Studies on the resting behaviour and host choice of Anopheles gambiae and An. arabiensis from Muleba, Tanzania.

The relative efficacy of a mechanical (Prokopack) collection method vs. manual aspiration in the collection of resting mosquitoes was evaluated in northern Tanzania before and after an intervention using indoor residual spraying and longlasting insecticide-treated nets. In smoke-free houses mosquitoes were collected from the roof and walls, but in smoky houses mosquitoes were found predominantly on the walls. Anopheles gambiae (Diptera: Culicidae) constituted 97.7% of the 312 An. gambiae complex specimens identified before but only 19.3% of the 183 identified after the intervention. A single sampling with the Prokopack collected a third of the available insects. Anopheles gambiae completed its gonotrophic development indoors, whereas Anopheles arabiensis did so outdoors. In both species gonotrophic development took 2 days. Most unfed resting An. arabiensis collected outdoors were virgins, whereas the majority of engorged insects were parous (with well-contracted sacs). Daily survival was estimated to be 80.0%. Only 9.4% of the engorged An. arabiensis collected outdoors and 47.1% of those collected indoors had fed on humans. Using the Prokopack sampler is more efficient than manual methods for the collection of resting mosquitoes. Malaria transmission may have been affected by a change in vector composition resulting from a change in feeding, rather than reduced survival. Monitoring the proportions of members of the An. gambiae complex may provide signals of an impending breakdown in control.

Effects of the spatial repellent metofluthrin on landing rates of outdoor biting anophelines in Cambodia, Southeast Asia.

The emergence of artemisinin-resistant malaria in Southeast Asia is a major problem. The fact that many people become infected with malaria when they are outside has prompted the development of 'spatial' rather than topical repellents. The respective effects of one or four slow-release emanators of metofluthrin, a pyrethroid, were tested in Pailin, Pursat and Koh Kong, Cambodia. Numbers of mosquitoes counted in outdoor landing catches when one or four emanators were suspended close to the collector were compared with control collections. In Pailin, the effects of emanators on catches in Furvela tent traps and Centers for Disease Control (CDC) light traps suspended underneath houses were also investigated. Rate ratios were used to determine differences. A total of 29 255 mosquitoes were collected over 2934 h of landing collections, 87 nights of tent trapping and 81 nights of light trap capture. In Pailin, landing rates were reduced by 48% by a single emanator and by 67% by four emanators (P < 0.001). Similar reductions were observed in the number of mosquitoes collected in tent traps and the number of anophelines only collected in light traps. Results were similar in Pursat, but, for unknown reasons, those in Koh Kong showed no difference between control and metofluthrin collections (P > 0.05). These findings suggest that although the product can produce a significant effect, it requires further improvement.

Analysis of the sporozoite ELISA for estimating infection rates in Mozambican anophelines.

Comparisons were undertaken to investigate cost-effective methods of implementing the enzyme-linked immunosorbent assay (ELISA) for sporozoite determination in anophelines when large numbers require processing. Comparisons between ELISA plate reader and visual assessments were performed with Anopheles funestus and Anopheles gambiae s.l. (Diptera: Culicidae), as were comparisons between whole-body mosquito samples, heads and thoraces, and abdomens alone. Rates obtained from pools of five or 10 mosquitoes were compared with those for individual mosquitoes, as were rates obtained using different sampling methods. A total of 41 792 An. funestus and 9431 An. gambiae s.l. collected in light traps, and 22 323 An. funestus and 6860 An. gambiae s.l. from exit collections were analysed. Visual assessments gave results similar to those of machine readings. Sporozoite rates were similar in both species, as were rates by collection method. The use of whole mosquitoes increased estimates of infection rate by 0.6%. Pool size did not affect infection rates of An. gambiae s.l., but rates were higher among individually tested An. funestus than among those tested in pools. For large-scale surveys, the use of whole mosquitoes in pools of 10 mosquitoes, with correction for overestimation, and the noting of results according to a simple three-stage visual assessment of positivity is the most cost-effective approach and is sufficient to obtain reliable data for comparative purposes.

