Introgression of a synthetic sex ratio distortion transgene into different genetic backgrounds of Anopheles coluzzii.

The development of genetically modified mosquitoes (GMM) and their subsequent field release offers innovative approaches for vector control of malaria. A non-gene drive self-limiting male-bias Ag(PMB)1 strain has been developed in a 47-year-old laboratory G3 strain of Anopheles gambiae s.l. When Ag(PMB)1 males are crossed to wild-type females, expression of the endonuclease I-PpoI during spermatogenesis causes the meiotic cleavage of the X chromosome in sperm cells, leading to fertile offspring with a 95% male bias. However, World Health Organization states that the functionality of the transgene could differ when inserted in different genetic backgrounds of Anopheles coluzzii which is currently a predominant species in several West-African countries and thus a likely recipient for a potential release of self-limiting GMMs. In this study, we introgressed the transgene from the donor Ag(PMB)1 by six serial backcrosses into two recipient colonies of An. coluzzii that had been isolated in Mali and Burkina Faso. Scans of informative Single Nucleotide Polymorphism (SNP) markers and whole-genome sequencing analysis revealed a nearly complete introgression of chromosomes 3 and X, but a remarkable genomic divergence in a large region of chromosome 2 between the later backcrossed (BC6) transgenic offspring and the recipient paternal strains. These findings suggested to extend the backcrossing breeding strategy beyond BC6 generation and increasing the introgression efficiency of critical regions that have ecological and epidemiological implications through the targeted selection of specific markers. Disregarding differential introgression efficiency, we concluded that the phenotype of the sex ratio distorter is stable in the BC6 introgressed An. coluzzii strains.

Bulk-up synchronization of successive larval cohorts of Anopheles gambiae and Anopheles coluzzii through temperature reduction at early larval stages: effect on emergence rate, body size and mating success.

BACKGROUND: Malaria persists as a huge medical and economic burden. Although the number of cases and death rates have reduced in recent years, novel interventions are a necessity if such gains are to be maintained. Alternative methods to target mosquito vector populations that involve the release of large numbers genetically modified mosquitoes are in development. However, their successful introduction will require innovative strategies to bulk-up mosquito numbers and improve mass rearing protocols for Anopheles mosquitoes. METHODS: The relationship between mosquito aquatic stage development and temperature was exploited so that multiple cohorts of mosquitoes, from separate egg batches, could be synchronized to 'bulk-up' the number of mosquitoes released. First instar larvae were separated into two cohorts: the first, maintained under standard insectary conditions at 27oC, the second subjected to an initial 5-day cooling period at 19oC. RESULTS: Cooling of 1st instars slowed the mean emergence times of Anopheles coluzzii and Anopheles gambiae by 2.4 and 3.5 days, respectively, compared to their 27oC counterparts. Pupation and emergence rates were good (> 85 %) in all conditions. Temperature adjustment had no effect on mosquito sex ratio and adult fitness parameters such as body size and mating success. CONCLUSIONS: Bulk-up larval synchronization is a simple method allowing more operational flexibility in mosquito production towards mark-release-recapture studies and mass release interventions.

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.

Addressing key gaps in implementation of mosquito larviciding to accelerate malaria vector control in southern Tanzania: results of a stakeholder engagement process in local district councils.

BACKGROUND: Larval source management was historically one of the most effective malaria control methods but is now widely deprioritized in Africa, where insecticide-treated nets (ITNs) and indoor residual spraying (IRS) are preferred. However, in Tanzania, following initial successes in urban Dar-es-Salaam starting early-2000s, the government now encourages larviciding in both rural and urban councils nationwide to complement other efforts; and a biolarvicide production-plant has been established outside the commercial capital. This study investigated key obstacles and opportunities relevant to effective rollout of larviciding for malaria control, with a focus on the meso-endemic region of Morogoro, southern Tanzania. METHODS: Key-informants were interviewed to assess awareness and perceptions regarding larviciding among designated health officials (malaria focal persons, vector surveillance officers and ward health officers) in nine administrative councils (n = 27). Interviewer-administered questionnaires were used to assess awareness and perceptions of community members in selected areas regarding larviciding (n = 490). Thematic content analysis was done and descriptive statistics used to summarize the findings. RESULTS: A majority of malaria control officials had participated in larviciding at least once over the previous three years. A majority of community members had neutral perceptions towards positive aspects of larviciding, but overall support for larviciding was high, although several challenges were expressed, notably: (i) insufficient knowledge for identifying relevant aquatic habitats of malaria vectors and applying larvicides, (ii) inadequate monitoring of programme effectiveness, (iii) limited financing, and (iv) lack of personal protective equipment. Although the key-informants reported sensitizing local communities, most community members were still unaware of larviciding and its potential. CONCLUSIONS: The larviciding programme was widely supported by both communities and malaria control officials, but there were gaps in technical knowledge, implementation and public engagement. To improve overall impact, it is important to: (i) intensify training efforts, particularly for identifying habitats of important vectors, (ii) adopt standard technical principles for applying larvicides or larval source management, (iii) improve financing for local implementation and (iv) improve public engagement to boost community awareness and participation. These lessons could also be valuable for other malaria endemic areas wishing to deploy larviciding for malaria control or elimination.

