Diel activity patterns of male Aedes albopictus in the laboratory.

This study was conducted to investigate the flight and dispersal behavior of male Aedes albopictus under laboratory conditions. Two different methods, the Observer and Ethovision software devices, were used to determine the total duration, the mean and maximum flight speed, and the distance covered by these mosquitoes. During 24 h, mosquitoes were more active from 0800 to 1200 h and from 1700 to 2100 h than during the rest of the day. Male Ae. albopictus displayed different activities at different times. The flight activity was 47 min and 57 sec over 24 h. During this period, sugar-fed males flew an average distance of 236.20 m at an average speed of 8.5 cm/sec. The unfed males flew significantly faster than recently fed males, with maximum flight speeds of 44.9 cm/sec and 33.6 cm/sec, respectively. The time used for resting (22 h 2 min +/- 13 min) was significantly higher (P < 0.0001) than walking and flying times (68 min +/- 10 sec and 49 min +/- 5 min, respectively). Overall, both methods allowed observations on flight activity, and the camera recordings allowed these activities to be quantified.

Influence of soil quality in the larval habitat on development of Anopheles gambiae Giles.

Larval ecology is an important aspect of the population dynamics of anopheline mosquitoes (Diptera: Culicidae), the vectors of malaria. Anopheles larvae live in pools of stagnant water and adult fitness may be correlated with the nutritional conditions under which larvae develop. A study was conducted in Mbita, Western Kenya, to investigate how properties of the soil substrate of Anopheles gambiae breeding pools can influence development of this mosquito species. An. gambiae eggs from an established colony were dispensed into experimental plastic troughs containing soil samples from a range of natural Anopheles larval habitats and filtered Lake Victoria water. The duration of larval development (8-15 days), pupation rate (0-79%), and adult body size (20.28-26.91 mm3) varied among different soil types. The total organic matter (3.61-21.25%), organic carbon (0.63-7.18%), and total nitrogen (0.06-0.58%) levels of the soils were positively correlated with pupation rate and negatively correlated with development time and adult body size.

Behavioural determinants of gene flow in malaria vector populations: Anopheles gambiae males select large females as mates.

BACKGROUND: Plasmodium-refractory mosquitoes are being rapidly developed for malaria control but will only succeed if they can successfully compete for mates when released into the wild. Pre-copulatory behavioural traits maintain genetic population structure in wild mosquito populations and mating barriers have foiled previous attempts to control malaria vectors through sterile male release. METHODS: Varying numbers of virgin male and female Anopheles gambiae Giles, from two strains of different innate sizes, were allowed to mate under standardized conditions in laboratory cages, following which, the insemination status, oviposition success and egg batch size of each female was assessed. The influence of male and female numbers, strain combination and female size were determined using logistic regression, correlation analysis and a simple mechanistic model of male competition for females. RESULTS: Male An. gambiae select females on the basis of size because of much greater fecundity among large females. Even under conditions where large numbers of males must compete for a smaller number of females, the largest females are more likely to become inseminated, to successfully oviposit and to produce large egg batches. CONCLUSIONS: Sexual selection, on the basis of size, could either promote or limit the spread of malaria-refractory genes into wild populations and needs to be considered in the continued development and eventual release of transgenic vectors. Fundamental studies of behavioural ecology in malaria vectors such as An. gambiae can have important implications for malaria control and should be prioritised for more extensive investigation in the future.

Estimation of malaria transmission from humans to mosquitoes in two neighbouring villages in south Cameroon: evaluation and comparison of several indices.

Malaria transmission from humans to mosquitoes was assessed in two neighbouring villages in a rural area near Yaoundé, Cameroon during high and low transmission seasons during 1998-2000, using several indices previously evaluated in different areas endemic for malaria but never directly compared. These indices were estimated from human parasitological data and mosquito infection rates and, for each individual, thick blood films were prepared at the same time as experimental infection of laboratory-bred mosquitoes. Among the 685 volunteers examined, the prevalence of Plasmodium falciparum gametocyte carriers was 16%, and 8% of individuals were able to infect mosquitoes. The percentage of mosquitoes that became infected by feeding on the infectious individuals was 21%. Children aged < 10 years contributed to about 75% of the infectious reservoir, although they constituted only 35% of the total population. Differences were found between the transmission seasons and the villages, and varied according to the index examined. Although there were more infectious individuals in one of the two villages, they were less infectious than those in the other village during the high transmission season. Comparative analysis of the transmission indices suggests the existence of functioning transmission-blocking immunity in one of the villages, which until now has been only hypothetically considered to play a role in malaria transmission in a natural setting. The epidemiological value of all the indices used and their accuracy in estimating the human infectious reservoir and its natural or induced variations are discussed.