Some like it cool: the effect of ambient temperature on the size of Anopheles funestus from southern Mozambique.

The wing lengths of 3,553 unfed, recently emerged, and 13,256 gravid female Anopheles funestus Giles from exit collections, undertaken between March 2004 and May 2005, were measured. Only in the warmest months were mean wing lengths of unfed females significantly smaller than gravid females. Mean wing lengths of unfed, females varied from 2.26 mm (Bootstrap 95% CI [2.25-2.27]) in January when mean air temperatures were 24.8 degrees C to 2.70 mm (Bootstrap 95% CI [2.68-2.72]) in July when mean temperatures were 8.4 degrees C cooler. Mean wing lengths increased by 0.05 mm for each degree decrease in air temperature. Temperature explained 60% of the variation in wing length.

Feeding frequency and survival of Anopheles gambiae in a rice-growing area in Ghana.

Mortality rates, determined by dissection, of predominantly M form female Anopheles gambiae (Diptera: Culicidae) were estimated. Mosquitoes were collected in tent traps and light traps in an irrigation project village in Ghana in June and July 2010, when much of the area was flooded. Both M and S form larvae were collected from rice fields (74 of 80 specimens were M form). Adults were collected in equal proportions from the two traps (90 of 107 specimens from the light trap and 106 of 116 specimens from the tent trap were M form). During the study, collection numbers rose from 105 to 972 per night. A total of 1787 of the 15 431 An. gambiae collected were dissected. Of these, 953 (53%) were found to have taken their first bloodmeal, either as virgins or following mating. The age profiles of mosquitoes collected alive and dead, respectively, were similar. Eighteen of 2933 (0.61 ± 0.49%) specimens were found to be positive for sporozoites in an enzyme-linked immunosorbent assay (ELISA). Lagged cross correlations among the different age groups implied that the mosquitoes fed on days 2 and 4 following emergence prior to oviposition and every 2.65 ± 0.17 days thereafter. The best model to describe the observed population patterns implied a daily mortality of 84%. The results are discussed in relation to possible mosquito control measures for the village.

Does moonlight influence the biting behaviour of Anopheles funestus?

The possible effect of moonlight on the biting behaviour of mosquitoes in southern Mozambique, in particular that of Anopheles funestus (Diptera: Culicidae), a primary vector of malaria, was investigated by comparing catches indoors and outdoors using CDC light traps and 'Furvela' tent traps, respectively, for 35 consecutive nights, from 9 September to 15 October 2008. Collections were separated into three 4-hourly samples each night. A total of 17 591 mosquitoes belonging to nine species were collected, 6747 in light traps and 10 844 in tent traps. Anopheles funestus (n = 7634) and Mansonia africana (n = 4859) were the most abundant species. Fluctuations in temperature and humidity were the two environmental variables associated with changes in relative abundance of mosquitoes. Most An. funestus were collected indoors, with the majority collected in the first 4 h of the night. This was most evident on nights when moonlight was present in the early part of the night. A total of 3488 An. funestus were dissected for gonotrophic age determination. Parous rates did not change with lunar phase, but estimated oviposition cycle length was significantly shorter on nights when moonlight was present at the time of oviposition. Moonlight at dusk did not, however, affect the proportion of newly emerged insects with mating plugs collected. Outdoor transmission of malaria, especially on moonlit nights, remains a problem for control programmes.

Studies on the behaviour of peridomestic and endophagic M form Anopheles gambiae from a rice growing area of Ghana.

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.

Cost-sharing strategies combining targeted public subsidies with private-sector delivery achieve high bednet coverage and reduced malaria transmission in Kilombero Valley, southern Tanzania.

