Entomological baseline data collection and power analyses in preparation of a mosquito swarm-killing intervention in south-western Burkina Faso.

BACKGROUND: Insecticides are currently the main tools used to reduce the transmission of malaria; therefore, the development of resistance to insecticides in malaria vectors is of major concern for malaria control. The resistance level to pyrethroids is particularly high in the Western region of Burkina Faso and may affect the efficacy of insecticidal bed nets and indoor residual spraying. Adult mosquito swarming and other nocturnal behaviours exhibit spatial and temporal patterns that suggest potential vulnerability to targeted space spraying with effective insecticides. Indeed, targeted space-spraying against adult mosquito swarms has been used to crash mosquito populations and disrupt malaria transmission. METHODS: Prior to impact assessment of swarm killing, a baseline data collection was conducted from June to November 2016 in 10 villages divided into two areas in western Burkina Faso. The data considered both ecological and demographic characteristics to monitor the key entomological parameters. RESULTS: The mean number of swarms observed was 35 per village, ranging from 25 to 70 swarms according to the village. Female density in both areas varied significantly as a function of the village and the period of collection. The human biting rate was significantly affected by the period of collection and depended upon whether the collection was carried out indoors or outdoors. Averages of parity rate were high in both areas for all periods of collection, ranging from 60 to 90%. These values ranged from 80 to 100% for inseminated females. Sporozoite rates ranged between 1.6 and 7.2% depending upon the village. The molecular identification of resting and swarming mosquitoes showed the presence of the three major malaria vectors in Burkina Faso, but in different proportions for each village. CONCLUSIONS: The distribution of the potential swarm markers and swarms in villages suggested that swarms are clustered across space, making intervention easier. Power simulations showed that the direct sampling of swarms provides the highest statistical power, thereby reducing the number of villages needed for a trial.

Comparison of swarming, mating performance and longevity of males Anopheles coluzzii between individuals fed with different natural fruit juices in laboratory and semi-field conditions.

BACKGROUND: It is assumed that malaria vectors feed on locally available nectar sources to obtain energy. Sugar feeding is energetically critical for the Anopheles male swarming and mating activities. However, little is known about the impact of local nectar feeding on male physiological development and its consequences on male mosquito life traits in the malaria control context. This study aimed to evaluate the influence of local fruit juices on the life traits of males Anopheles coluzzii. METHODS: Swarming characteristics (number of males in swarm, number of mating pairs, and swarm duration) in semi-field conditions; mating rate and longevity in a laboratory setting were compared between males An. coluzzii fed exclusively with mango, papaya or banana juices. The trophic preference was investigated in semi-field conditions. RESULTS: The results of this study showed that in the laboratory, mosquitoes fed with papaya juices lived on average longer (10 days) than those fed with banana or mango juices (5 days) and had higher a mating rate (53%) than those fed with banana juice (40%). In the semi-field, the swarm size of mosquitoes fed with banana juice (85 males) was larger than that of mosquitoes fed with mango juice (60 males). The number of mating pairs formed from banana-fed male swarms (17 mating pairs) was higher than that formed from mango-fed male swarm (8 mating pairs). There was no difference in swarming duration between male treatments. Male mosquitoes had a preference for papaya and banana juices. CONCLUSIONS: The results indicate that the origin of plant-derived feeding is an important factor in the survival and reproduction of mosquitoes. This calls for further investigations of chemical contents of nectars and their impact on the physiological development of mosquitoes.

Mating competitiveness of sterile male Anopheles coluzzii in large cages.