Infectivity of Plasmodium falciparum gametocytes in patients attending rural health centres in western Kenya.

BACKGROUND: Experimentally studying the transmission of the malaria parasite and its regulating factors requires availability of human blood donors carrying infectious gametocytes. The difficulty of identifying gametocyte carriers from the community is often limited due to financial and human resources constraints. The available alternative is rural health centres where malaria patients go for treatment. In this study, the potential of recruiting volunteers and acquiring infectious blood for experimental infections from rural health centers in malaria endemic area was examined through routine patient diagnosis. OBJECTIVE: To examine the patients presenting at rural health centers for the potential to carry sexual stage malaria parasite and test their infectivity to Anopheles gambiae mosquitoes. SETTING: Mbita Health Centre, Mbita Town Ship, Suba District, western Kenya. METHODOLOGY: Routine survey of all patients attending Mbita Health Centre with suspected malaria. Patients were examined for Plasmodium falciparum trophozoites and gametocytes. Gametocyte-positive volunteers were recruited for their potential to infect Anopheles mosquitoes via membrane feeding. RESULTS: Three thousand nine hundred and eighty seven patients were screened between May 2000 and April 2001. Plasmodium falciparum was the predominant parasite species and P. malariae being the only minor species, accounting for 0.9% of malaria cases. Clinical malaria varied with age and prevailed throughout the year with a slight seasonality. Gametocyte prevalence was low (0.9-6.6%), and gametocyte densities were generally very low with a geometric mean of 39 gametocytes per microl blood. Children aged > 5 years constituted 67% of all gametocyte carriers. Only 22 volunteers with mean gametocytes density of 39.62 per microl blood (range: 16-112) were recruited for study of parasite infectiousness to laboratory-reared mosquitoes. Only two patients infected 1% of 1099 mosquitoes with one or two oocysts. CONCLUSION: The low gametocyte densities or other possible host and vector related factors regulating infectivity of gametocyte carriers to mosquitoes may have caused the poor infections of mosquitoes. This study indicates that rural health centers in malaria-endemic areas may not be suitable for recruiting infectious gametocyte donors for studies of vector competence. They are suitable for passive clinical case surveillance and for evaluation of the effects of control measures.

[Levels of malaria transmission: methods and parameters]. / L'évaluation des niveaux de transmission palustre: méthodologies et paramètres.

Evaluation of malaria transmission levels is necessary to compare ecologically diverse areas and to assess the effectiveness of efforts to control the disease. The purpose of this report is to describe useful techniques for descriptive epidemiology and potentially pertinent indicators regarding the three links in the epidemiological chain: transmission from mosquito to man, transmission from man to mosquito, and sporogonic cycle. Standards for evaluation of transmission from mosquito to man are now well established. Techniques and resulting data, mostly entomological, have been validated in numerous multicenter and multidiscipline studies before and after implementation of control measurements. Evaluation of transmission from man to mosquito has not yet been extensively studied. Gametocyte index does not appear to be a good indicator of infectivity in mosquitoes. Two other parameters that have been proposed in the literature are rate of human infectivity to mosquitoes and probability that a bloodmeal will be infectious. However these evaluation techniques have been neither subjected to comparative study nor validated in epidemiological surveys. The third factor for evaluation of malaria transmission levels involves sexual development of the gametocytes (sporogonic cycle) in the vector. Two indicators that might be useful in this regard are quantification of early-stage parasites in the stomach of the mosquito and study of blood factors in subjects in whom inhibition transmission has been documented. Since these methods have been used only sporadically, further study will be needed to validate this approach to evaluate transmission level.