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.

Behavioural and Electrophysiological Responses of Female Anopheles gambiae Mosquitoes to Volatiles from a Mango Bait.

Attractive Toxic Sugar Baits (ATSB) are used in a "lure-and-kill" approach for management of the malaria vector Anopheles gambiae, but the active chemicals were previously unknown. Here we collected volatiles from a mango, Mangifera indica, juice bait which is used in ATSBs in Tanzania and tested mosquito responses. In a Y-tube olfactometer, female mosquitoes were attracted to the mango volatiles collected 24-48 h, 48-72 h and 72-96 h after preparing the bait but volatiles collected at 96-120 h were no longer attractive. Volatile analysis revealed emission of 23 compounds in different chemical classes including alcohols, aldehydes, alkanes, benzenoids, monoterpenes, sesquiterpenes and oxygenated terpenes. Coupled GC-electroantennogram (GC-EAG) recordings from the antennae of An. gambiae showed robust responses to 4 compounds: humulene, (E)-caryophyllene, terpinolene and myrcene. In olfactometer bioassays, mosquitoes were attracted to humulene and terpinolene. (E)-caryophyllene was marginally attractive while myrcene elicited an avoidance response with female mosquitoes. A blend of humulene, (E)-caryophyllene and terpinolene was highly attractive to females (P < 0.001) when tested against a solvent blank. Furthermore, there was no preference when this synthetic blend was offered as a choice against the natural sample. Our study has identified the key compounds from mango juice baits that attract An. gambiae and this information may help to improve the ATSBs currently used against malaria vectors.

Seasonal malaria vector and transmission dynamics in western Burkina Faso.

BACKGROUND: In the context of widespread mosquito resistance to currently available pesticides, novel, precise genetic vector control methods aimed at population suppression or trait replacement are a potentially powerful approach that could complement existing malaria elimination interventions. Such methods require knowledge of vector population composition, dynamics, behaviour and role in transmission. Here were characterized these parameters in three representative villages, Bana, Pala and Souroukoudingan, of the Sudano-Sahelian belt of Burkina Faso, a region where bed net campaigns have recently intensified. METHODS: From July 2012 to November 2015, adult mosquitoes were collected monthly using pyrethroid spray catches (PSC) and human landing catches (HLC) in each village. Larval habitat prospections assessed breeding sites abundance at each site. Mosquitoes collected by PSC were identified morphologically, and then by molecular technique to species where required, to reveal the seasonal dynamics of local vectors. Monthly entomological inoculation rates (EIR) that reflect malaria transmission dynamics were estimated by combining the HLC data with mosquito sporozoite infection rates (SIR) identified through ELISA-CSP. Finally, population and EIR fluctuations were fit to locally-collected rainfall data to highlight the strong seasonal determinants of mosquito abundance and malaria transmission in this region. RESULTS: The principal malaria vectors found were in the Anopheles gambiae complex. Mosquito abundance peaked during the rainy season, but there was variation in vector species composition between villages. Mean survey HLC and SIR were similar across villages and ranged from 18 to 48 mosquitoes/person/night and from 3.1 to 6.6% prevalence. The resulting monthly EIRs were extremely high during the rainy season (0.91-2.35 infectious bites/person/day) but decreased substantially in the dry season (0.03-0.22). Vector and malaria transmission dynamics generally tracked seasonal rainfall variations, and the highest mosquito abundances and EIRs occurred in the rainy season. However, despite low residual mosquito populations, mosquitoes infected with malaria parasites remained present in the dry season. CONCLUSION: These results highlight the important vector control challenge facing countries with high EIR despite the recent campaigns of bed net distribution. As demonstrated in these villages, malaria transmission is sustained for large parts of the year by a very high vector abundance and high sporozoite prevalence, resulting in seasonal patterns of hyper and hypo-endemicity. There is, therefore, an urgent need for additional vector control tools that can target endo and exophillic mosquito populations.

