ELISA absorbance cut-off method affects malaria sporozoite rate determination in wild Afrotropical Anopheles

ABSTRACT. Malaria sporozoite infection rates in a mixed species group of 244 Anopheles gambiae Giles sensu lato and 115 An. funestus Giles wild female mosquitoes were compared using three methods to determine cutoff absorbance values for positivity of a Plasmodium falciparum Welch enzyme-linked immunosorbent assay (ELISA). Positive controls were based on P. falciparum circumsporozoite protein. As negative controls, four wild male Anopheles were included on each microtitre plate; tests were repeated on four consecutive days for each plate.
Infection rates were estimated at 13.1–22.8% using the mean absorbance value of negative controls plus three standard deviations, 11.7–12.8% using double the mean and 12.5–13.6% using the fixed cut-off value of 0.20 (allowing for 20% variation in negative control absorbance values).
Observed agreement for positivity or negativity among samples tested four times was 98.6% for the 2× mean method, 97.2% for the fixed cut-off 0.20 value, but only 82.7% for the mean +3 SD method. It was concluded that the 2× mean cut-off method is most reliable for field studies. P. falciparum sporozoite rates of 12.2% in An. funestus and 11.9% in An. gambiae s. l . were thus determined on the basis of the 2× mean cut-off method.
This comparative evaluation demonstrates that vector infectivity rates can be seriously over-estimated from sporozoite ELISA tests, by as much as 87% in one case considered here, depending on the absorbance cut-off method applied for negative controls.     

Quantitation of malaria sporozoites in the salivary glands of wild Afrotropical Anopheles

The number of malaria sporozoites in the salivary glands was determined microscopically for 1137 wild, naturally infected Anopheles from western Kenya. Infective Anopheles gambiae Giles sensu lato (n = 874) contained a geometric mean (GM) of 962 sporozoites and An.funestus Giles (n = 263) contained 812. No significant differences were detected in geometric mean numbers of sporozoites between species, collection techniques or sites. Of the infective An.gambiae, 1.7% (15/874) contained more than 41,830 sporozoites, the maximum observed for An.funestus. Microscopic techniques were found to be more sensitive than enzyme-linked immunosorbent assays (ELISA) for detecting low-grade sporozoite infections in salivary glands. Salivary gland sporozoites from 83.6% of the 1137 gland infections were identified by ELISA as either Plasmodium falciparum Welch (n = 910), P.ovale Stephens (n = 7), P.malariae Grassi & Feletti (n = 3) or mixed (n = 30). The 187 gland infections which could not be identified by ELISA contained significantly fewer sporozoites (GM = 242) than those which could be identified (GM = 1200).     

Quantitation of malaria sporozoites transmitted in vitro during salivation by wild Afrotropical Anopheles

Abstract. The malaria transmission potential of wild, infective Anopheles from western Kenya was evaluated by determining the number of sporozoites transmitted in vitro by salivation when their mouthparts were inserted into capillary tubes containing either sucrose or blood. With sucrose, 86.6% of 102 infective Anopheles transmitted a geometric mean (GM) of 3.84 sporozoites (range 1–34). With blood, 23.1% of 104 infective Anopheles , tested on the day of collection, transmitted a GM of 2.30 sporozoites (range 1–117). For Anopheles held 5 days postcapture before testing with blood, 53.6% of 56 transmitted a GM of 6.04 sporozoites (range 1–420). Transmitting Anopheles contained significantly more salivary gland sporozoites than non-transmitters. No significant differences were detected between Anopheles gambiae Giles sensu lato and Anopheles funestus Giles in sporozoite transmission by individuals with sporozoites in their salivary glands.
Sporozoites were detected microscopically in the salivary duct from heads in 80.3% of 117 infective Anopheles (GM=11.2, range 1–71). Sporozoite detection in mosquito heads by ELISA was 25% less efficient than microscopic detection.
Over 98% of the infective Anopheles transmitted less than twenty-five sporozoites. Transmitted sporozoites represented only about 3% of the total sporozoites in the salivary glands suggesting that sporozoite transmission may be restricted to sporozoites in the salivary duct at the time of feeding. Results are discussed in relation to anti-sporozoite vaccine development.     

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.     