Occurrence of natural Anopheles arabiensis swarms in an urban area of Bobo-Dioulasso City, Burkina Faso, West Africa.

The swarming behaviour of natural populations of Anopheles arabiensis was investigated by conducting transect surveys on 10 consecutive days, around dusk, from March to April and from September to October 2012 in Dioulassoba, a district of Bobo-Dioulasso city in Burkina Faso (West Africa). Swarms were observed outside, around identified larval breeding sites on the banks of the Houet River, as well as in the open-air courtyards found at the centre of many homes in the region. Swarms were found to occur in open sunlit spaces, mostly located above physical or visual cues somehow visually distinct from the surrounding area. Overall 67 and 78 swarms were observed, respectively, during the dry season (March-April) and the rainy season (September-October) of 2012, between 1.5m and 4.5m above the ground at their centre. 964 mosquitoes were collected and analysed from dry season swarms, of which most were male, and all were An. arabiensis, as were the few resting mosquitoes collected indoors. Larvae collected from breeding sites found on the banks of the Houet River mostly consisted of An. arabiensis and only a minority of Anopheles coluzzii (formerly identified as An. gambiae M form). Of 1694 mosquitoes analysed from 78 swarms in the rainy season collections, a few An. gambiae (formerly known as An. gambiae S form) males were identified, and the remainders were An. arabiensis. The majority of larvae collected during the wet season from the same breeding sites were identified as An. arabiensis followed by An. coluzzii and An. gambiae. The same pattern of species composition was observed in resting mosquitoes, though the proportion of An. arabiensis was less overwhelming. These data support the conclusion that An. arabiensis is the most prevalent species in this area, though the difference in species composition when using different population sampling techniques is noteworthy. Further studies are required for more detailed investigations of male dispersal, feeding behaviour and mating patterns in this urban setting.

Swarming behaviour in natural populations of Anopheles gambiae and An. coluzzii: review of 4 years survey in rural areas of sympatry, Burkina Faso (West Africa).

The swarming behaviour of natural populations of Anopheles gambiae and An. coluzzii (formerly known as An. gambiae S and M forms, respectively) were investigated through longitudinal surveys conducted between July 2006 and October 2009 in two rural areas of south-western Burkina Faso where these forms are sympatric. In both sites, the majority of swarms were recorded above visual markers localised among houses. In Soumousso, a wooded area of savannah, 108 pairs caught in copula from 205 swarms were sampled; in VK7, a rice growing area, 491 couples from 250 swarms were sampled. If segregated swarms were the norm in both sites, many visual markers were shared by the two forms of An. gambiae. Furthermore, mixed swarms were collected annually in frequencies varying from one site to another, though no mixed inseminations were recorded, corroborating the low hybrid rate previously reported in the field. The occurrence of inter-specific mate-recognition mechanisms, which allow individuals to avoid hybridisation, is discussed.

Role of heat-labile serum factor or host complement in the inhibition of Plasmodium falciparum sporogonic stages in Anopheles stephensi by gametocyte carriers' serological factors.

This study investigated the significance of serum complement on transmission-reducing activity (TRA) of field sera from 24 infected Plasmodium falciparum gametocyte carriers (from Cameroon) against cultured NF54 P. falciparum. Laboratory-reared Anopheles stephensi were given infectious blood meals prepared either with sera from naïve Dutch donor (AB type) or pair-matched field serum samples, both with and without active complement. TRA of serum factors and host complement on mosquito infection rate and oocyst intensity were divided into the various components involved in the early stages of sporogony. The majority (>80%) of sera tested showed positive antibody titres to Pfs230, the relevant complement-dependent target of transmission-reducing mechanisms. Regardless of the presence of active complement, bloodmeals with field sera exhibited significantly lower infection rates and oocyst intensity than the control group. Serological reactivity in Capture-ELISA against Pfs230 was significantly correlated with the reduction of parasite infectivity. Contrary to our expectation, the presence of active complement in the mosquito bloodmeal did not increase parasite losses and therefore the magnitude of transmission reduction by individual immune sera. Our findings on P. falciparum are consistent with previous studies on animal hosts of Plasmodium, indicating that early P. falciparum sporogonic stages may be insensitive to the antibody-dependent pathways of complement in human serum.