Assessment of the developmental success of Anopheles coluzzii larvae under different nutrient regimes: effects of diet quality, food amount and larval density.

BACKGROUND: In a context of increasing resistance of both vectors toward main classes of insecticides used in public health and parasites toward anti-malarial drugs, development of new and complementary molecules or control approaches is fundamental to achieve the objective of controlling or even eliminating malaria. Concerning vector control, the sterile insect technique and other genetic control approaches are among promising complementary tools in an integrated management strategy for malaria control. These approaches rely not only on a good understanding of vector biology (especially during larval stages), but also on the availability of adequate supplies and protocols for efficient mosquito rearing. The aim of this study was to assess the factors impacting the life history of Anopheles coluzzii mosquitoes at the larval stage, in the context of genetic and sterile insect approaches to control malaria vectors. METHODS: The effect of different larval diets and larval rearing densities on the development of An. coluzzii were evaluated in the laboratory. Emergence rate (ER), pre-imaginal developmental time (DT) and adult wing length (WL) were measured under different food regimes. Four diets were tested among which three were provided by the Insect Pest Control Laboratory (IPCL) of the FAO/IAEA Joint division. RESULTS: Data showed significant differences in the quality of the different diets and suggested a negative density dependence in all three life history parameters measured under tested rearing conditions. ER and WL increased with food availability, but decreased with increasing larval density. Conversely DT was shortened with increasing food availability but increased with larval density. These data demonstrates intraspecific larval competition modulated by food amount and space availability. Of the four diets tested, the one made of a mix of tuna meal, bovine liver powder, brewer's yeast, squid liver powder and vitamin mix (diet 2) yielded the best results as it produced a good balance between ER, DT and WL. Food availability for optimal development (highest survival at shortest time) was in the range of 180-400 µg/larvae/day for the three diets provided by the IPCL. CONCLUSION: There is an interaction between diet type, diet concentration and larval density. Best results in terms of optimal larvae development parameters happen when moderately high values of those three variables are observed.

Experimental swap of Anopheles gambiae's assortative mating preferences demonstrates key role of X-chromosome divergence island in incipient sympatric speciation.

Although many theoretical models of sympatric speciation propose that genes responsible for assortative mating amongst incipient species should be associated with genomic regions protected from recombination, there are few data to support this theory. The malaria mosquito, Anopheles gambiae, is known for its sympatric cryptic species maintained by pre-mating reproductive isolation and its putative genomic islands of speciation, and is therefore an ideal model system for studying the genomic signature associated with incipient sympatric speciation. Here we selectively introgressed the island of divergence located in the pericentric region of the X chromosome of An. gambiae s.s. into its sister taxon An. coluzzii through 5 generations of backcrossing followed by two generations of crosses within the introgressed strains that resulted in An. coluzzii-like recombinant strains fixed for the M and S marker in the X chromosome island. The mating preference of recombinant strains was then tested by giving virgin recombinant individuals a choice of mates with X-islands matching and non-matching their own island type. We show through genetic analyses of transferred sperm that recombinant females consistently mated with matching island-type males thereby associating assortative mating genes with the X-island of divergence. Furthermore, full-genome sequencing confirmed that protein-coding differences between recombinant strains were limited to the experimentally swapped pericentromeric region. Finally, targeted-genome comparisons showed that a number of these unique differences were conserved in sympatric field populations, thereby revealing candidate speciation genes. The functional demonstration of a close association between speciation genes and the X-island of differentiation lends unprecedented support to island-of-speciation models of sympatric speciation facilitated by pericentric recombination suppression.

Killer bee molecules: antimicrobial peptides as effector molecules to target sporogonic stages of Plasmodium.