Chromosomal evidence of incipient speciation in the Afrotropical malaria mosquito Anopheles funestus

The analysis of chromosomal polymorphism of paracentric inversions in anopheline mosquitoes has often been instrumental to the discovery of sibling species complexes and intraspecific genetic heterogeneities associated with incipient speciation processes. To investigate the population structure of Anopheles funestus Giles (Diptera: Culicidae), one of the three most important vectors of human malaria in sub-Saharan Africa, a three-year survey of chromosomal polymorphism was carried out on 4,638 karyotyped females collected indoors and outdoors from two villages of central Burkina Faso. Large and temporally stable departures from Hardy-Weinberg equilibrium due to significant deficits of heterokaryotypes were found irrespective of the place of capture, and of the spatial and temporal units chosen for the analysis. Significant linkage disequilibrium was observed among inversion systems on independently assorting chromosomal arms, indicating the existence of assortative mating phenomena. Results were consistent with the existence of two chromosomal forms characterized by contrasting degrees of inversion polymorphism maintained by limitations to gene flow. This hypothesis was supported by the reestablishment of Hardy-Weinberg and linkage equilibria when individual specimens were assigned to each chromosomal form according to two different algorithms. This pattern of chromosomal variability is suggestive of an incipient speciation process in An. funestus populations from Burkina Faso.     

Evaluation of monoclonal antibodies against Plasmodium vivax sporozoites for ELISA development

Nine monoclonal antibodies (MAbs) developed against Plasmodium vivax (Grassi & Feletti) salivary gland sporozoites were evaluated for use in an enzyme-linked immunosorbent assay (ELISA), using sporozoites developed in Anopheles dirus Peyton & Harrison An. gambiae Giles and An.maculatus Theobald. Four of the antibodies were unsuitable due to the low sensitivity of the resulting assays or the requirement for high concentrations of capture antibody. An additional two MAbs were rejected because they resulted in assays with high background absorbance, attributed to self-binding. Of the three remaining MAbs, the use of Navy vivax sporozoite (NVS) 3 resulted in an ELISA with the highest sensitivity and the lowest concentration requirement for capture antibody. Assay sensitivity varied with sporozoite strain indicating possible quantitative epitope heterogeneity. None of the MAbs cross-reacted with the heterologous sporozoites tested by immunofluorescence antibody assay (IFA). The IFA activity was not an indicator of ELISA sensitivity. The use of MAb NVS 3 in a standardized ELISA method resulted in an assay 10 times more sensitive than reported previously for P. vivax sporozoites, with a detection limit of fewer than 100 sporozoites per mosquito.     

Malaria transmission potential of Anopheles mosquitoes in the Mwea-Tebere irrigation scheme, Kenya

1. Anopheles arabiensis Patton and An. funestus Giles were identified as vectors of Plasmodium falciparum malaria in the Mwea-Tebere irrigation scheme, Kenya. An. arabiensis was the only member of the An. gambiae complex identified from chromosome characteristics. Other Anopheles species found included An. pharoensis Theobald, An. rufipes Gough and An. coustani Laveran. Survival rates per gonotrophic cycle for An. arabiensis averaged 0.37 during the short rains (October-November), 0.49 during the dry season (February) and 0.78 during the long rains (May-June). Vectorial capacities were correspondingly low due to low survival rates and a high degree of zoophily. The average duration of infective life for P. falciparum was 0.2 days for both An. arabiensis and An. funestus. In contrast, entomological inoculation rates were comparatively high: 6-8 infective bites/man/month. An. pharoensis averaged 110 bites/man/night during the short rains; 1/999 (0.1%) was positive by ELISA for P. falciparum circumsporozoite antigen, but the ELISA evidence is not conclusive for vector incrimination. In correspondence with clinical observations, the transmission of P. malariae and P. ovale is unlikely due to the low vector survival rates. The observed anomaly between low vectorial capacities and high entomological inoculation rates demonstrates the importance of accurately estimating vector sporozoite rates to monitor unstable malaria transmission in irrigated areas.     

冈比亚按蚊嗅觉结合蛋白候选基因cDNA的克隆、鉴定及其表达型分析

疟蚊主要依靠嗅觉发现寄主。非洲疟蚊冈比亚按蚊Anopheles gambiae是一种嗜吸人血的疟疾传播媒介昆虫。该文作者基于其全基因组序列,采用RT-PCR和标准分子克隆技术获得2个嗅觉结合蛋白候选基因agLZ3788和agLZ9988。测序分析结果表明,它们具有嗅觉结合蛋白的标志性结构域。进一步采用半定量RT-PCR技术研究了它们的空间表达型,结果发现它们不但在雌蚊触角中表达,也在其他部位(尤其是蚊虫足部)有强的表达。这一发现说明疟蚊嗅觉结合蛋白可能具有更广的功能,也为进一步重组表达和功能研究提供了重要依据。     