BACKGROUND: Understanding the factors that account for male mating competitiveness is critical to the development of the sterile insect technique (SIT). Here, the effects of partial sterilization with 90 Gy of radiation on sexual competitiveness of Anopheles coluzzii allowed to mate in different ratios of sterile to untreated males have been assessed. Moreover, competitiveness was compared between males allowed one versus two days of contact with females. METHODS: Sterile and untreated males four to six days of age were released in large cages (~1.75 sq m) with females of similar age at the following ratios of sterile males: untreated males: untreated virgin females: 100:100:100, 300:100:100, 500:100:100 (three replicates of each) and left for two days. Competitiveness was determined by assessing the egg hatch rate and the insemination rate, determined by dissecting recaptured females. An additional experiment was conducted with a ratio of 500:100:100 and a mating period of either one or two days. Two controls of 0:100:100 (untreated control) and 100:0:100 (sterile control) were used in each experiment. RESULTS: When males and females consort for two days with different ratios, a significant difference in insemination rate was observed between ratio treatments. The competitiveness index (C) of sterile males compared to controls was 0.53. The number of days of exposure to mates significantly increased the insemination rate, as did the increased number of males present in the untreated: sterile male ratio treatments, but the number of days of exposure did not have any effect on the hatch rate. DISCUSSION: The comparability of the hatch rates between experiments suggest that An. coluzzii mating competitiveness experiments in large cages could be run for one instead of two days, shortening the required length of the experiment. Sterilized males were half as competitive as untreated males, but an effective release ratio of at least five sterile for one untreated male has the potential to impact the fertility of a wild female population. However, further trials in field conditions with wild males and females should be undertaken to estimate the ratio of sterile males to wild males required to produce an effect on wild populations.

Towards a transactional medicine approach to combating global emerging pathogens: the case of COVID-19.

When the COVID-19 pandemic struck and China reported the first case to the World Health Organization in December 2019, there was no evidence-based treatment to combat it. With the catastrophic situation that followed, materialised by a considerable number of deaths, researchers, doctors, traditional healers, and governments of all nations committed themselves to find therapeutic solutions, including preventive and curative. There are effective treatments offered both by modern medicine and traditional medicine for COVID-19 today. However, other therapeutic proposals have not been approved due to the lack of effectiveness and scientific rigour during their development process. Proponents of modern medicine prefer biomedical therapies while in some countries, traditional treatments are used regularly because of their availability, affordability and satisfaction they bring to the population. In this paper, we propose a transactional medicine approach where the interaction between traditional and modern medicine produces a change. With this approach, the promoters of traditional medicine and those of modern medicine will be able to acquire knowledge through the experience produced by their encounters. Transactional medicine aims to be a model for decolonising medicine and recognising the value of both traditional and modern medicine in the fight against COVID-19 and other global emerging pathogens.

Characterization of the swarming behavior of Anopheles coluzzii and Anopheles gambiae (Diptera: Culicidae) populations in a hybrid zone of Senegal.

Anopheles gambiae and Anopheles coluzzii, often found in sympatry and synchronous, have undergone a premating reproductive isolation across their distribution range. However, in the Western coast of Africa, unexpected hybridization zones have been observed, and little is known about swarming behavior of these cryptic taxa. Here, we characterized the swarming behavior of An. coluzzii and An. gambiae to investigate its role in the high hybridization level in Senegal. The study was conducted in the south and central Senegal during the 2018 rainy season. Mating swarms of malaria vectors were surveyed at sunset and collected using an insect net. Meanwhile, indoor resting populations of malaria vectors were collected by pyrethrum spray catches. Upon collection, specimens were identified morphologically, and then members of the An. gambiae complex were identified at the species level by polymerase chain reaction (PCR). An. gambiae swarmed mainly over bare ground, whereas An. coluzzii were found swarming above various objects creating a dark-light contrast with the bare ground. The swarms height varied from 0.5 to 2.5 m. Swarming starting time was correlated with sunset whatever the months for both species, and generally lasted about 10 min. No mixed swarm of An. gambiae and An. coluzzii was found even in the high hybridization area. These results indicated a premating isolation between An. coluzzii and An. gambiae. However, the high hybridization rate in the sympatric area suggests that heterogamous mating is occurring, thus stressing the need for further extensive studies.

Perfect association between spatial swarm segregation and the X-chromosome speciation island in hybridizing Anopheles coluzzii and Anopheles gambiae populations.