A new generation of strategies is evolving that aim to block malaria transmission by employing genetically modified vectors or mosquito pathogens or symbionts that express anti-parasite molecules. Whilst transgenic technologies have advanced rapidly, there is still a paucity of effector molecules with potent anti-malaria activity whose expression does not cause detrimental effects on mosquito fitness. Our objective was to examine a wide range of antimicrobial peptides (AMPs) for their toxic effects on Plasmodium and anopheline mosquitoes. Specifically targeting early sporogonic stages, we initially screened AMPs for toxicity against a mosquito cell line and P. berghei ookinetes. Promising candidate AMPs were fed to mosquitoes to monitor adverse fitness effects, and their efficacy in blocking rodent malaria infection in Anopheles stephensi was assessed. This was followed by tests to determine their activity against P. falciparum in An. gambiae, initially using laboratory cultures to infect mosquitoes, then culminating in preliminary assays in the field using gametocytes and mosquitoes collected from the same area in Mali, West Africa. From a range of 33 molecules, six AMPs able to block Plasmodium development were identified: Anoplin, Duramycin, Mastoparan X, Melittin, TP10 and Vida3. With the exception of Anoplin and Mastoparan X, these AMPs were also toxic to an An. gambiae cell line at a concentration of 25 µM. However, when tested in mosquito blood feeds, they did not reduce mosquito longevity or egg production at concentrations of 50 µM. Peptides effective against cultured ookinetes were less effective when tested in vivo and differences in efficacy against P. berghei and P. falciparum were seen. From the range of molecules tested, the majority of effective AMPs were derived from bee/wasp venoms.

Differential effects of inbreeding and selection on male reproductive phenotype associated with the colonization and laboratory maintenance of Anopheles gambiae.

BACKGROUND: Effective mating between laboratory-reared males and wild females is paramount to the success of vector control strategies aiming to decrease disease transmission via the release of sterile or genetically modified male mosquitoes. However mosquito colonization and laboratory maintenance have the potential to negatively affect male genotypic and phenotypic quality through inbreeding and selection, which in turn can decrease male mating competitiveness in the field. To date, very little is known about the impact of those evolutionary forces on the reproductive biology of mosquito colonies and how they ultimately affect male reproductive fitness. METHODS: Here several male reproductive physiological traits likely to be affected by inbreeding and selection following colonization and laboratory rearing were examined. Sperm length, and accessory gland and testes size were compared in male progeny from field-collected females and laboratory strains of Anopheles gambiae sensu stricto colonized from one to over 25 years ago. These traits were also compared in the parental and sequentially derived, genetically modified strains produced using a two-phase genetic transformation system. Finally, genetic crosses were performed between strains in order to distinguish the effects of inbreeding and selection on reproductive traits. RESULTS: Sperm length was found to steadily decrease with the age of mosquito colonies but was recovered in refreshed strains and crosses between inbred strains therefore incriminating inbreeding costs. In contrast, testes size progressively increased with colony age, whilst accessory gland size quickly decreased in males from colonies of all ages. The lack of heterosis in response to crossing and strain refreshing in the latter two reproductive traits suggests selection for insectary conditions. CONCLUSIONS: These results show that inbreeding and selection differentially affect reproductive traits in laboratory strains overtime and that heterotic 'supermales' could be used to rescue some male reproductive characteristics. Further experiments are needed to establish the exact relationship between sperm length, accessory gland and testes size, and male reproductive success in the laboratory and field settings.

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.

Attributes of Anopheles gambiae swarms in South Central Uganda.

BACKGROUND: Anopheles gambiae continues to be widespread and an important malaria vector species complex in Uganda. New approaches to malaria vector control are being explored including population suppression through swarm reductions and genetic modification involving gene drives. Designing and evaluating these new interventions require good understanding of the biology of the target vectors. Anopheles mosquito swarms have historically been hard to locate in Uganda and therefore have remained poorly characterized. In this study we sought to identify and characterize An. gambiae s.l mosquito swarms in three study sites of high An. gambiae s.l prevalence within Central Uganda. METHODS: Nine sampling visits were made to three villages over a 2-year period. Sampling targeted both wet and dry seasons and was done for 2 days per village during each trip, using sweep nets. All swarm data were analysed using the JMP 14 software (SAS Institute, Inc., Cary, NC, USA), parametrically or non-parametrically as appropriate. RESULTS: Most of the An. gambiae s.s. swarms sampled during this study were single-species swarms. However, some mixed An. gambiae s.s. and Culex spp. mosquito swarms were also observed. Swarms were larger in the wet season than in the dry season. Mean swarm height ranged from 2.16 m to 3.13 m off the ground and only varied between villages but not by season. Anopheles gambiae mosquitoes were present in all three villages, preferred to swarm over bare ground markers, and could be effectively sampled by field samplers. CONCLUSIONS: This study demonstrated that An. gambiae s.l swarms could be effectively located and sampled in South Central Uganda and provided in-depth descriptions of hitherto poorly understood aspects of An. gambiae local swarm characteristics. Swarms were found close to inhabited households and were greater in size and number during the rainy season. Anopheles gambiae s.s swarms were significantly associated with bare ground markers and were sometimes at heights over 4 m above the ground, showing a necessity to develop tools suitable for swarm sampling at these heights. While mixed species swarms have been reported before elsewhere, this is the first documented instance of mixed genus swarms found in Uganda and should be studied further as it could have implications for swarm sampling explorations where multiple species of mosquitoes exist.

