Understanding the impact of altitude and ecological heterogeneity at a fine scale on the populations of malaria vectors is essential to better understand and anticipate eventual epidemiological changes. It could help to evaluate the spread of alleles conferring resistance to insecticides and also determine any increased entomological risk of transmission in highlands due to global warming. We used microsatellite markers to measure the effect of altitude and distance on the population genetic structure of Anopheles funestus and Anopheles gambiae s.s. in the Muheza area in the north-eastern part of Tanzania (seven loci for each species). Our analysis reveals strong gene flow between the different populations of An. funestus from lowland and highland areas, as well as between populations of An. gambiae sampled in the lowland area. These results highlight for An. funestus the absence of a significant spatial subpopulation structuring at small-scale, despite a steep ecological and altitudinal cline. Our findings are important in the understanding of the possible spread of alleles conferring insecticide resistance through mosquito populations. Such information is essential for vector control programmes to avoid the rapid spread and fixation of resistance in mosquito populations.

Control of the malaria vector An. gambiae is still largely obtained through chemical intervention using pyrethroids, such as permethrin. However, strains of An. gambiae that are resistant to the toxic effects of pyrethroids have become widespread in several endemic areas over the last decade. The objective of this study was to assess differences in five life-history traits (larval developmental time and the body weight, fecundity, hatch rate, and longevity of adult females) and energy metabolism between a strain of An. gambiae that is resistant to permethrin (RSP), due to knockdown resistance and enhanced metabolic detoxification, and a permethrin susceptible strain reared under laboratory conditions. We also quantified the expression levels of five antioxidant enzyme genes: GSTe3, CAT, GPXH1, SOD1, and SOD2. We found that the RSP strain had a longer developmental time than the susceptible strain. Additionally, RSP adult females had higher wet body weight and increased water and glycogen levels. Compared to permethrin susceptible females, RSP females displayed reduced metabolic rate and mitochondrial coupling efficiency and higher mitochondrial ROS production. Furthermore, despite higher levels of GSTe3 and CAT transcripts, RSP females had a shorter adult life span than susceptible females. Collectively, these results suggest that permethrin resistance alleles might affect energy metabolism, oxidative stress, and adult survival of An. gambiae. However, because the strains used in this study differ in their genetic backgrounds, the results need to be interpreted with caution and replicated in other strains to have significant implications for malaria transmission and vector control.

Short and long dry spells occur throughout the year and limit the access of gravid mosquitoes to oviposition sites. Using simulations in laboratory conditions, we explored the possible consequences of a long duration of egg retention on reproductive capacity, trophic behaviour, life expectancy and gonotrophic cycle in a context of breeding-site absence. KISUMU and wild gravid females of Anopheles gambiae were subjected to a delay in egg-laying following the preset modalities as immediate egg-laying versus retention for 5, 10 and 15 days. The egg batch size and the hatching rate were measured. In addition, blood-feeding patterns and life expectancy were investigated. Our study showed that oviposition occurs much faster in the standard susceptible laboratory strain An. gambiae KISUMU with a lapse of 2.2 days than in the wild females of An. gambiae with an average of 5 days between the second blood meal and egg-laying. In this experiment, the egg-retention time did not significantly affect the number of eggs laid. In the absence of breeding sites, the rate of egg-hatching decreased in proportion as the retention time became longer. The study also showed that gravid females deprived of oviposition sites continue to take blood meals. The average longevity of non-gravid females was estimated to be lower than that of gravid females. The oviposition-site deprivation has no impact on female fecundity and blood feeding, but influences egg-hatching.