Discriminative feeding behaviour of Anopheles gambiae s.s. on endemic plants in western Kenya.

Anopheles gambiae Giles s.s. (Diptera: Culicidae) is known to feed on plant sugars, but this is the first experimental study to consider whether it discriminates between plant species. Thirteen perennial plant species were selected on the basis of their local availability within the vicinity of human dwellings and larval habitats of An. gambiae s.s. in western Kenya. Groups of 100 or 200 mosquitoes were released into cages either with a cutting of one plant type at a time (single-plant assay) or with cuttings of all 13 plants simultaneously (choice assay), respectively, and left overnight. In the choice assay, direct observations of the percentages of mosquitoes perching or feeding on each plant were recorded over four 1-h periods each night. For both types of assay, mosquitoes were recaptured and the percentage that had fed on plants was assessed by testing them individually for the presence of fructose. To identify which plants the choice-assay mosquitoes had fed on, gas chromatography (GC) profiles of samples of mosquito homogenates were compared with GC profiles of extracts from relevant parts of each plant. Four of the plants that were observed to have been fed on most frequently in the choice assay (Parthenium hysterophorus L., Tecoma stans L., Ricinus communis L., and Senna didymobotrya Fresen) were also shown to have been ingested most often by mosquitoes in both types of assay, suggesting that An. gambiae is differentially responsive to this range of plants, regardless of whether the plants were presented singly or mixed together. Significantly more females than males fed on plants, with the exception of P. hysterophorus L., one of the plants most frequently fed on. For most plant species (ten of 13), GC profiles indicated that An. gambiae obtained sugars primarily from flowers. The exceptions were P. hysterophorus L., Lantana camara L. and R. communis L., on which An. gambiae fed more often from leaves and stems than from flowers.

The early sporogonic cycle of Plasmodium falciparum in laboratory-infected Anopheles gambiae: an estimation of parasite efficacy.

This study investigated the successive losses in the parasite densities of Plasmodium falciparum stages during the early sporogony in laboratory-reared Anopheles gambiae infected by membrane feeding with blood from naturally infected gametocyte carriers (>50 gametocytes/mm3). The developmental stages of P. falciparum in the mosquito were studied from zygote to oocyst, by immunofluorescent method using monoclonal antibodies against the Pfs25 protein present on the surface of newly formed gametes. This method allows for assessment of the various sporogonic stages before, during and after passage of the midgut wall. Parasite densities were determined within the entire blood meal at 3 h (zygotes and macrogametes) and 24 h (ookinetes) post-infection. At 48 h after the mosquito blood meal, midguts were checked for the presence of early oocysts. For the mid-size oocysts count, classic microscopy examination was used at day 7 postinfection. The parasite efficacy was estimated by following successive losses in parasite densities between different early stages of the sporogonic cycle in A. gambiae. Thirty-seven experimental infections were realized with high gametocyte densities, ranging from 64 to 2392 gametocytes/mm3. All gametocyte carriers showed infection with round forms 100%; ookinetes were found in 91.9%. The prevalences of infections with oocysts were 48.6% at day 2 (young oocyst) and 37.8% at day 7 (mid-size oocyst). The mean densities per mosquito for each parasite stage were 12.6 round forms, 5.5 ookinetes, 1.8 young oocyst and 2 mid-size oocysts. Significant correlations were found between two consecutive parasite stages (round forms/ookinetes, ookinetes/young oocysts, young oocysts/mid-size oocysts) and between round forms and mid-size oocysts. The mean parasite density significantly decreased between round forms and ookinetes (yield Y1 = 41.6%) and between ookinetes and young oocysts (Y2 = 61.4%). By contrast, no significant decrease was observed between young oocysts and mid-size oocysts (Y3 = 91.2%). The overall yield of the early sporogonic cycle (from round form to oocyst at day 7) was equal to 25.7%, indicating that almost 3/4 of the total parasites were lost during the early step of the sporogonic cycle, from 3 h post-infection to day 7.