The sibling species An. coluzzii and An. gambiae s.s. are major malaria vectors thought to be undergoing sympatric speciation with gene flow. In the absence of intrinsic post-zygotic isolation between the two taxa, speciation is thought possible through the association of assortative mating and genomic regions protected from gene flow by recombination suppression. Such genomic islands of speciation have been described in pericentromeric regions of the X, 2L and 3L chromosomes. Spatial swarm segregation plays a major role in assortative mating between sympatric populations of the two species and, given their importance for speciation, genes responsible for such pre-mating reproductive barriers are expected to be protected within divergence islands. In this study 2063 male and 266 female An. coluzzii and An. gambiae s.s. individuals from natural swarms in Burkina Faso, West Africa were sampled. These were genotyped at 16 speciation island SNPs, and characterized as non-hybrid individuals, F1 hybrids or recombinant F1+n backcrossed individuals. Their genotypes at each speciation island were associated with their participation in An. coluzzii and An. gambiae-like swarms. Despite extensive introgression between the two species, the X-island genotype of non-hybrid individuals (37.6%), F1 hybrids (0.1%) and F1+n recombinants (62.3%) of either sex perfectly associated to each swarm type. Associations between swarm type and the 3L and 2L speciation islands were weakened or broken down by introgression. The functional demonstration of a close association between spatial segregation behaviour and the X speciation island lends further support to sympatric speciation models facilitated by pericentric recombination suppression in this important species complex.

Comparison of entomological impacts of two methods of intervention designed to control Anopheles gambiae s.l. via swarm killing in Western Burkina Faso.

Outdoor biting constitutes a major limitation of current vector control based primarily on long-lasting insecticidal nets and indoor residual spraying, both of which are indoor interventions. Consequently, malaria elimination will not be achieved unless additional tools are found to deal with the residual malaria transmission and the associated vector dynamics. In this study we tested a new vector control approach for rapidly crashing mosquito populations and disrupting malaria transmission in Africa. This method targets the previously neglected swarming and outdoor nocturnal behaviors of both male and female Anopheles mosquitoes. It involved accurate identification and targeted spraying of mosquito swarms to suppress adult malaria vector populations and their vectorial capacities. The impact of targeted spraying was compared to broadcast spraying and evaluated simultaneously. The effects of the two interventions were very similar, no significant differences between targeted spraying and broadcast spraying were found for effects on density, insemination or parity rate. However, targeted spraying was found to be significantly more effective than broadcast spraying at reducing the number of bites per person. As expected, each intervention had a highly significant impact upon all parameters measured, but the targeted swarm spraying required less insecticide.

Rapid age-grading and species identification of natural mosquitoes for malaria surveillance.

The malaria parasite, which is transmitted by several Anopheles mosquito species, requires more time to reach its human-transmissible stage than the average lifespan of mosquito vectors. Monitoring the species-specific age structure of mosquito populations is critical to evaluating the impact of vector control interventions on malaria risk. We present a rapid, cost-effective surveillance method based on deep learning of mid-infrared spectra of mosquito cuticle that simultaneously identifies the species and age class of three main malaria vectors in natural populations. Using spectra from over 40, 000 ecologically and genetically diverse An. gambiae, An. arabiensis, and An. coluzzii females, we develop a deep transfer learning model that learns and predicts the age of new wild populations in Tanzania and Burkina Faso with minimal sampling effort. Additionally, the model is able to detect the impact of simulated control interventions on mosquito populations, measured as a shift in their age structures. In the future, we anticipate our method can be applied to other arthropod vector-borne diseases.

Cuticular hydrocarbons are associated with mating success and insecticide resistance in malaria vectors.

Anopheles coluzzii females, important malaria vectors in Africa, mate only once in their lifetime. Mating occurs in aerial swarms with a high male-to-female ratio, where traits underlying male mating success are largely unknown. Here, we investigated whether cuticular hydrocarbons (CHCs) influence mating success in natural mating swarms in Burkina Faso. As insecticides are widely used in this area for malaria control, we also determined whether CHCs affect insecticide resistance levels. We find that mated males have higher CHC abundance than unmated controls, suggesting CHCs could be determinants of mating success. Additionally, mated males have higher insecticide resistance under pyrethroid challenge, and we show a link between resistance intensity and CHC abundance. Taken together, our results suggest that CHC abundance may be subject to sexual selection in addition to selection by insecticide pressure. This has implications for insecticide resistance management, as these traits may be sustained in the population due to their benefits in mating even in the absence of insecticides.