Identification of the major loci responsible for insecticide resistance in malaria vectors would aid the development and implementation of effective resistance management strategies, which are urgently needed to tackle the growing threat posed by resistance to the limited insecticides available for malaria control. Genome-wide association studies in the major malaria vector, Anopheles gambiae, have been hindered by the high degree of within-population structuring and very low levels of linkage disequilibrium hence we revisited the use of quantitative trait loci (QTL) mapping to study resistance phenotypes in this vector species. Earlier work, identified two major QTL associated with pyrethroid resistance in A. gambiae s.s. from East Africa using genetic crossing of laboratory-colonized resistant and susceptible strains. In this study, we report the results from genetic mapping of pyrethroid resistance in three isofemale pedigrees established from wild-caught female A. gambiae s.s. mosquitoes from Benin. We identified two QTL on chromosomes 2L and 3R in these field populations, in similar genomic locations to the QTL identified in laboratory strains. The relative merits of two alternative study designs are discussed and suggestions made for future genetic mapping studies of insecticide resistance in mosquitoes.

The ‘paddy paradox’, the occurrence of large populations of vectors but low amounts of malaria transmission where irrigated rice is grown, was investigated in a village in Ghana where M form Anopheles gambiae are common. Peridomestic and indoor host-seeking mosquitoes were collected in tent traps and light traps over 21 consecutive nights at the start of the rainy season in June 2009 when the population increased exponentially from less than 100 per night to over 1000. Infection rates in the overall mosquito population were 0.3% and in the estimated parous population were 1.9%. Numbers of An. gambiae in the tent trap peaked between midnight and 02:40 am. The majority of insects were taking their first blood meal, as virgins or shortly after mating. More than expected were collected in the light trap during a rainstorm at the start of the rains but overall numbers were not affected. Fewer than expected were collected after a subsequent storm. Recruitment to the adult population decreased over the following days. It is hypothesised that the ‘paddy paradox’ is due to young pre-gravid insects dispersing more widely than gravid ones, not necessarily to low survival in the mosquito.

There has been a recent shift in the literature on mosquito/Plasmodium interactions with an increasingly large number of theoretical and experimental studies focusing on their population biology and evolutionary processes. Ecological immunology of mosquito-malaria interactions – the study of the mechanisms and function of mosquito immune responses to Plasmodium in their ecological and evolutionary context – is particularly important for our understanding of malaria transmission and how to control it. Indeed, describing the processes that create and maintain variation in mosquito immune responses and parasite virulence in natural populations may be as important to this endeavor as describing the immune responses themselves. For historical reasons, Ecological Immunology still largely relies on studies based on non-natural model systems. There are many reasons why current research should favour studies conducted closer to the field and more realistic experimental systems whenever possible. As a result, a number of researchers have raised concerns over the use of artificial host-parasite associations to generate inferences about population-level processes. Here I discuss and review several lines of evidence that, I believe, best illustrate and summarize the limitations of inferences generated using non-natural model systems.

The effects of sex, age of the human host, patency of asexual and sexual stages and seasonality on infectiousness of Plasmodium falciparum to mosquitoes were investigated in a rural village in southern Tanzania between 1992 and 1994. Villagers from randomized subgroups of households were surveyed for malaria parasites. Gametocyte and trophozoite prevalences were age dependent and fluctuated without any clear pattern of seasonality. A sample of 107 participants, selected to include an excess of gametocyte carriers, slept under bednets with holes cut into the sides for 3 weeks. A total of 3837 Anopheles gambiae s.l. and 5403 A. funestus recovered from these bednets, was examined for all oocysts 5–7 days after feeding or for oocysts less than 17·5 µn in diameter 2–3 days after feeding. Additional blood slides from participants were taken twice weekly. The 5–7 day oocyst rates were 12·1% in A. gambiae s.l. and 10·9% in A. funestus and 2–3 day rates were 3·6 and 4·9%, respectively. The higher rates using the former method were attributed to previous infection. There were strong correlations in the levels of infection in both vectors when they fed on the same hosts. However, patent gametocytaemia was only weakly associated with the development of oocysts in the mosquito. Infectiousness was not related to host age, sex, or the season.

The influence of adult female body size of Anopheles gambiae s.l. on development of midgut and salivary gland infections by the parasite Plasmodium falciparum was investigated in a field study carried out in Tanzania. The proportion of mosquitoes infected during a blood meal was independent of size. However, the number of oocysts harboured by infected mosquitoes increased with size of the mosquito. The proportion of mosquitoes with sporozoites, and thus potentially infective to humans, was highest in intermediate-sized mosquitoes, whereas the largest and smallest mosquitoes were less likely to have sporozoites. This pattern is interpreted as a combination of high survival rate of large, uninfected mosquitoes and of low survival rate of mosquitoes infected with many oocysts.

