Entomological characterization of malaria in northern Colombia through vector and parasite species identification, and analyses of spatial distribution and infection rates

Background: Malaria remains a worldwide public health concern and, in Colombia, despite the efforts to stop malaria transmission, the incidence of cases has increased over the last few years. In this context, it is necessary to evaluate vector diversity, infection rates, and spatial distribution, to better understand disease transmission dynamics. This information may contribute to the planning and development of vector control strategies. Results: A total of 778 Anopheles mosquitoes were collected in fifteen localities of Córdoba from August 2015 to October 2016. Six species were identified and overall, Anopheles albimanus was the most widespread and abundant species (83%). Other species of the Nyssorhynchus subgenus were collected, including Anopheles triannulatus (13%), Anopheles nuneztovari (1%), Anopheles argyritarsis (< 1%) and two species belonging to the Anopheles subgenus: Anopheles pseudopunctipennis (3%) and Anopheles neomaculipalpus (< 1%). Four species were found naturally infected with two Plasmodium species: Anopheles nuneztovari was detected naturally infected with Plasmodium falciparum and Anopheles pseudopunctipennis with Plasmodium vivax, whereas An. albimanus and An. triannulatus were found infected with both parasite species and confirmed by nested PCR. Conclusions: In general, the obtained results were contrasting with previous studies in terms of the most abundant and widespread collected species, and regarding infection rates, which were higher than those previously reported. A positive relationship between mosquito local abundance at the locality level and human infection at the municipality level was found. Mosquito local abundance and the number of houses with mosquitoes in each village are factors explaining malaria human cases in these villages. The obtained results suggest that other factors related to the apparent variation in malaria eco-epidemiology in northern Colombia, must be identified, to provide health authorities with better decision tools aiming to design control and prevention strategies

A new species in the major malaria vector complex sheds light on reticulated species evolution

Complexes of closely related species provide key insights into the rapid and independent evolution of adaptive traits. Here, we described and studied Anopheles fontenillei sp.n., a new species in the Anopheles gambiae complex that we recently discovered in the forested areas of Gabon, Central Africa. Our analysis placed the new taxon in the phylogenetic tree of the An. gambiae complex, revealing important introgression events with other members of the complex. Particularly, we detected recent introgression, with Anopheles gambiae and Anopheles coluzzii, of genes directly involved in vectorial capacity. Moreover, genome analysis of the new species allowed us to clarify the evolutionary history of the 3La inversion. Overall, An. fontenillei sp.n. analysis improved our understanding of the relationship between species within the An. gambiae complex, and provided insight into the evolution of vectorial capacity traits that are relevant for the successful control of malaria in Africa

Highly Efficient Site-Specific Mutagenesis in Malaria Mosquitoes Using CRISPR.

Anopheles mosquitoes transmit at least 200 million annual malaria infections worldwide. Despite considerable genomic resources, mechanistic understanding of biological processes in Anopheles has been hampered by a lack of tools for reverse genetics. Here, we report successful application of the CRISPR/Cas9 system for highly efficient, site-specific mutagenesis in the diverse malaria vectors Anopheles albimanus, A. coluzzii, and A. funestus When guide RNAs (gRNAs) and Cas9 protein are injected at high concentration, germline mutations are common and usually biallelic, allowing for the rapid creation of stable mutant lines for reverse genetic analysis. Our protocol should enable researchers to dissect the molecular and cellular basis of anopheline traits critical to successful disease transmission, potentially exposing new targets for malaria control

Urban agriculture and Anopheles habitats in Dar es Salaam, Tanzania.

A cross-sectional survey of agricultural areas, combined with routinely monitored mosquito larval information, was conducted in urban Dar es Salaam, Tanzania, to investigate how agricultural and geographical features may influence the presence of Anopheles larvae. Data were integrated into a geographical information systems framework, and predictors of the presence of Anopheles larvae in farming areas were assessed using multivariate logistic regression with independent random effects. It was found that more than 5% of the study area (total size 16.8 km2) was used for farming in backyard gardens and larger open spaces. The proportion of habitats containing Anopheles larvae was 1.7 times higher in agricultural areas compared to other areas (95% confidence interval = 1.56-1.92). Significant geographic predictors of the presence of Anopheles larvae in gardens included location in lowland areas, proximity to river, and relatively impermeable soils. Agriculture-related predictors comprised specific seedbed types, mid-sized gardens, irrigation by wells, as well as cultivation of sugar cane or leafy vegetables. Negative predictors included small garden size, irrigation by tap water, rainfed production and cultivation of leguminous crops or fruit trees. Although there was an increased chance of finding Anopheles larvae in agricultural sites, it was found that breeding sites originated by urban agriculture account for less than a fifth of all breeding sites of malaria vectors in Dar es Salaam. It is suggested that strategies comprising an integrated malaria control effort in malaria-endemic African cities include participatory involvement of farmers by planting shade trees near larval habitats

Sustainable control of Anopheles mosquito population

Despite the widespread use of insecticides, community engagement programmes and preventive measures mosquito borne diseases are growing and new tools to prevent the spread of disease are urgently needed. An alternative control measure for the eradication of Anopheles mosquitoes is suggested by the use of a Sustainable Control Model, which demonstrates the capability of Odonata, a natural beneficial predator, to exercise control over Anopheles mosquitoes in less than 140 days