Assessment of the ecologically dependent post-zygotic isolation between Anopheles coluzzii and Anopheles gambiae.

Within the Anopheles gambiae complex, the sibling species An. coluzzii and An. gambiae are undergoing sympatric speciation. These species are characterized by rare hybrids in most of their geographical distribution. A strong assortative mating mediated by spatial swarm segregation has been shown whereas no intrinsic post-zygotic barriers have been found in laboratory conditions. To test the role of the hybridisation in reproductive isolation in natural populations transplant experiment are therefore needed to establish the significance of post-zygotic barriers. Previous studies indicated that predation is one of the major forces driving ecological divergence between An. gambiae and An. coluzzii. Here we extended these studies to their hybrids. Parental species and their F1 hybrids from reciprocal crosses were generated by the forced-mating technique as follows: female An. coluzzii × male An. coluzzii; female An. coluzzii × male An. gambiae; female An. gambiae × male An. coluzzii and female An. gambiae × Male An. gambiae. First instar larvae of each group from the crossing (here after An. coluzzii, Hybrid COL/GAM, Hybrid GAM/COL and An. gambiae, respectively) were transplanted in a field experiment with predation effect. Emergence success, development time of larvae and body size of the newly emerging adults were estimated as fitness components and then compared between parental species and F1 hybrids in absence and in presence of predators. Our findings confirm that An. coluzzii had higher fitness than An. gambiae in presence of predators versus in absence of predators. Moreover, the fitness of the F1 hybrid COL/GAM whose female parent was An. coluzzii matched that of An. coluzzii while that of the F1 reciprocal hybrid GAM/COL was similar to An. gambiae.

Multiple insecticide resistance and Plasmodium infection in the principal malaria vectors Anopheles funestus and Anopheles gambiae in a forested locality close to the Yaoundé airport, Cameroon.

Background: Reducing the burden of malaria requires better understanding of vector populations, particularly in forested regions where the incidence remains elevated. Here, we characterized malaria vectors in a locality near the Yaoundé international airport, Cameroon, including species composition, abundance, Plasmodium infection rate, insecticide resistance profiles and underlying resistance mechanisms. Methods: Blood-fed adult mosquitoes resting indoors were aspirated from houses in April 2019 at Elende, a village located 2 km from the Yaoundé-Nsimalen airport. Female mosquitoes were forced to lay eggs to generate F 1 adult progeny. Bioassays were performed to assess resistance profile to insecticides. The threshold of insecticide susceptibility was defined above 98% mortality rate and mortality rates below 90% were indicative of confirmed insecticide resistance. Furthermore, the molecular basis of resistance and Plasmodium infection rates were investigated. Results: Anopheles funestus s.s. was most abundant species in Elende (85%) followed by Anopheles gambiae s.s. (15%) with both having a similar sporozoite rate. Both species exhibited high levels of resistance to pyrethroids (<40% mortality). An. gambiae s.s. was also resistant to DDT (9.9% mortality) and bendiocarb (54% mortality) while susceptible to organophosphate. An. funestus s.s. was resistant to dieldrin (1% mortality), DDT (86% mortality) but susceptible to carbamates and organophosphates. The L119F-GSTe2 resistance allele (8%) and G119S ace-1 resistance allele (15%) were detected in An. funestus s.s. and An. gambiae s.s., respectively . Furthermore, the high pyrethroid/DDT resistances in An. gambiae s.s. corresponded with an increase frequency of 1014F kdr allele (95%). Transcriptional profiling of candidate cytochrome P450 genes reveals the over-expression of CYP6P5, CYP6P9a and CYP6P9b. Conclusion: The resistance to multiple insecticide classes observed in these vector populations alongside the high Plasmodium sporozoite rate highlights the challenges that vector control programs encounter in sustaining the regular benefits of contemporary insecticide-based control interventions in forested areas.

