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.

Studies on the bionomics of male Anopheles gambiae Giles and male Anopheles funestus Giles from southern Mozambique.

Little is known about the fitness of wild male mosquitoes, the females of which are vectors of malaria. The problem of studying male biology has been exacerbated by difficulties associated with catching them. In southern Mozambique, however, almost the entire adult population of An. funestus and An. gambiae s.l. rest inside houses. They leave in a dusk exodus, which makes them easy to collect. In 8,348 exit collections from a village from 2003 to 2009, 567,195 male An. funestus and 34,591 male An. gambiae s.l. were collected. During the study, numbers of An. funestus increased but numbers of An. gambiae s.l. declined to the point of extinction. Overall numbers of An. gambiae s.l. were positively correlated with temperature, whilst the relationship between temperature and numbers of An. funestus changed from an initially positive one in the first three years of the study to a negative one in the last three years. Marked males were recaptured up to 300 m from the release site, with most recaptures occurring within 150 m. Estimated mean daily survival of male An. funestus was 0.86 (95% C.I. 0.869-0.850). For the years 2003-2007, estimated mean daily survival of male An gambiae s.l. was 0.660 (95% C.I. 0.682-0.638). For either species, there was no relationship between mean weekly temperature and estimated daily survival. These results imply that males of An. funestus live as long as females but have a relatively short flight range. They are discussed in the light of possible release strategies of sterile or genetically modified mosquitoes.

Do developing malaria parasites manipulate their mosquito host? Evidence from infected Anopheles funestus (Giles) from Mozambique.

Mosquito survival is linked to the activities performed in each oviposition cycle, whilst development of malaria parasites in them is largely temperature dependent. Extending the oviposition cycle of the mosquito, even as a side effect of normal development of the parasite, may enhance malaria transmission. A study was therefore undertaken to compare the time spent before returning to feed among infected and uninfected host seeking Anopheles funestus from a village in southern Mozambique. The stomachs of 2073 parous mosquitoes (1289 with sacs, indicative of a rapid return to host-seeking and 784 without sacs indicating a delay in host-seeking) from four extended sampling periods were examined for oocysts. From three of these, and overall, significantly more mosquitoes without sacs had oocysts than mosquitoes with sacs (Fishers Exact Test P=0.0051). This is the first time that possible effects on oviposition cycle length among infected vectors of human malaria have been demonstrated.

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.

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.

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.

Mating does not affect the biting behaviour of Anopheles gambiae from the islands of São Tomé and Príncipe, West Africa.

To determine if mating or gonotrophic age influenced the biting behaviour of Anopheles gambiae s.s., a series of all-night landing captures was performed on the islands of São Tomé and Príncipe in the Gulf of Guinea. On São Tomé 49% and on Príncipe 56% of the newly emerged An. gambiae taking their first bloodmeal were virgins. On each island, with the exception of recently mated insects on Príncipe, all age-groups had similar biting cycles. The biting cycle on Príncipe resembled that observed on continental Africa, with a peak in the latter part of the night. Peak biting on São Tomé, however, occurred before midnight. Estimated daily survival rates were 0.77 and 0.29 for São Tomé and Príncipe, respectively. Mating does not affect the biting behaviour of An. gambiae on these islands.

An island within an island: genetic differentiation of Anopheles gambiae in São Tomé, West Africa, and its relevance to malaria vector control.

Islands are choice settings for experimental studies of vector control strategies based on transgenic insects. Before considering this approach, knowledge of the population structure of the vector is essential. Genetic variation at 12 microsatellite loci was therefore studied in samples of the malaria vector Anopheles gambiae s.s., collected from six localities of São Tomé island (West Africa). The objectives were (i) to assess the demographic stability and effective population size of A. gambiae from these sites, (ii) to determine population differentiation and (iii) to relate the observed patterns of population structure with geographic, ecological and historical aspects of the vector on the island. Significant population differentiation, revealed by FST and RST statistics, was found between the southernmost site, Porto Alegre, and northern localities. The observed patterns of population substructure are probably a result of restrictions to gene flow in the less inhabited, more densely forested and mountainous south. In all localities surveyed, A. gambiae appeared to be experiencing a demographic expansion, consistent with a relatively recent (ca. 500 years) founder effect. The results are discussed with respect to current and future prospects of malaria vector control.

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.

Genetic structure of Anopheles gambiae (Diptera: Culicidae) in São Tomé and Príncipe (West Africa): implications for malaria control.

The impact of a vector eradication programme, conducted in the 1980s, on Anopheles gambiae populations from the islands of São Tomé and Príncipe, was evaluated by microsatellite DNA analysis. Significant genetic differentiation was observed within and between the two islands and between the islands and a population from Gabon, suggesting a degree of isolation between them. Large estimates of long-term N(e) suggested that the control programme did not affect the effective population size of the vector. Heterozygosity tests were also not consistent with a recent bottleneck.

Male size does not affect mating success (of Anopheles gambiae in São Tomé).

For malaria control, the utility of transgenic vector Anopheles mosquitoes (Diptera: Culicidae) refractory to Plasmodium transmission, will depend on their interbreeding with the wild vector population. In many species, larger males are more successful in obtaining mates. In São Tomé island, we determined that size did not affect mating success of male Anopheles gambiae Giles sensu stricto, the main malaria vector in tropical Africa. Also we showed that larval intraspecific competition is probably insignificant in this population of An. gambiae. Thus, the potential success of transgenic An. gambiae is unlikely to be affected by size selection under field conditions.

The swarming and mating behaviour of Anopheles gambiae s.s. (Diptera: Culicidae) from São Tomé Island.

The swarming and mating behaviours of the forest cytoform of Anopheles gambiae s.s. were investigated on 194 evenings and 14 mornings between April 1997 and November 1999 in a peri-urban area of the island of São Tomé, West Africa. Males swarmed 2-3 m above markers of horizontal contrast such as those formed between grass areas and footpaths, or bushes. Evening swarms started 2 min before sunset in sheltered sites and a minute or two later in exposed ones. It took approximately 5 mins from the arrival of the first male for the swarm to reach estimated maximum numbers. Mating pairs were first seen 7 min after the start of swarming. Maximum numbers of pairs in copula were observed 8 min later. Up to 270 pairings were seen in the 20 min period before darkness. Removal of males had no effect on the number of females arriving at the swarm. Males were attracted to sounds that approximated the female flight tone but not to filter paper samples of squashed virgin females swung through the swarm. A much-reduced amount of swarming and a single mating were recorded at dawn. The same locations for swarming were used at different times and at different heights by ants, Culicoides sp. and Culex quinquefasciatus.

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.

Dogs as a favored host choice of Anopheles gambiae sensu stricto (Diptera: Culicidae) of São Tomé West Africa.

The host source and human blood index (HBI) of an exophilic population of the "forest" cytoform of Anopheles gambiae Giles sensu stricto, from a peri-urban area of the island of São Tomé were assessed. Blood meals of 434 An. gambiae females from all-night indoor light-trap collections, 193 from indoor and 422 from outdoor resting collections, were determined by ELISA. Significant differences were found in the HBI estimates from insects collected indoors (0.93) and outdoors (0.27). Blood-fed insects collected resting outdoors provided the most representative sample for host determination. Dogs were the predominant hosts, followed by humans and pigs. Of all human feeds, it was estimated that 81.5% were taken inside houses. The low HBI of 0.27 for the An. gambiae population explains the low sporozoite rate and the meso-endemicity of malaria in the island.

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.