In an area of holoendemic malaria in Northern Tanzania, Anopheles gambiae s.l. females were age-graded by Polovodova's method and dissected for sporozoites. Age-specific sporozoite rates implied that mosquitoes acquired new infections at all ages. The extrinsic period lasted just over 3 gonotrophic cycles (9–11 days). Very high sporozoite rates in the oldest females implied the absence or rarity of genetic refractoriness to infection. A method is described for estimating the proportion of bloodmeals which result in mosquito infection. This method makes relatively few assumptions about mosquito behaviour, and could be useful for evaluating transmission-blocking interventions. Overall, it is estimated that about 21% of meals are infectious. This is much higher than previous estimates derived either from experimental mosquito feeding studies or from similar age-grading data collected from the same area in 1962. Various alternative explanations are considered, and it is concluded that there has been a 2.5-fold increase in human infectiousness in the last 25 years. This is partly attributable to suppression of human infectiousness by widespread chloroquine usage during the 1960s, followed by removal of this effect by drug resistance. It is argued that chloroquine would be expected to select for increased infectivity in the parasite, and this may also have contributed to the observed increase.

The aim of this study was to document what water types were most stimulatory to oviposition by the malaria mosquito, Anopheles gambiae Giles s.s. In laboratory choice tests, dishes of black cotton and red laterite soils from around Kisumu, Kenya, equally stimulated An. gambiae oviposition when saturated with water. When infused into either soil type, water from Lake Victoria was significantly more stimulatory than any other type of water or infusion tested. Water from natural An. gambiae puddle habitats, borehole water, cow dung infusion, Bermuda grass infusion and distilled water followed in declining rank order. Relative to An. gambiae puddle-habitat water and distilled water, Lake Victoria water appears to provide the strongest chemical cues for An. gambiae oviposition recorded to date. This work suggests that this important vector of human malaria may oviposit in and around Lake Victoria, as well as in its usual puddle habitats. Lake Victoria should be explored for its possible contribution to malaria mosquito populations.

Root bark extracts of five Meliaceae species (Turraea abyssinica Hochst., Turraea wakefeldii Oliv., Turraea mombassana Hiern ex C.DC., Trichilia roka (Forsk) Chiov. and Melia volkensii Guerke.) and different fractions thereof (following chloroform extraction and column chromatographic separation) were compared for their immediate toxicity and long-term effects on Anopheles gambiae Giles sensu stricto (Diptera: Culicidae). Larvicidal effects of the extracts appeared to be largely associated with limonoids of medium polarity with M. volkensii and T. mombassana extracts being more potent than those of the other Meliaceae. Long-term (6–8 days post-exposure) observations at lower doses showed that 100% cumulative mortality can be achieved with some extracts (particularly those of T. mombassana and M. volkensii) with interesting growth-inhibition effects. Follow-up studies that are needed and practical implications of the results in terms of development of larval control strategies for African malaria vectors are discussed.

The survival of female Anopheles gambiae s.s. mosquitoes inside two village house types (grass-thatched and iron-roofed) was studied in relation to diet and ambient indoor microclimatic conditions. Two batches of 20–30, 1-day-old laboratory-bred mosquitoes were maintained inside cages in the grass-thatched (n=2) and iron-roofed (n=2) houses and fed daily, one group on 10% glucose and the other on human blood. Throughout the experiments, indoor temperature and relative humidity of the houses were recorded, and mortality of mosquitoes monitored daily until all had died. The experiments were replicated thrice. There was no significant variation in the overall mean temperature (P=0.93) or relative humidity profiles (P=0.099) between the two house types, although the iron-roofed houses recorded higher temperature peaks. A Kaplan–Meier survival analysis showed that the mean survival times of mosquitoes were 8 and 10 days in the two grass-thatched huts and 7 and 10 days in the two iron-roof houses for mosquitoes feeding on blood and sugar meals, respectively. The mean survival times of mosquitoes maintained inside similar house types differed only due to diet. In the proportionality of hazards model (Cox regression), the dietary regimes significantly influenced the probability of survival (P=0.0001), with mosquitoes surviving longer on sugar meals than on blood. Microclimatic factors inside houses also significantly influenced mosquito survival. Although higher peak temperatures were recorded in corrugated iron-roofed houses, the survival of the mosquitoes resting in them did not differ significantly from that in grass-thatched houses. However, the impact of these temperatures on the development of malaria parasites inside the vector needs to be investigated.