Stage-specific effects of host plasma factors on the early sporogony of autologous Plasmodium falciparum isolates within Anopheles gambiae.

Summary Quantitatively assessing the impact of naturally occurring transmission-blocking (TB) immunity on malaria parasite sporogonic development may provide a useful interpretation of the underlying mechanisms. Here, we compare the effects of plasma derived from 23 naturally infected gametocyte carriers (OWN) with plasma from donors without previous malaria exposure (AB) on the early sporogonic development of Plasmodium falciparum in Anopheles gambiae. Reduced parasite development efficiency was associated with mosquitoes taking a blood meal mixed with the gametocyte carriers' own plasma, whereas replacing autologous plasma with non-immune resulted in the highest level of parasite survival. Seven days after an infective blood meal, 39.1% of the gametocyte carriers' plasma tested showed TB activity as only a few macrogametocytes ingested along with immune plasma ended up as ookinetes but subsequent development was blocked in the presence of immune plasma. In other experiments (60.9%), the effective number of parasites declined dramatically from one developmental stage to the next, and resulted in an infection rate that was two-fold lower in OWN than in AB infection group. These findings are in agreement with those in other reports and go further by quantitatively examining at which transition stages TB immunity exerts its action. The transitions from macrogametocytes to gamete/zygote and from gamete/zygote to ookinete were identified as main targets. However, the net contribution of host plasma factors to these interstage parasite reductions was low (5-20%), suggesting that irrespective of the host plasma factors, mosquito factors might also lower the survival level of parasites during the early sporogonic development.

The use of anti-Pfs 25 monoclonal antibody for early determination of Plasmodium falciparum oocyst infections in Anopheles gambiae: comparison with the current technique of direct microscopic diagnosis.

Experimental infections of laboratory-reared anopheline mosquitoes were carried out with 57 Plasmodium falciparum gametocyte carriers from Cameroon. Prevalence of infected mosquitoes and oocyst intensity were determined by two independent methods. Young P. falciparum oocysts were detected on day 2 after feeding using an immunofluorescent assay, and the results were compared with direct microscopic examination of midgut oocysts on day 7 postinfection. The immunofluorescent assay was based on a FITC-labeled anti-25-kDa monoclonal antibody, while the direct microscopy was performed on midguts stained with 2% mercurochrome. Young oocysts were easily detected by their typical and bright green-fluorescing Pfs25 positive coat and their characteristic pattern of pigment granules under transmitted white light examination. The agreement between the results of the two methods was assessed using the Kappa coefficient on prevalences of positive infections and the interclass correlation coefficient on arithmetic mean oocyst load per infected midgut. The results indicated a low agreement between the two methods for the comparison of prevalences of infected mosquitoes. However, this agreement was near perfect for the comparison of mean oocyst intensities. Prevalences of positive infections and the overall number of parasites per positive gut were significantly correlated for both methods. Thus, the immunofluorescent test could be an appropriate tool for early determination of malaria infection in mosquitoes, particularly under laboratory conditions. The possible applications of this immuno-fluorescent technique are discussed.

Plasmodium falciparum malaria disease manifestations in humans and transmission to Anopheles gambiae: a field study in Western Kenya.

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.

Treatment failure of pyrimethamine-sulphadoxine and induction of Plasmodium falciparum gametocytaemia in children in western Kenya.

Sub-Saharan Africa faces increasing levels of resistance of Plasmodium falciparum parasites to the first-line drug pyrimethamine-sulphadoxine (SP). Successful treatment with SP is reported to induce gametocytes and drug resistance may further increase gametocytaemia after treatment. Treatment success, gametocyte prevalence and gametocyte density were determined in 224 asymptomatic children in western Kenya on day 7 after treatment with SP. Treatment failure (R2 or R3 resistance) was observed in 22% of the children. The relative risk to show gametocytes on day 7 after treatment in children with treatment failure was 4.1 (95% CI 1.4-11.6) times higher compared to children with a sensitive infection, after adjustment for age and trophozoite density at the start of treatment. In addition, the gametocyte density was also higher upon SP treatment failure. These findings are reason for concern, as the increased gametocyte prevalence and density after SP treatment failure may increase the spread of SP-resistant strains in the population.