Semi-field and indoor setups to study malaria mosquito swarming behavior.

BACKGROUND: The recent resurgence of interest in sterile insect techniques to control vector mosquitoes has renewed interest in novel methods for observing mating behavior. Malarial vectors of the Anopheles gambiae complex are known to mate in swarms at specific locations at dawn and dusk. Most knowledge of mosquito swarming behavior is derived from field observations and a few experimental studies designed to assess critical parameters that affect mosquito swarming. However, such studies are difficult to implement in the field because of uncontrollable environmental factors and mosquito conditions. Here, we present two experimental setups specifically designed to analyze mosquito swarming behavior and provide evidence that swarming behavior of mosquitoes can be generated and accurately assessed under both semi-field and laboratory conditions. METHODS: The Mosquito Ecology Research Facility setup is a semi-field enclosure made of 12 compartments (10.0 × 6.0 × 4.5 m L × W × H each) exposed to ambient meteorological and lighting conditions. The laboratory setup consists of a windowless room (5.1 × 4.7 × 3.0 m) in which both environmental and mosquito conditions can be controlled. In the two setups, 300 3-6-days-old An. coluzzii virgin males were released and some swarm characteristics were recorded such as the time at which the swarm started, the number of mosquitoes in the swarm and the height. Climatic conditions in the semi-field setup were also recorded. RESULTS: In both setups, An. coluzzii males displayed stereotyped and consistent swarming behavior day after day; males gradually gather into a swarm over a ground marker at sunset, flying in loops in relation to specific visual features on the ground. Although semi-field climatic conditions were slightly different from outdoors conditions, they did not impede swarming behavior and swarm characteristics were similar to those observed in the field. CONCLUSIONS: Swarm characteristics and their consistency across days provide evidences that these facilities can be used confidently to study swarming behavior. These facilities come to complement existing semi-field setups and pave the way for new experimental studies which will enhance our understanding of mating behavior but also mosquito ecology and evolution, a prerequisite for application of genetic approaches to malaria control.

Prediction of mosquito species and population age structure using mid-infrared spectroscopy and supervised machine learning.

Despite the global efforts made in the fight against malaria, the disease is resurging. One of the main causes is the resistance that Anopheles mosquitoes, vectors of the disease, have developed to insecticides. Anopheles must survive for at least 10 days to possibly transmit malaria. Therefore, to evaluate and improve malaria vector control interventions, it is imperative to monitor and accurately estimate the age distribution of mosquito populations as well as their population sizes. Here, we demonstrate a machine-learning based approach that uses mid-infrared spectra of mosquitoes to characterise simultaneously both age and species identity of females of the African malaria vector species Anopheles gambiae and An. arabiensis, using laboratory colonies. Mid-infrared spectroscopy-based prediction of mosquito age structures was statistically indistinguishable from true modelled distributions. The accuracy of classifying mosquitoes by species was 82.6%. The method has a negligible cost per mosquito, does not require highly trained personnel, is rapid, and so can be easily applied in both laboratory and field settings. Our results indicate this method is a promising alternative to current mosquito species and age-grading approaches, with further improvements to accuracy and expansion for use with wild mosquito vectors possible through collection of larger mid-infrared spectroscopy data sets.

Targeting male mosquito swarms to control malaria vector density.