Laboratory studies were carried out to investigate the role of larval habitat-derived microorganisms in the production of semiochemicals for oviposition site selection by Anopheles gambiae Giles sensu stricto mosquitoes. Dual-choice bioassays with gravid females were conducted in standard mosquito cages. Field-collected or laboratory-reared mosquitoes, individually or in groups, were offered a choice between unmodified (water or soil from a natural breeding site) or modified substrates (filtered water, autoclaved soil or sterile media to which bacterial suspensions had been added). Egg counts were used to assess oviposition preferences. Mosquitoes preferred to oviposit on unmodified substrates from natural larval habitats containing live microorganisms rather than on sterilized ones. Variable responses were observed when sterile substrates were inoculated with bacteria isolated from water and soil from natural habitats. We conclude that microbial populations in breeding sites produce volatiles that serve as semiochemicals for gravid An. gambiae. These signals, in conjunction with other (non-olfactory) chemical and physical cues, may be used by the female to assess the suitability of potential larval habitats in order to maximize the fitness of her offspring.

Transmission of the malaria parasite Plasmodium is influenced by many different host, vector and parasite factors. Here we conducted a field study at Mbita, an area of endemic malaria in Western Kenya, to test whether parasite transmission to mosquitoes is influenced by the severity of malaria infection in its human host at the time when gametocytes, the transmission forms, are present in the peripheral blood. We examined the infectivity of 81 Plasmodium falciparum gametocyte carriers to mosquitoes. Of these, 21 were patients with fever and other malaria-related symptoms, and 60 were recruited among apparently healthy volunteers. Laboratory-reared Anopheles gambiae s.s. (local strain) were experimentally infected with blood from these gametocyte carriers by membrane-feeding. The severity of the clinical symptoms was greater in febrile patients. These symptomatic patients had higher asexual parasitaemia and lower gametocyte densities (P=0·05) than healthy volunteers. Ookinete development occurred in only 6 out of the 21 symptomatic patients, of which only 33·3% successfully yielded oocysts. The oocyst prevalence was only 0·6% in the 546 mosquitoes that were fed on blood from this symptomatic group, with mean oocyst intensity of 0·2 (range 0–2) oocysts per mosquito. In contrast, a higher proportion (76·7%) of healthy gametocyte carriers yielded ookinetes, generating an oocyst rate of 12% in the 1332 mosquitoes that fed on them (mean intensity of 6·3, range: 1–105 oocysts per mosquito). Statistical analysis indicated that the increased infectivity of asymptomatic gametocyte carriers was not simply due to their greater gametocyte abundance, but also to the higher level of infectivity of their gametocytes, possibly due to lower parasite mortality within mosquitoes fed on blood from healthy hosts. These results suggest that blood factors and/or conditions correlated with illness reduce P. falciparum gametocyte infectivity.

Experiments were carried out to determine the factors that influence the feeding behaviour of Culex tigripes, a mosquito whose larvae are predators of other mosquito larvae. The results indicated that the efficiency of predatory activities of Cx tigripes larvae increased as they developed from 1st to 4th instar with regard to the number of mosquito prey larvae they consumed in a given period of time. Fourth instars of Cx tigripes are the most voracious of all the stages and are better adapted for catching and consuming all stages of mosquito larvae. The mean number of mosquito larvae consumed in 24 hours increased with increasing prey density. When offered a mixture of different prey species and larval stages, Cx tigripes larvae consistently selected 4th instars of mosquitoes over pupae and preferred Aedes aegypti larvae to the larvae of Cx quinquefasciatus and Anopheles gambiae. Prey size, density and mobility were also found to influence prey selection by Cx tigripes.

