mosquitoes Through Metabarcoding of Bulk Samples: A Case Study for Non-Model Culicids With Unique Ecologies

Abstract

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.

Taxocenose e morfologia de mosquitos (Diptera: Culicidae) com ênfase na tribo Mansoniini em áreas de aproveitamento hidrelétrico na Amazônia, Estado de Rondônia - Brasil

“…As Mansonia spp. são vorazes durante a hematofagia e são capazes de causar problemas médicos e sociais quando em alta densidade (REHENA & MATDOAN, 2020;PEDRO et al, 2021). Essas espécies têm hábitos noturnos, mas podem ser encontradas abundantemente durante o dia, picando a qualquer hora, nas matas próximas aos seus habitats larvais (LIMA, 1929;.…”

Nondestructive Methods of Pathogen Detection: Importance of Mosquito Integrity in Studies of Disease Transmission and Control

“…Molecular tools and analyses are especially important to distinguish morphologically similar species. The banding pattern of chromosomes, isoenzyme profiles, DNA probes, the analysis of DNA restriction fragments, cytogenetic physical methods, the analysis of nucleotide sequences of target genes, and barcoding and metabarcoding have been employed as routine molecular methods [ 6 , 15 , 17 ].…”

“…For adult mosquitoes, the D2 rDNA approach was comparable to morphological diagnosis. However, several immature forms of specimens of the genus Mansonia could not be classified into species [ 15 ]. Researchers in Canada, using DNA metabarcoding based on the COI gene, were able to identify and also estimate the number of captured mosquitoes.…”

“…Some alternative surveillance programs for mosquitoes have been proposed, including citizen science [ 11 , 12 , 13 ] and the metabarcoding of bulk samples [ 14 , 15 , 16 ], for assessing and monitoring vector density at a given location, time, or season. Mosquito metabarcoding faces challenges regarding the cost per sample, as well as an absence of sequences for known taxa, or sequences recorded for unknown taxa, in addition to the standardization of datasets [ 17 , 18 ].…”

Metabarcoding mosquitoes: MinION sequencing of bulk samples gives accurate species profiles for vector surveillance (Culicidae)

“…Mosquito identification is also limited by the availability and/or quality of published dichotomous taxonomic keys and/or local species checklists (15,17,19). Furthermore, in recent years, insect taxonomic expertise is becoming scarce and requires substantial investment in training manpower (21)(22)(23)(24). These issues are major hurdles for frequent and routine specieslevel identification, especially in many tropical countries that suffer from high incidences of mosquito-borne disease transmission and where mosquitoes are also the most biodiverse but understudied (25).…”