Improved Use of Drone Imagery for Malaria Vector Control through Technology-Assisted Digitizing (TAD)

Drones have the potential to revolutionize malaria vector control initiatives through rapid and accurate mapping of potential malarial mosquito larval habitats to help direct field Larval Source Management (LSM) efforts. However, there are no clear recommendations on how these habitats can be extracted from drone imagery in an operational context. This paper compares the results of two mapping approaches: supervised image classification using machine learning and Technology-Assisted Digitising (TAD) mapping that employs a new region growing tool suitable for non-experts. These approaches were applied concurrently to drone imagery acquired at seven sites in Zanzibar, United Republic of Tanzania. Whilst the two approaches were similar in processing time, the TAD approach significantly outperformed the supervised classification approach at all sites (t = 5.1, p < 0.01). Overall accuracy scores (mean overall accuracy 62%) suggest that a supervised classification approach is unsuitable for mapping potential malarial mosquito larval habitats in Zanzibar, whereas the TAD approach offers a simple and accurate (mean overall accuracy 96%) means of mapping these complex features. We recommend that this approach be used alongside targeted ground-based surveying (i.e., in areas inappropriate for drone surveying) for generating precise and accurate spatial intelligence to support operational LSM programmes.

Comprehensive Genomic Discovery of Non-Coding Transcriptional Enhancers in the African Malaria Vector Anopheles coluzzii

Almost all regulation of gene expression in eukaryotic genomes is mediated by the action of distant non-coding transcriptional enhancers upon proximal gene promoters. Enhancer locations cannot be accurately predicted bioinformatically because of the absence of a defined sequence code, and thus functional assays are required for their direct detection. Here we used a massively parallel reporter assay, Self-Transcribing Active Regulatory Region sequencing (STARR-seq), to generate the first comprehensive genome-wide map of enhancers in Anopheles coluzzii, a major African malaria vector in the Gambiae species complex. The screen was carried out by transfecting reporter libraries created from the genomic DNA of 60 wild A. coluzzii from Burkina Faso into A. coluzzii 4a3A cells, in order to functionally query enhancer activity of the natural population within the homologous cellular context. We report a catalog of 3,288 active genomic enhancers that were significant across three biological replicates, 74% of them located in intergenic and intronic regions. The STARR-seq enhancer screen is chromatin-free and thus detects inherent activity of a comprehensive catalog of enhancers that may be restricted in vivo to specific cell types or developmental stages. Testing of a validation panel of enhancer candidates using manual luciferase assays confirmed enhancer function in 26 of 28 (93%) of the candidates over a wide dynamic range of activity from two to at least 16-fold activity above baseline. The enhancers occupy only 0.7% of the genome, and display distinct composition features. The enhancer compartment is significantly enriched for 15 transcription factor binding site signatures, and displays divergence for specific dinucleotide repeats, as compared to matched non-enhancer genomic controls. The genome-wide catalog of A. coluzzii enhancers is publicly available in a simple searchable graphic format. This enhancer catalogue will be valuable in linking genetic and phenotypic variation, in identifying regulatory elements that could be employed in vector manipulation, and in better targeting of chromosome editing to minimize extraneous regulation influences on the introduced sequences.Importance: Understanding the role of the non-coding regulatory genome in complex disease phenotypes is essential, but even in well-characterized model organisms, identification of regulatory regions within the vast non-coding genome remains a challenge. We used a large-scale assay to generate a genome wide map of transcriptional enhancers. Such a catalogue for the important malaria vector, Anopheles coluzzii, will be an important research tool as the role of non-coding regulatory variation in differential susceptibility to malaria infection is explored and as a public resource for research on this important insect vector of disease.

Using Bayesian state-space models to understand the population dynamics of the dominant malaria vector, Anopheles funestus in rural Tanzania

Background: It is often assumed that the population dynamics of the malaria vector Anopheles funestus, its role in malaria transmission and the way it responds to interventions are similar to the more elaborately characterized An. gambiae. However, An. funestus has several unique ecological features that could generate distinct transmission dynamics and responsiveness to interventions. The objectives of this work were to develop a model which will; 1) reconstruct the population dynamics, survival, and fecundity of wild An. funestus populations in southern Tanzania, 2) quantify impacts of density dependence on the dynamics, and 3) assess seasonal fluctuations in An. funestus demography. Through quantifying the population dynamics of An. funestus, this model will enable analysis of how their stability and response to interventions may different from that of An. gambiae s.l.Methods: A Bayesian State Space Model (SSM) based on mosquito life history was fit to time series data on the abundance of female An. funestus s.s. collected over 2 years in southern Tanzania. Prior values of fitness and demography were incorporated from empirical data on larval development, adult survival and fecundity from laboratory-reared first generation progeny of wild caught An. funestus. The model was structured to allow larval and adult fitness traits to vary seasonally in response to environmental covariates (i.e. temperature and rainfall), and for density dependency in larvae. We measured the effects of density dependence and seasonality through counterfactual examination of model fit with or without these covariates.Results: The model accurately reconstructed the seasonal population dynamics of An. funestus and generated biologically-plausible values of their survival larval, development and fecundity in the wild. This model suggests that An-funestus survival and fecundity annual pattern was highly variable across the year, but did not show consistent seasonal trends either rainfall or temperature. While the model fit was somewhat improved by inclusion of density dependence, this was a relatively minor effect and suggests that this process is not as important for An. funestus as it is for An. gambiae populations.Conclusion: The model's ability to accurately reconstruct the dynamics and demography of An. funestus could potentially be useful in simulating the response of these populations to vector control techniques deployed separately or in combination. The observed and simulated dynamics also suggests that An. funestus could be playing a role in year-round malaria transmission, with any apparent seasonality attributed to other vector species.