BACKGROUND: Cost-sharing schemes incorporating modest targeted subsidies have promoted insecticide-treated nets (ITNs) for malaria prevention in the Kilombero Valley, southern Tanzania, since 1996. Here we evaluate resulting changes in bednet coverage and malaria transmission. METHODS: Bednets were sold through local agents at fixed prices representing a 34% subsidy relative to full delivery cost. A further targeted subsidy of 15% was provided to vulnerable groups through discount vouchers delivered through antenatal clinics and regular immunizations. Continuous entomological surveys (2,376 trap nights) were conducted from October 2001 to September 2003 in 25 randomly-selected population clusters of a demographic surveillance system which monitored net coverage. RESULTS: Mean net usage of 75% (11,982/16,086) across all age groups was achieved but now-obsolete technologies available at the time resulted in low insecticide treatment rates. Malaria transmission remained intense but was substantially reduced: Compared with an exceptionally high historical mean EIR of 1481, even non-users of nets were protected (EIR [fold reduction] = 349 infectious bites per person per year [x4]), while the average resident (244 [x6]), users of typical nets (210 [x7]) and users of insecticidal nets (105 [x14]) enjoyed increasing benefits. CONCLUSION: Despite low net treatment levels, community-level protection was equivalent to the personal protection of an ITN. Greater gains for net users and non-users are predicted if more expensive long-lasting ITN technologies can be similarly promoted with correspondingly augmented subsidies. Cost sharing strategies represent an important option for national programmes lacking adequate financing to fully subsidize comprehensive ITN coverage.

Dry season refugia of malaria-transmitting mosquitoes in a dry savannah zone of east Africa.

Dry season survival of Anopheles funestus, Anopheles gambiae and Anopheles arabiensis in the Kilombero valley a dry savannah zone of east Africa, was investigated with over 400 collections from 23 areas, covering 300 sq km of the valley. Anopheles gambiae was found only in association with humans, in forested areas of high annual rainfall, while An. funestus occurred at high densities at the valley edge where large non-moving bodies of water remained. A large population of An. arabiensis was present along the river system throughout the middle of the valley, and mosquitoes probably derived from this population were occasionally caught in villages bordering the valley. No evidence was obtained of aestivation in any mosquito species. Anopheles gambiae was the most long lived, 6.3% compared to 2.0% of the An. arabiensis and 4% of the An. funestus surviving for four or more gonotrophic cycles, the approximate duration of the extrinsic cycle of most malaria parasites. Oocysts of malaria parasites were found in 5.4% of An. funestus and 2.3% of An. arabiensis from villages. Oocyst rates in An. funestus differed significantly between areas but not between houses within areas. Anopheles funestus is the most important dry season malaria vector in the valley, and remains in foci closely associated with groups of houses. All three species survive at high densities but as otherwise hidden refugia populations.

Incidence of Plasmodium falciparum infection in infants in relation to exposure to sporozoite-infected anophelines.

The relationship of the incidence of Plasmodium falciparum infection to entomologic inoculation rates (EIRs) was studied in 163 children less than one year of age in a Tanzanian village to determine likely effects of transmission-reducing interventions on infection incidence. A total of 66,727 Anopheles gambiae s.l. and 17,620 An. funestus mosquitoes were caught in 1,056 light trap collections from 139 houses over a period of more than two years. Time period-specific human biting rates were estimated for 11 village neighborhoods. Sporozoites were detected by ELISA in 4.4% of the An. funestus and 2.5% of the An. gambiae s.l. Eight hundred seventeen pairs of blood slides with approximately two-week intervals between slides were used to estimate incidence of parasitemia by fitting reversible catalytic models to parasite positivity data. Estimated EIRs during the four weeks preceding each intersurvey interval averaged 1.6 (SD = 2.1) per adult per night. Parasites were present at the end of 31% of the 443 intervals that commenced with a parasite-negative slide. Attack rates were comparable with those in western Kenya, and the proportion of bites resulting in human infections was strongly dependent on mosquito density. Incidence of infection increased with the EIR up to approximately one bite from a sporozoite-carrying mosquito per adult per night. However, higher levels of transmission observed locally in the wet season did not result in a correspondingly higher incidence. These data suggest that transmission-reducing measures cannot be expected to reduce incidence of infection at the highest levels of EIR.

