The understanding of malaria vector species in association with their bionomic traits is vital for targeting malaria interventions and measuring effectiveness. Many entomological studies rely on morphological identification of mosquitoes, limiting recognition to visually distinct species/species groups. Anopheles species assignments based on ribosomal DNA ITS2 and mitochondrial DNA COI were compared to morphological identifications from Luangwa and Nyimba districts in Zambia. The comparison of morphological and molecular identifications determined that interpretations of species compositions, insecticide resistance assays, host preference studies, trap efficacy, and Plasmodium infections were incorrect when using morphological identification alone. Morphological identifications recognized eight Anopheles species while 18 distinct sequence groups or species were identified from molecular analyses. Of these 18, seven could not be identified through comparison to published sequences. Twelve of 18 molecularly identified species (including unidentifiable species and species not thought to be vectors) were found by PCR to carry Plasmodium sporozoites - compared to four of eight morphological species. Up to 15% of morphologically identified Anopheles funestus mosquitoes in insecticide resistance tests were found to be other species molecularly. The comprehension of primary and secondary malaria vectors and bionomic characteristics that impact malaria transmission and intervention effectiveness are fundamental in achieving malaria elimination.

Abstract. The success of mosquito-based malaria control is dependent upon susceptible bionomic traits in local malaria vectors. It is crucial to have accurate and reliable methods to determine mosquito species composition in areas subject to malaria. An unexpectedly diverse set of Anopheles species was collected in the western Kenyan highlands, including unidentified and potentially new species carrying the malaria parasite Plasmodium falciparum. This study identified 2,340 anopheline specimens using both ribosomal DNA internal transcribed spacer region 2 and mitochondrial DNA cytochrome oxidase subunit 1 loci. Seventeen distinct sequence groups were identified. Of these, only eight could be molecularly identified through comparison to published and voucher sequences. Of the unidentified species, four were found to carry P. falciparum by circumsporozoite enzyme-linked immunosorbent assay and polymerase chain reaction, the most abundant of which had infection rates comparable to a primary vector in the area, Anopheles funestus. High-quality adult specimens of these unidentified species could not be matched to museum voucher specimens or conclusively identified using multiple keys, suggesting that they may have not been previously described. These unidentified vectors were captured outdoors. Diverse and unknown species have been incriminated in malaria transmission in the western Kenya highlands using molecular identification of unusual morphological variants of field specimens. This study demonstrates the value of using molecular methods to compliment vector identifications and highlights the need for accurate characterization of mosquito species and their associated behaviors for effective malaria control.

The main method of malaria control is based on a simple premise: avoid mosquito bites by killing the mosquitoes. This concept relies on spraying insecticides indoors and sleeping under insecticide-treated bed nets because it is assumed that malaria mosquitoes spend most of their time indoors and feed at night. That is, until now. A recent study has identified new species of mosquitoes that prefer to be outdoors and to feed earlier in the evening. These behavior patterns could render current control practices ineffective. New malaria control methods need to be developed according to the specific behavior of all the different vectors.

BackgroundThe accurate monitoring and evaluation of malaria vectors requires efficient sampling. The objective of this study was to compare methods for sampling outdoor-biting Anopheles mosquitoes in Cambodia.MethodsIn the Cambodian provinces of Pursat, Preah Vihear, and Ratanakiri, six different mosquito trapping methods were evaluated: human landing collection (HLC), human-baited tent (HBT), cow-baited tent (CBT), CDC miniature light trap (LT), CDC miniature light trap baited with molasses and yeast (LT-M), and barrier fence (F) in a Latin square design during four or six consecutive nights at the height of the malaria transmission season.ResultsUsing all traps, a total of 507, 1175, and 615 anophelines were collected in Pursat, Preah Vihear, and Ratanakiri, respectively. CBTs captured 10- to 20-fold more anophelines per night than the other five sampling methods. All 2297 Anopheles mosquitoes were morphologically identified and molecularly typed using standard morphological keys and sequencing the rDNA ITS2 region to distinguish cryptic species, respectively. Overall, an extremely diverse set of 27 known Anopheles species was sampled. CBTs captured the same molecular species that HLCs and the other four traps did, as well as additional species. Nine specimens representing five Anopheles species (Anopheles hyrcanus, Anopheles barbirostris sensu stricto, Anopheles barbirostris clade III, Anopheles nivipes, and Anopheles peditaeniatus) were infected with Plasmodium falciparum and were exclusively captured in CBTs.ConclusionsThese data indicate that cow-baited tents are highly effective in sampling diverse Anopheles malaria vectors in Cambodia. This sampling method captured high numbers of anophelines with limited sampling effort and greatly reduced human exposure to mosquito bites compared to the gold-standard human landing collection.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-016-1488-y) contains supplementary material, which is available to authorized users.