A review of applications and limitations of using aquatic macroinvertebrate predators for biocontrol of the African malaria mosquito, Anopheles gambiae sensu lato.

Macroinvertebrate predators such as backswimmers (Heteroptera: Notonectidae), dragonflies (Odonata: Aeshnidae), and predatory diving beetles (Coleoptera: Dytiscidae) naturally inhabit aquatic ecosystems. Some aquatic ecosystems inhabited by these macroinvertebrate predator taxa equally form malaria vector larval habitats. The presence of these predators in malaria vector larval habitats can negatively impact on development, adult body size, fecundity, and longevity of the malaria vectors, which form important determinants of their fitness and future vectorial capacity. These potential negative impacts caused by aquatic macroinvertebrate predators on malaria vectors warrant their consideration as biocontrol agents in an integrated program to combat malaria. However, the use of these macroinvertebrate predators in malaria biocontrol is currently constrained by technical bottlenecks linked to their generalist predatory tendencies and often long life cycles, demanding complex rearing systems. We reviewed the literature on the use of aquatic macroinvertebrate predators for biocontrol of malaria vectors from the An. gambiae s.l. complex. The available information from laboratory and semi-field studies has shown that aquatic macroinvertebrates have the potential to consume large numbers of mosquito larvae and could thus offer an additional approaches in integrated malaria vector management strategies. The growing number of semi-field structures available in East and West Africa provides an opportunity to conduct ecological experimental studies to reconsider the potential of using aquatic macroinvertebrate predators as a biocontrol tool. To achieve a more sustainable approach to controlling malaria vector populations, additional, non-chemical interventions could provide a more sustainable approach, in comparison with the failing chemical control tools, and should be urgently considered for integration with the current mosquito vector control campaigns.

Entomological survey of sibling species in the Anopheles funestus group in Tanzania confirms the role of Anopheles parensis as a secondary malaria vector.

BACKGROUND: Malaria transmission in Tanzania is driven by mosquitoes of the Anopheles gambiae complex and Anopheles funestus group. The latter includes An. funestus s.s., an anthropophilic vector, which is now strongly resistant to public health insecticides, and several sibling species, which remain largely understudied despite their potential as secondary vectors. This paper provides the initial results of a cross-country study of the species composition, distribution and malaria transmission potential of members of the Anopheles funestus group in Tanzania. METHODS: Mosquitoes were collected inside homes in 12 regions across Tanzania between 2018 and 2022 using Centres for Disease Control and Prevention (CDC) light traps and Prokopack aspirators. Polymerase chain reaction (PCR) assays targeting the noncoding internal transcribed spacer 2 (ITS2) and 18S ribosomal DNA (18S rDNA) were used to identify sibling species in the An. funestus group and presence of Plasmodium infections, respectively. Where DNA fragments failed to amplify during PCR, we sequenced the ITS2 region to identify any polymorphisms. RESULTS: The following sibling species of the An. funestus group were found across Tanzania: An. funestus s.s. (50.3%), An. parensis (11.4%), An. rivulorum (1.1%), An. leesoni (0.3%). Sequencing of the ITS2 region in the nonamplified samples showed that polymorphisms at the priming sites of standard species-specific primers obstructed PCR amplification, although the ITS2 sequences closely matched those of An. funestus s.s., barring these polymorphisms. Of the 914 samples tested for Plasmodium infections, 11 An. funestus s.s. (1.2%), and 2 An. parensis (0.2%) individuals were confirmed positive for P. falciparum. The highest malaria transmission intensities [entomological inoculation rate (EIR)] contributed by the Funestus group were in the north-western region [108.3 infectious bites/person/year (ib/p/y)] and the south-eastern region (72.2 ib/p/y). CONCLUSIONS: Whereas An. funestus s.s. is the dominant malaria vector in the Funestus group in Tanzania, this survey confirms the occurrence of Plasmodium-infected An. parensis, an observation previously made in at least two other occasions in the country. The findings indicate the need to better understand the ecology and vectorial capacity of this and other secondary malaria vectors in the region to improve malaria control.