Relationships of malaria morbidity with exposure to Plasmodium falciparum in young children in a highly endemic area.

To study incidence of clinical Plasmodium falciparum malaria in relation to exposure to parasites, attendance of children less than eighteen months old at a village dispensary in a highly endemic area of Tanzania was recorded. Entomologic inoculation rates (EIRs), estimated as a function of time period and place of residence, exceeded one sporozoite positive bite per adult per night in some village neighborhoods during the wet season. Incidence of clinical P. falciparum malaria, defined either as fever with parasitemia or as fever with hyperparasitemia, increased with the EIR over the whole range of exposures. Each 10-fold increase in the EIR corresponded to a 1.6-fold increase in incidence of fever plus parasitemia (95% confidence interval = 1.4-2.0). Therefore reduction of human-vector contacts will probably reduce morbidity incidence even at very high exposures. Incidence showed little relationship to estimated cumulative numbers of inoculations since birth, but decreased steeply with estimated cumulative time infected with trophozoites. This suggests that clinical immunity depends mainly on the extent of exposure to blood-stage antigens, not on the diversity of inocula seen, and thus temporary reductions in human-vector contacts are unlikely to result in subsequent increases in morbidity.

Observations on the swarming and mating behaviour of Anopheles funestus from southern Mozambique.

BACKGROUND: Control of malaria by the release of genetically modified mosquitoes refractory to transmission is now becoming a possibility. In many areas of Africa, Anopheles gambiae is found together with an equally important vector, An. funestus. Given their sympatry and the likelihood of a similar mating period some aspects of the mating behaviour of An. gambiae s.l. and An. funestus are likely to differ. We therefore attempted to characterise the swarming behaviour of An. funestus and to determine if any aspects of the observed behaviour differed from that recorded for the M form of An. gambiae from São Tomé. METHODS: In March - May 2002 the swarming, mating, house exiting and resting behaviour of Anopheles funestus was studied by direct observation in Mozambique. Swarming males and insects in copula were collected by sweep net. Wing lengths of males collected resting, exiting houses, swarming and mating were measured and the wingbeat frequency distribution of individual insects, in free flight confined inside netting covered paper cups, was also determined. RESULTS: Mono-specific swarms occurred at sunset in relatively open areas close to houses used for resting. Mating pairs were seen 11 +/- 3.7 min after the start of swarming. The number of total pairs observed being inversely proportional to the time difference between the start of swarming and the first pairing. The great majority of females mated before feeding. Male or female size did not appear to affect mating success or other behaviours. During the study, ambient temperatures decreased and female, but not male, wing size increased. At 516 Hz, the flight tone of female An. funestus was similar to the 497 Hz of the local An. gambiae. Males dispersed if light or dark artificial horizontal markers were placed underneath naturally occurring swarms. CONCLUSION: Differential response to markers would be sufficient for swarming in An. funestus and An. gambiae s.l. to occur in distinct sites.

Estimation of the infectious reservoir of Plasmodium falciparum in natural vector populations based on oocyst size.

A method for determining the infectious reservoir of malaria (K) and vector survival rate (P) by measuring oocyst size and discriminating between the most recent and other infections is described. In the laboratory the mean diameter of 3 d oocysts in Anopheles gambiae, kept at 26 degrees C, was 11.5 microns and the mean diameter at day 5 was 24.5 microns. Oocyst sizes in wild caught mosquitoes from southern Tanzania, that had fed on the occupants of bed nets with holes in the sides, were more variable. 2060 A. gambiae s.l. and 1982 A. funestus were examined for oocysts 3 d after feeding; 796 and 654 oocysts from the 153 and 170 infected females, respectively, were measured. Because of misclassification errors, the use of a simple cut-off model, in which all oocysts less than 17.5 microns in diameter were considered to have arisen from the most recent feed, was thought to overestimate K and underestimate P. A statistical model which allows for overlap in the oocyst size distributions is described. Estimates of the infectious reservoir derived from this model were 2.8% for A. gambiae s.l. and 4.2% for A. funestus, and the estimated survival rates per gonotrophic cycle were 65.5% and 52.9%, respectively. The utility of measuring oocyst size in naturally infected mosquitoes is discussed.

