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.

Artemisinin-resistant Plasmodium falciparum parasites are rapidly spreading in Southeast Asia, yet nothing is known about their transmission. This knowledge gap, and the possibility that these parasites will spread to Africa, endangers global efforts to eliminate malaria. Here we produce gametocytes from parasite clinical isolates that displayed artemisinin resistance in patients and in vitro, and use them to infect native and non-native mosquito vectors. We show that contemporary artemisinin-resistant isolates from Cambodia develop and produce sporozoites in two Southeast Asian vectors, Anopheles dirus and An. minimus, and the major African vector, An. coluzzii (formerly An. gambiae M). The ability of artemisinin-resistant parasites to infect such highly diverse Anopheles species, combined with their higher gametocyte prevalence in patients, may explain the rapid expansion of these parasites in Cambodia and neighboring countries, and further compromise efforts to prevent their global spread.

The development of a sterilizing vaccine against malaria remains one of the highest priorities for global health research. While sporozoite vaccines targeting the pre-erythrocytic stage show great promise, it has not been possible to maintain efficacy long-term, likely due to an inability of these vaccines to maintain effector memory T cell responses in the liver. Vaccines based on human cytomegalovirus (HCMV) might overcome this limitation since vectors based on rhesus CMV (RhCMV), the homologous virus in rhesus macaques (RM), elicit and indefinitely maintain high frequency, non-exhausted effector memory T cells in extralymphoid tissues, including the liver. Moreover, RhCMV strain 68–1 elicits CD8+ T cells broadly recognizing unconventional epitopes exclusively restricted by MHC-II and MHC-E. To evaluate the potential of these unique immune responses to protect against malaria, we expressed four Plasmodium knowlesi (Pk) antigens (CSP, AMA1, SSP2/TRAP, MSP1c) in RhCMV 68–1 or in Rh189-deleted 68–1, which additionally elicits canonical MHC-Ia-restricted CD8+ T cells. Upon inoculation of RM with either of these Pk Ag expressing RhCMV vaccines, we obtained T cell responses to each of the four Pk antigens. Upon challenge with Pk sporozoites we observed a delayed appearance of blood stage parasites in vaccinated RM consistent with a 75–80% reduction of parasite release from the liver. Moreover, the Rh189-deleted RhCMV/Pk vectors elicited sterile protection in one RM. Once in the blood, parasite growth was not affected. In contrast to T cell responses induced by Pk infection, RhCMV vectors maintained sustained T cell responses to all four malaria antigens in the liver post-challenge. The delayed appearance of blood stage parasites is thus likely due to a T cell-mediated inhibition of liver stage parasite development. As such, this vaccine approach can be used to efficiently test new T cell antigens, improve current vaccines targeting the liver stage and complement vaccines targeting erythrocytic antigens.

Anopheles is a diverse genus of mosquitoes comprising over 500 described species, including all known human malaria vectors. While a limited number of key vector species have been studied in detail, the goal of malaria elimination calls for surveillance of all potential vector species. Here, we develop a multilocus amplicon sequencing approach that targets 62 highly variable loci in the Anopheles genome and two conserved loci in the Plasmodium mitochondrion, simultaneously revealing both the mosquito species and whether that mosquito carries malaria parasites. We also develop a cheap, nondestructive, and high‐throughput DNA extraction workflow that provides template DNA from single mosquitoes for the multiplex PCR, which means specimens producing unexpected results can be returned to for morphological examination. Over 1000 individual mosquitoes can be sequenced in a single MiSeq run, and we demonstrate the panel’s power to assign species identity using sequencing data for 40 species from Africa, Southeast Asia, and South America. We also show that the approach can be used to resolve geographic population structure within An. gambiae and An. coluzzii populations, as the population structure determined based on these 62 loci from over 1000 mosquitoes closely mirrors that revealed through whole genome sequencing. The end‐to‐end approach is quick, inexpensive, robust, and accurate, which makes it a promising technique for very large‐scale mosquito genetic surveillance and vector control.

BackgroundIndonesia is home to a variety of malaria vectors whose specific bionomic traits remain largely uncharacterized. Species-specific behaviours, such as host feeding preferences, impact the dynamics of malaria transmission and the effectiveness of vector control interventions.MethodsTo examine species-specific host attraction and feeding behaviours, a Latin square design was used to compare Anopheles mosquitoes attracted to human, cow, and goat-baited tents. Anopheles mosquitoes were collected hourly from the inside walls of each baited tent. Species were morphologically and then molecularly identified using rDNA ITS2 sequences. The head and thorax of individual specimens were analysed for Plasmodium DNA using PCR. Bloodmeals were identified using a multiplex PCR.ResultsA total of 1024, 137, and 74 Anopheles were collected over 12 nights in cow, goat, and human-baited tents, respectively. The species were identified as Anopheles kochi, Anopheles farauti s.s., Anopheles hackeri, Anopheles hinesorum, Anopheles indefinitus, Anopheles punctulatus, Anopheles tessellatus, Anopheles vagus, and Anopheles vanus, many of which are known to transmit human malaria. Molecular analysis of blood meals revealed a high level of feeding on multiple host species in a single night. Anopheles kochi, An. indefinitus, and An. vanus were infected with Plasmodium vivax at rates comparable to primary malaria vectors.ConclusionsThe species distributions of Anopheles mosquitoes attracted to human, goat, and cow hosts were similar. Eight of nine sporozoite positive samples were captured with animal-baited traps, indicating that even predominantly zoophilic mosquitoes may be contributing to malaria transmission. Multiple host feeding and flexibility in blood feeding behaviour have important implications for malaria transmission, malaria control, and the effectiveness of intervention and monitoring methods, particularly those that target human-feeding vectors.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-017-1950-5) contains supplementary material, which is available to authorized users.