Environmentally friendly tool to control mosquito populations without risk of insecticide resistance: the Lehmann's funnel entry trap.

BACKGROUND: Current malaria control strategies have cut down the malaria burden in many endemic areas, however the emergence and rapid spread of insecticide and drug resistance undermine the success of these efforts. There is growing concern that malaria eradication will not be achieved without the introduction of novel control tools. One approach that has been developed in the last few years is based on house screening to reduce indoor mosquito vector densities and consequently decrease malaria transmission. Here screening and trapping were combined in one tool to control mosquito populations. The trap does not require an insecticide or even an attractant, yet it effectively collects incoming resistant and susceptible mosquitoes and kills them. RESULTS: Performance of the funnel entry trap was tested in low and high malaria vector density areas. An overall reduction of 70 to 80% of mosquito density was seen in both. Species and molecular forms of Anopheles gambiae identification indicated no variation in the number of Anopheles arabiensis and the molecular forms of An. gambiae between houses and traps. Mosquitoes collected in the traps and in houses were highly resistant to pyrethroids (0.9 kdr-based mechanism). CONCLUSION: There is a global consensus that new intervention tools are needed to cross the last miles in malaria elimination/eradication. The funnel entry trap showed excellent promise in suppressing mosquito densities even in area of high insecticide resistance. It requires no chemicals and is self-operated.

Forensic DNA Analysis of Mixed Mosquito Blood Meals: STR Profiling for Human Identification.

Mosquito vectors captured at a crime scene are forensically valuable since they feed on human blood, and hence, human DNA can be recovered to help identify the victim and/or the suspect. This study investigated the validity of obtaining the human short tandem repeats (STRs) profile from mixed blood meals of the mosquito, Culex pipiens L. (Diptera, Culicidae). Thus, mosquitoes were membrane-feed on blood from six different sources: a human male, a human female, mixed human male-female blood, mixed human male-mouse blood, mixed human female-mouse blood, and mixed human male-female-mouse blood. DNA was extracted from mosquito blood meals at 2 h intervals up to 72 h post-feeding to amplify 24 human STRs. Data showed that full DNA profiles could be obtained for up to 12 h post-feeding, regardless of the type of blood meal. Complete and partial DNA profiles were obtained up to 24 h and 36 h post-feeding, respectively. The frequencies of STR loci decreased over time after feeding on mixed blood until they became weakly detectable at 48 h post-feeding. This may indicate that a blood meal of human blood mixed with animal blood would contribute to maximizing DNA degradation and thus affects STR identification beyond 36 h post-feeding. These results confirm the feasibility of human DNA identification from mosquito blood meals, even if it is mixed with other types of non-human blood, for up to 36 h post-feeding. Therefore, blood-fed mosquitoes found at the crime scene are forensically valuable, as it is possible to obtain intact genetic profiles from their blood meals to identify a victim, a potential offender, and/or exclude a suspect.

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.

Visual and olfactory associative learning in the malaria vector Anopheles gambiae sensu stricto.

BACKGROUND: Memory and learning are critical aspects of the ecology of insect vectors of human pathogens because of their potential effects on contacts between vectors and their hosts. Despite this epidemiological importance, there have been only a limited number of studies investigating associative learning in insect vector species and none on Anopheline mosquitoes. METHODS: A simple behavioural assays was developed to study visual and olfactory associative learning in Anopheles gambiae, the main vector of malaria in Africa. Two contrasted membrane qualities or levels of blood palatability were used as reinforcing stimuli for bi-directional conditioning during blood feeding. RESULTS: Under such experimental conditions An. gambiae females learned very rapidly to associate visual (chequered and white patterns) and olfactory cues (presence and absence of cheese or Citronella smell) with the reinforcing stimuli (bloodmeal quality) and remembered the association for up to three days. Associative learning significantly increased with the strength of the conditioning stimuli used. Importantly, learning sometimes occurred faster when a positive reinforcing stimulus (palatable blood) was associated with an innately preferred cue (such as a darker visual pattern). However, the use of too attractive a cue (e.g. Shropshire cheese smell) was counter-productive and decreased learning success. CONCLUSIONS: The results address an important knowledge gap in mosquito ecology and emphasize the role of associative memory for An. gambiae's host finding and blood-feeding behaviour with important potential implications for vector control.