Mapping the densities of malaria vectors within a single village.

Small scale spatial variation and temporal heterogeneity in mosquito densities can have important consequences for disease transmission, but the extreme variation which is observed in populations of malaria vectors makes it difficult to obtain good predictions of densities for short time periods over limited areas. We have applied Bayesian techniques derived for use in cancer epidemiology in order to map densities of Anopheles gambiae s.l. and A. funestus in a Tanzanian village where there is intense transmission of Plasmodium falciparum malaria. Estimates derived in this way should prove useful in vector population biology and in improving estimates of exposure-response relationships of the human host to malaria. The same methods can be applied in other fields of animal ecology.

A field trial with Lambda-cyhalothrin (ICON) for the intradomiciliary control of malaria transmitted by Anopheles darlingi root in Rondonia, Brazil.

A two stage field trial comparing the effects of Lambdacyhalothrin (ICON) and DDT when used as residual sprays on the inside surfaces of houses, was conducted in the Machadinho and Jaru areas of Rodonia, Brazil, in 1987 and 1988. In 1987 houses along two 16 km contiguous stretches of a main and a side road were sprayed and the effects on malaria vectors monitored for the succeeding year. In the second stage approximately 55,000 houses in both districts were sprayed with ICON and the effect on malaria incidence measured by passive case detection. Of the eleven species of Anopheles caught in indoor and peridomiciliary collections A. darlingi was the commonest and is recognised as the most important vector in Brazil. ICON at either of two concentrations in bioassays killed more mosquitoes than DDT at each test from seven to twelve months after spraying. A rise in the number of A. darlingi collected eight months after spraying with DDT was not so marked in the ICON areas. Side effects of the insecticide were limited. The number of reported Plasmodium falciparum cases in the second phase declined 76% in Machadinho after spraying with ICON to 2851 cases. In Jaru there was a 28% reduction. The observed efficacy of the insecticide, its ready acceptance by the local populace, and its cost effectiveness make it a more useful insecticide for anti-malaria campaigns than DDT.

Prospective risk of morbidity in relation to multiplicity of infection with Plasmodium falciparum in São Tomé.

The prospective risk of acute morbidity was analysed in relation to multiplicity of Plasmodium falciparum infection in 491 individuals in a peri-urban community in São Tomé. In an initial cross-sectional survey, 40.5% of individuals were recorded by microscopy as infected with P. falciparum, and by PCR 60.5%, with the maximum prevalence in children aged 5-10 years. PCR-RFLP typing of the msp-2 gene of P. falciparum found a mean of 2.4 parasite genotypes per infected person, with little age dependence in this multiplicity and a total of 43 different msp-2 alleles identified. None of these were unique for São Tomé. Study participants were encouraged to report to a project worker whenever they suffered a febrile illness. During the 3 months following the parasitological survey the recorded incidence rates decreased with increasing baseline msp-2 multiplicity, both for P. falciparum-positive episodes and for fever without parasitaemia. While this is consistent with suggestions that multiple P. falciparum infections may protect against super-infecting parasites, confounding by patterns of health service usage is an alternative explanation. The incidence of clinical malaria episodes was only a little higher in children than in adults. This weak age-dependence in clinical immunity might be a consequence of a cohort effect resulting from resurgence of the disease after the breakdown of malaria control programs in the 1980s.

The impact of indoor residual spraying with malathion on malaria in refugee camps in eastern Sudan.