Current status of insecticide resistance in malaria vectors in the Asian countries: a systematic review

Background: The application of insecticides for malaria vector control has led to a global problem, which is the current trend of increased resistance against these chemicals. This study aimed to review the insecticide resistance status was previously determined in Asia and how to implement the necessary interventions. Moreover, the implications of resistance in malaria vector control in this region were studied. Methods: This systematic review was conducted using a predefined protocol based on PRISMA-retrieved articles from four science databases, namely ProQuest, Science Direct, EBSCO, and PubMed in the last ten years (2009 to 2019). The searching process utilized four main combinations of the following keywords: malaria, vector control, insecticide, and Asia. In ProQuest, malaria control, as well as an insecticide, were used as keywords. The following criteria were included in the filter, namely full text, the source of each article, scholarly journal, Asia, and publication date as in the last ten years. Results: There were 1408 articles retrieved during the initial search (ProQuest=722, Science Direct=267, EBSCO=50, PubMed=285, and Scopus=84). During the screening, 27 articles were excluded because of duplication, 1361 based on title and abstract incompatibility with the inclusion criteria, and 20 due to content differences. In the final screening process, 15 articles were chosen to be analyzed. From the 15 articles, it is known that there was organochlorine (DDT), organophosphate (malathion), and pyrethroids resistance in several Anopheles species with a less than 80% mortality rate. Conclusion: This review found multiple resistance in several Anopheles includes resistance to pyrethroid. The reports of pyrethroid resistance were quite challenging because it is considered effective in the malaria vector control. Several countries in Asia are implementing an insecticide resistance management (IRM) strategy against malaria vectors following the Global Plan for IRM.

Integration of whole genome sequencing and transcriptomics reveals a complex picture of the reestablishment of insecticide resistance in the major malaria vector Anopheles coluzzii

Insecticide resistance is a major threat to gains in malaria control, which have been stalling and potentially reversing since 2015. Studies into the causal mechanisms of insecticide resistance are painting an increasingly complicated picture, underlining the need to design and implement targeted studies on this phenotype. In this study, we compare three populations of the major malaria vector An. coluzzii: a susceptible and two resistant colonies with the same genetic background. The original colonised resistant population rapidly lost resistance over a 6-month period, a subset of this population was reselected with pyrethroids, and a third population of this colony that did not lose resistance was also available. The original resistant, susceptible and re-selected colonies were subject to RNAseq and whole genome sequencing, which identified a number of changes across the transcriptome and genome linked with resistance. Firstly, an increase in the expression of genes within the oxidative phosphorylation pathway were seen in both resistant populations compared to the susceptible control; this translated phenotypically through an increased respiratory rate, indicating that elevated metabolism is linked directly with resistance. Genome sequencing highlighted several blocks clearly associated with resistance, including the 2Rb inversion. Finally, changes in the microbiome profile were seen, indicating that the microbial composition may play a role in the resistance phenotype. Taken together, this study reveals a highly complicated phenotype in which multiple transcriptomic, genomic and microbiome changes combine to result in insecticide resistance.

Anopheline species (Diptera: Culicidae) from the Maracá Ecological Station, Roraima State, Brazil

Anopheline species were sampled in different ecotones at Maracá Ecological Station and in Cantá municipality, both in Roraima State, Brazil, in June 2016. Nine species were recorded. Six species belong to the subgenus Nyssorhynchus and three species to the subgenus Anopheles. The most abundant species collected were Anopheles triannulatus s.l., Anopheles nuneztovari s.l. and Anopheles oswaldoi species B. The remaining species had frequencies lower than 10%. While A. triannulatus s.l. was the most frequent and the most widely distributed species in the studied area, Anopheles darlingi Root, the main malaria vector in the Brazilian Amazon region, was captured in two different ecotones and in low frequency. Our findings suggest that the risk of malaria outbreaks at Maracá Ecological Station may be low. This study also describes the first record of Anopheles costai Fonseca & Ramos in Maracá Ecological Station, Roraima State, increasing the distribution range of this taxon.