There is some evidence that pathology induced by heavy malaria infections (many oocysts) increases mortality of infected mosquitoes. However, there is little or no published evidence that documented changes in feeding behaviour associated with malaria infection also contribute to higher mortality of infected mosquitoes relative to uninfected individuals. We show here for the first time that, in a natural situation, infection by the sporozoites of the malaria parasite Plasmodium falciparum significantly reduced survival of blood-feeding Anopheles gambiae, the major vector of malaria in sub-Saharan Africa. To estimate feeding-associated mortality of infected mosquitoes, we compared the percentage of sporozoite infection in host-seeking mosquitoes caught before and after feeding. The infection rate was 12% for mosquitoes caught during the night as they were entering a tent to feed; however, only 7·5% of the surviving members of the same cohort caught after they had had the opportunity to feed were infected. Thus, Plasmodium falciparum sporozoites increased the probability of dying during the night-time feeding period by 37·5%. The increase in mortality was probably due to decreased efficiency in obtaining blood and by increased feeding activity of the sporozoite-infected mosquitoes that elicited a greater degree of defensive behaviour of hosts under attack.

Out of 85 Bacillus thuringiensis strains isolated from Nigerian soils, 18 were larvicidal to Aedes aegypti, Culex quinquefasciatus and Anopheles gambiae. At 10−6 dilution of the final whole culture (FWC), the 11 most active strains, code-named GSC1, GSC3, GNA13, GNA14, OGL8, BUS4, BUS5, OBG1, OBG4, OBG8 and BAR3 produced mortality of 11.7–61.7% and 3.3–48.3% in Ae. aegypti and in C. quinquefasciatus fourth instars respectively. At 0.5 × 10−5 dilution of the FWC, the B.t. strains caused mortality in An. gambiae larvae ranging from 31.7 to 94.1%. All the 18 mosquitocidal isolates belonged to the subspecies israelensis, i.e. serotype 14. Microscopic examination revealed all the isolates to be typically Bacillus, with irregular or amorphous crystals of varying sizes.

Rodent and avian malaria parasites have been reported to have an adverse affect upon the reproductive fitness of mosquitoes. In order to determine whether fecundity reduction occurs in Anopheles gambiae s. l. infected with human malaria a study of wild-caught mosquitoes was undertaken in the Muheza district of north east Tanzania. Fully engorged, indoor resting females were collected daily for 4 months and maintained for 5 days. A sporozoite rate of 11·5% was detected for the whole collection and of those females alive on day 6 an additional 17·5% were infected with oocysts alone. Oocyst, but not sporozoite, infection resulted in a 17·5% reduction in egg production. Fecundity reduction was not caused by a reduction in bloodmeal size in infected females and no size difference was detected between oocyst-infected and uninfected females although sporozoite-positive females were significantly larger. Comparisons in parity between uninfected and infected groups indicate that infection does not affect survival beyond the first gonotrophic cycle as no changes in survivorship occurred as a result of sporozoite infection.

Organosoluble extracts from 55 Turkish medicinal plants were tested under standardised conditions for biological activity against third-instar larvae of mosquitoes Aedes aegypti and Anopheles gambiae. Eight extracts demonstrated significant larvicidal activity, with An. gambiae being more susceptible than Ae. aegypti in all cases. The possibility of using indigenous plants for mosquito control is discussed.

Gas Liquid Chromatography was used to obtain profiles of cuticular hydrocarbons from single larvae of two species belonging to the Anopheles gambiae complex: A. gambiae and A. arabiensis. By applying discriminant analysis of hydrocarbons it was possible to separate the two populations with a 95% success rate.

The same analysis was used to achieve an average of 94% segregation among their strains. This analysis does not require preservation of specimen by liquid nitrogen or refrigeration and further identification by other procedures can be carried out on the specimens after cuticular hydrocarbon analysis. More importantly the technique permits analysis of single rather than pooled specimens used by earlier investigators.