Malaria control programs are being jeopardized by the spread of insecticide resistance in mosquito vector populations. It has been estimated that the spread of resistance could lead to an additional 120000 deaths per year, and interfere with the prospects for sustained control or the feasibility of achieving malaria elimination. Another complication for the development of resistance management strategies is that, in addition to insecticide resistance, mosquito behavior evolves in a manner that diminishes the impact of LLINs and IRS. Mosquitoes may circumvent LLIN and IRS control through preferential feeding and resting outside human houses and/or being active earlier in the evening before people go to sleep. Recent developments in our understanding of mosquito swarming suggest that new tools targeting mosquito swarms can be designed to cut down the high reproductive rate of malaria vectors. Targeting swarms of major malaria vectors may provide an effective control method to counteract behavioral resistance developed by mosquitoes. Here, we evaluated the impact of systematic spraying of swarms of Anopheles gambiae s.l. using a mixed carbamate and pyrethroid aerosol. The impact of this intervention on vector density, female insemination rates and the age structure of males was measured. We showed that the resulting mass killing of swarming males and some mate-seeking females resulted in a dramatic 80% decrease in population size compared to a control population. A significant decrease in female insemination rate and a significant shift in the age structure of the male population towards younger males incapable of mating were observed. This paradigm-shift study therefore demonstrates that targeting primarily males rather than females, can have a drastic impact on mosquito population.

Does extreme asymmetric dominance promote hybridization between Anopheles coluzzii and Anopheles gambiae s.s. in seasonal malaria mosquito communities of West Africa?

BACKGROUND: Anopheles gambiae s.s. and An. coluzzii are two of the most important malaria vector species in sub-Saharan Africa. These recently-diverged sibling species do not exhibit intrinsic post-zygotic barriers to reproduction and are thought to be separated by strong assortative mating combined with selection against hybrids. At present, little is known about the ecological conditions that determine hybridization and introgression between these cryptic taxa. METHODS: Swarm segregation and assortative mating were studied in Western Burkina Faso in the villages of Vallée du Kou (VK7) and Soumousso which differed in terms of which sibling species was much rarer than the other, and in Bana where both occurred in similar proportions. Swarms and pairs in copula were collected and genotyped, the proportion of intra and interspecific mating determined, and interspecific sperm transfer checked genetically. Females were collected through larval and adult indoor collections and genotyped or sexed-and-genotyped via a novel multiplex PCR. RESULTS: A total of 3,687 males and 220 females were collected and genotyped from 109 swarms. Only 3 swarms were composed of males from both species, and these were from the village of VK7 where An. gambiae s.s. was comparatively rare. Mixed-species pairs captured in copula were only detected in that area and made for 3.62 % and 100 % of mating pairs involving An. coluzzii and An. gambiae s.s. individuals, respectively. The high An. gambiae s.s. cross-mating rate was mirrored by high rates of hybridizations estimated from female larvae and adults indoor collections. This contrasted with Soumousso, where despite being much less common than An. gambiae s.s., An. coluzzii males did not form mixed swarms, females were not found in interspecific swarms or copula and hybridization rates were low in both sibling species. CONCLUSIONS: These data suggest that ecological conditions leading to rare An. gambiae s.s. in populations dominated by An. coluzzii may promote a breakdown of spatial swarm segregation and assortative mating between the two species. The lower overall hybridization rates observed at the larval and adult indoor stages compared to cross-mating rates support the idea that post-mating selection processes acting against hybrids may occur mostly prior to and/or at the early larval stages.

Role of nutritional reserves and body size in Anopheles gambiae males mating success.

A better knowledge of the different parameters that account for male mating success in the wild is critical to the development of genetic control strategies. In this study, we measured energy budgets (total sugar and glycogen) as the daily energetic investment in swarming males of An. gambiae s.s. M and S molecular forms from two different field locations, VK7 and Soumousso. We also looked at the difference between energetic reserves in mated males compared to unmated ones, and assessed wing length in both molecular forms to explore whether this phenotypic trait was involved in swarming behavior or mating success. The current study showed that the energetic cost of 25 min of swarming was around 50% of the male's sugar (M form: 48.5%, S form: 56.2%) and glycogen (M form: 53.1%, S form: 59%) reserves. However, no difference in carbohydrate content was observed between mated and unmated males. Mated males were found to be bigger than unmated ones, while intermediate size of males is advantageous in mating system, both in M and S molecular forms and when collected in two different locations. Regardless of the collection location, no difference in wing size was observed in swarming males collected early or late during a particular swarm. The results are discussed in the context sexual selection in different ecological locations.