Using marine cargo traffic to identify countries in Africa with greatest risk of invasion by Anopheles stephensi

Anopheles stephensi is an efficient malaria vector commonly found in South Asia and the Arabian Peninsula, but in recent years it has established as an invasive species in the Horn of Africa (HoA). In this region An. stephensi was first detected in a livestock quarantine station near a major seaport in Djibouti in 2012, in Ethiopia in 2016, in Sudan in 2018 and Somalia in 2019. Anopheles stephensi often uses artificial containers as larval habitats, which may facilitate introduction through maritime trade as has been seen with other invasive container breeding mosquitoes. If An. stephensi is being introduced through maritime traffic, prioritization exercises are needed to identify locations at greatest risk of An. stephensi introduction for early detection and rapid response, limiting further invasion opportunities. Here, we use UNCTAD maritime trade data to 1) identify coastal African countries which were most highly connected to select An. stephensi endemic countries in 2011, prior to initial detection in Africa, 2) develop a ranked prioritization list of countries based on likelihood of An. stephensi introduction for 2016 and 2020 based on maritime trade alone and maritime trade and habitat suitability, and 3) use network analysis to describe intracontinental maritime trade and eigenvector centrality to determine likely paths of further introduction on the continent if An. stephensi is detected in a new location. Our results show that in 2011, Sudan and Djibouti were ranked as the top two countries with likelihood of An. stephensi introduction based on maritime trade alone, and these were indeed the first two coastal countries in the HoA where An. stephensi was detected. Trade data from 2020 with Djibouti and Sudan included as source populations identify Egypt, Kenya, Mauritius, Tanzania, and Morocco as the top five countries with likelihood of An. stephensi introduction. When factoring in habitat suitability, Egypt, Kenya, Tanzania, Morocco, and Libya are ranked highest. Network analysis revealed that the countries with the highest eigenvector centrality scores, and therefore highest degrees of connectivity with other coastal African nations were South Africa (0.175), Mauritius (0.159), Ghana (0.159), Togo (0.157), and Morocco (0.044) and therefore detection of An. stephensi in any one of these locations has a higher potential to cascade further across the continent via maritime trade than those with lower eigenvector centrality scores. Taken together, these data could serve as tools to prioritize efforts for An. stephensi surveillance and control in Africa. Surveillance in seaports of countries at greatest risk of introduction may serve as an early warning system for the detection of An. stephensi, providing opportunities to limit further introduction and expansion of this invasive malaria vector in Africa.

Multiple Novel Clades of Anopheline Mosquitoes Caught Outdoors in Northern Zambia

Residual vector populations that do not come in contact with the most frequently utilized indoor-directed interventions present major challenges to global malaria eradication. Many of these residual populations are mosquito species about which little is known. As part of a study to assess the threat of outdoor exposure to malaria mosquitoes within the Southern and Central Africa International Centers of Excellence for Malaria Research, foraging female anophelines were collected outside households in Nchelenge District, northern Zambia. These anophelines proved to be more diverse than had previously been reported in the area. In order to further characterize the anopheline species, sequencing and phylogenetic approaches were utilized. Anopheline mosquitoes were collected from outdoor light traps, morphologically identified, and sent to Johns Hopkins Bloomberg School of Public Health for sequencing. Sanger sequencing from 115 field-derived samples yielded mitochondrial COI sequences, which were aligned with a homologous 488 bp gene segment from known anophelines (n = 140) retrieved from NCBI. Nuclear ITS2 sequences (n = 57) for at least one individual from each unique COI clade were generated and compared against NCBI’s nucleotide BLAST database to provide additional evidence for taxonomical identity and structure. Molecular and morphological data were combined for assignment of species or higher taxonomy. Twelve phylogenetic groups were characterized from the COI and ITS2 sequence data, including the primary vector species Anopheles funestus s.s. and An. gambiae s.s. An unexpectedly large proportion of the field collections were identified as An. coustani and An. sp. 6. Six phylogenetic groups remain unidentified to species-level. Outdoor collections of anopheline mosquitoes in areas frequented by people in Nchelenge, northern Zambia, proved to be extremely diverse. Morphological misidentification and underrepresentation of some anopheline species in sequence databases confound efforts to confirm identity of potential malaria vector species. The large number of unidentified anophelines could compromise the malaria vector surveillance and malaria control efforts not only in northern Zambia but other places where surveillance and control are focused on indoor-foraging and resting anophelines. Therefore, it is critical to continue development of methodologies that allow better identification of these populations and revisiting and cleaning current genomic databases.