An exploratory trial of the efficacy of indoor spraying with malathion on morbidity and mortality in refugee camps in eastern Sudan was conducted during the rainy season of 1997. The interior walls of houses from a randomly selected group of five camps were sprayed with malathion in mid-September and morbidity and mortality rates in the camps for the months October to December compared with rates in five controls. Pyrethrum spray collection and human landing catches were performed in two collection rounds. An exophagic but endophilic population of Anopheles arabiensis was the most common mosquito collected. The mean human blood index of 242 mosquitoes from eight camps was 0.51. Only two of 1040 mosquitoes examined harboured sporozoites. Blood samples of 83 putative malaria patients were examined for parasites by PCR. Mortality rates in the 3 months following spraying were significantly lower in sprayed camps although differences in clinical malaria incidence between sprayed and non-sprayed camps were not significant.

Malaria in São Tomé and Príncipe: parasite prevalences and vector densities.

A cross-sectional survey was carried out in 16 localities on the island of São Tomé and three on the island of Príncipe, at the end of the rainy season of 1997, to determine malaria prevalence and vector densities. Blood samples from 664 inhabitants of all ages were examined by optical microscopy (OM) and PCR. Mosquito collections were made by outdoor landing captures from 21:00-23:00 h. Great differences were found between OM and PCR readings. OM had a sensitivity of 66%, a specificity of 79% and failed to reveal any mixed-infections. Overall prevalence, determined by PCR, was higher in São Tomé (53%) than in Príncipe (35%). It was highest in children below 16 years-old. All four human Plasmodium species occurred in São Tomé but P. ovale was not detected in Príncipe. The human population was largely asymptomatic. Bednet users had lower prevalence than did non-users. The FOREST form of Anopheles gambiae s.s., identified by PCR and cytogenetics, was the only vector on the islands. The sporozoite rate in São Tomé, assessed by ELISA, was 0.5%. Parasite prevalence and vector densities were positively correlated in São Tomé, where malaria transmission must occur predominantly in the more populated coastal areas.

Absence of seasonal variation in malaria parasitaemia in an area of intense seasonal transmission.

Parasitological surveys carried out in two villages of the Kilombero district of Tanzania indicated a very high prevalence of Plasmodium falciparum parasitaemia throughout the year (all ages mean prevalence = 69.2%) and a low, unstable prevalence of P. malariae (all ages mean prevalence = 4.5%). Fevers (temperature > or = 37.5 degrees C) in both children and adults showed irregular changes in prevalence over time, but there was no seasonal pattern. Neither was there seasonal variation in either P. falciparum parasite prevalence or parasite densities. This was despite marked seasonality in vectors caught in CDC light-traps and in estimated sporozoite inoculations determined by ELISA. The estimated mean annual inoculation rate was extremely high, over 300 infectious bites per person per year, the main vectors being members of the A. gambiae complex and Anopheles funestus. There was considerable variation between houses but even in houses with relatively low mosquito numbers the inoculation rate was sufficient to maintain a maximal P. falciparum prevalence. Heterogeneities in exposure cannot explain why the parasite prevalence is not always 100%. In areas of such high transmission, parasitaemias are likely to be determined mainly by the interaction of schizogony and anti-blood stage immunity, since parasites arising from new inoculations generally comprise only a small proportion of the total in the circulation. In any one individual, this will lead to periodic fluctuations in levels of parasitaemia. These are unlikely to show a close relationship to either seasonal variation in inoculations or to differences between households in the local inoculation rate.

Polymerase chain reaction used to describe larval habitat use by Anopheles gambiae complex (Diptera: Culicidae) in the environs of Ifakara, Tanzania.

Larvae of the Anopheles gambiae complex were collected in and around the town of Ifakara, southern Tanzania during the wet season of 1994 and identified to species by polymerase chain reaction. All but 1 surface pool contained mixed populations of An. gambiae and An. arabiensis larvae. The 2 species varied among locations rather than types of water. An. arabiensis predominated in pools close to cattle. The numbers of identified early instars of both species were similar, but more An. gambiae 4th instars were identified, perhaps indicating that An. gambiae were able to survive heavy rainfall better than A. arabiensis.