Interventions that effectively target Anopheles funestus mosquitoes could significantly improve control of persistent malaria transmission in south–eastern Tanzania

Malaria is transmitted by many Anopheles species whose proportionate contributions vary across settings. We re-assessed the roles of Anopheles arabiensis and Anopheles funestus, and examined potential benefits of species-specific interventions in an area in south-eastern Tanzania, where malaria transmission persists, four years after mass distribution of long-lasting insecticide-treated nets (LLINs). Monthly mosquito sampling was done in randomly selected households in three villages using CDC light traps and back-pack aspirators, between January-2015 and January-2016, four years after the last mass distribution of LLINs in 2011. Multiplex polymerase chain reaction (PCR) was used to identify members of An. funestus and Anopheles gambiae complexes. Enzyme-linked immunosorbent assay (ELISA) was used to detect Plasmodium sporozoites in mosquito salivary glands, and to identify sources of mosquito blood meals. WHO susceptibility assays were done on wild caught female An. funestus s.l, and physiological ages approximated by examining mosquito ovaries for parity. A total of 20,135 An. arabiensis and 4,759 An. funestus were collected. The An. funestus group consisted of 76.6% An. funestus s.s, 2.9% An. rivulorum, 7.1% An. leesoni, and 13.4% unamplified samples. Of all mosquitoes positive for Plasmodium, 82.6% were An. funestus s.s, 14.0% were An. arabiensis and 3.4% were An. rivulorum. An. funestus and An. arabiensis contributed 86.21% and 13.79% respectively, of annual entomological inoculation rate (EIR). An. arabiensis fed on humans (73.4%), cattle (22.0%), dogs (3.1%) and chicken (1.5%), but An. funestus fed exclusively on humans. The An. funestus populations were 100% susceptible to organophosphates, pirimiphos methyl and malathion, but resistant to permethrin (10.5% mortality), deltamethrin (18.7%), lambda-cyhalothrin (18.7%) and DDT (26.2%), and had reduced susceptibility to bendiocarb (95%) and propoxur (90.1%). Parity rate was higher in An. funestus (65.8%) than An. arabiensis (44.1%). Though An. arabiensis is still the most abundant vector species here, the remaining malaria transmission is predominantly mediated by An. funestus, possibly due to high insecticide resistance and high survival probabilities. Interventions that effectively target An. funestus mosquitoes could therefore significantly improve control of persistent malaria transmission in south–eastern Tanzania

The phylogeny of Anophelinae revisited: inferences about the origin and classification of Anopheles (Diptera: Culicidae)

© 2015 Royal Swedish Academy of Sciences. "This is the pre-peer reviewed version of the following article: Harbach, R. E. and I. J. Kitching (2016). "The phylogeny of Anophelinae revisited: inferences about the origin and classification of Anopheles (Diptera: Culicidae)." Zoologica Scripta 45(1): 34-47, which has been published in final form at http://onlinelibrary.wiley.com/doi/10.1111/zsc.12137/full. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.

Complex genome evolution in Anopheles coluzzii associated with increased insecticide usage in Mali.

In certain cases, a species may have access to important genetic variation present in a related species via adaptive introgression. These novel alleles may interact with their new genetic background, resulting in unexpected phenotypes. In this study, we describe a selective sweep on standing variation on the X chromosome in the mosquito Anopheles coluzzii, a principal malaria vector in West Africa. This event may have been influenced by the recent adaptive introgression of the insecticide resistance gene known as kdr from the sister species Anopheles gambiae. Individuals carrying both kdr and a nearly fixed X-linked haplotype, encompassing at least four genes including the P450 gene CYP9K1 and the cuticular protein CPR125, have rapidly increased in relative frequency. In parallel, a reproductively isolated insecticide-susceptible A. gambiae population (Bamako form) has been driven to local extinction, likely due to strong selection from increased insecticide-treated bed net usage

Diversity, differentiation, and linkage disequilibrium: prospects for association mapping in the malaria vector anopheles arabiensis

Association mapping is a widely applied method for elucidating the genetic basis of phenotypic traits. However, factors such as linkage disequilibrium and levels of genetic diversity influence the power and resolution of this approach. Moreover, the presence of population subdivision among samples can result in spurious associations if not accounted for. As such, it is useful to have a detailed understanding of these factors before conducting association mapping experiments. Here we conducted whole-genome sequencing on 24 specimens of the malaria mosquito vector, Anopheles arabiensis, to further understanding of patterns of genetic diversity, population subdivision and linkage disequilibrium in this species. We found high levels of genetic diversity within the An. arabiensis genome, with ~800,000 high-confidence, single- nucleotide polymorphisms detected. However, levels of nucleotide diversity varied significantly both within and between chromosomes. We observed lower diversity on the X chromosome, within some inversions, and near centromeres. Population structure was absent at the local scale (Kilombero Valley, Tanzania) but detected between distant populations (Cameroon vs. Tanzania) where differentiation was largely restricted to certain autosomal chromosomal inversions such as 2Rb. Overall, linkage disequilibrium within An. arabiensis decayed very rapidly (within 200 bp) across all chromosomes. However, elevated linkage disequilibrium was observed within some inversions, suggesting that recombination is reduced in those regions. The overall low levels of linkage disequilibrium suggests that association studies in this taxon will be very challenging for all but variants of large effect, and will require large sample sizes