Temperature-related duration of aquatic stages of the Afrotropical malaria vector mosquito Anopheles gambiae in the laboratory

Vector abundance is an important factor governing disease risk and is often employed when modelling disease transmission. The longevity of the aquatic stages of mosquitoes (Diptera: Culicidae) dictates the rate of production of adults and hence the intensity of disease transmission. We examined how temperature influences the survival of larval stages (larvae and pupae) of Anopheles gambiae Giles sensu stricto and subsequent adult production of this most efficient malaria vector. Groups of 30 mosquitoes were reared at constant temperatures (from 10 to 40 degrees C) from the first instar and observed until death or metamorphosis of the last individual. Larvae developed into adults at temperatures ranging from 16 to 34 degrees C. Larval survival was shortest (< 7 days) at 10-12 degrees C and 38-40 degrees C, and longest (> 30 days) at 14-20 degrees C. Within the temperature range at which adults were produced, larval mortality was highest at the upper range 30-32 degrees C, with death (rather than adult emergence) representing over 70% of the terminal events. The optimal survival temperatures were lower than the temperatures at which development was quickest, suggesting a critical relationship between temperature and the life cycle of the insect. These data provide fundamental information about An. gambiae s.s. adult productivity at different temperatures, which may facilitate the construction of process-based models of malaria risk in Africa and the development of early warning systems for epidemics.     

Isolation of leishmanial parasites from a wild caught Anopheles gambiae mosquito in Kenya

A total of 232 mosquitoes were collected and dissected for leishmanial parasites in the Baringo District, Kenya. Anopheles gambiae sensu lato comprised 90.9% of the sample. One female A. gambiae was found to be infected with leishmanial promastigotes. The parasites when injected into Balb C mice caused skin lesions characterized by heavy amastigote infections. The average size of the parasite was: body length, 11.7 ± 0.19 μm; width, 1.3 ± 0.04 μm; flagellum length, 15.5 ± 0.28 μm.     

Comparison of sampling anopheline mosquitoes by light-trap and human-bait collections indoors at Bagamoyo, Tanzania

Abstract. The mosquito sampling efficiency of CDC miniature light-traps, relative to night-biting collections, was evaluated indoors at two sites in coastal Tanzania. We found that the total number of anophelines captured overnight by light-traps (hung beside a bednet in use) was 1.23 times the number of anophelines captured by human-bait collections. This relationship was not affected significantly by changes in the mosquito density, order of trapping method, date of sampling, or number of household occupants. Malaria sporozoite rates were twice as high among mosquitoes captured by light-trap as compared to those captured by night-biting collection. This was attributed to the tendency of light-traps to capture a larger proportion of gravid mosquitoes, which also had high sporozoite rates. The differences in sporozoites rates according to abdominal stage indicates that unfed mosquitoes captured by light-traps may define more precisely the human-biting activity and sporozoite rates as seen by night-biting collections. Our study shows that light-traps, when used in combination with night-biting collections, can be an effective and sensitive means for measuring human-biting activity and the sporozoite rate.     

Development of an exposure-free bednet trap for sampling Afrotropical malaria vectors

An exposure-free bednet trap (the 'Mbita trap') for sampling of Afrotropical malaria vectors was developed during preliminary studies of mosquito behaviour around human-occupied bednets. Its mosquito sampling efficiency was compared to the CDC miniature light-trap and human landing catches under semi-field conditions in a screen-walled greenhouse using laboratory-reared Anopheles gambiae Giles sensu stricto (Diptera: Culicidae). When compared in a competitive manner (side by side), the Mbita trap caught 4.1+/-0.5 times as many mosquitoes as the CDC light-trap, hung beside an occupied bednet (P < 0.000 1) and 43.2+/-10% the number caught by human landing catches (P < 0.0001). The ratio of Mbita trap catches to those of the CDC light trap increased with decreasing mosquito density. Mosquito density did not affect the ratio of Mbita trap to human-landing catches. In a non-competitive comparison (each method independent of the other), the Mbita trap caught 89.7+/-10% the number of mosquitoes caught by human landing catches (P < 0.0001) and 1.2+/-0.1 times more mosquitoes than the CDC light trap (P = 0.0008). Differences in Mbita trap performance relative to the human landing catch under noncompetitive vs. competitive conditions were explained by the rate at which each method captured mosquitoes. Such bednet traps do not expose people to potentially infectious mosquito bites and operate passively all night without the need for skilled personnel. This trap is specifically designed to catch host-seeking mosquitoes only and may be an effective, sensitive, user-friendly and economic alternative to existing methods for mosquito surveillance in Africa.     

A novel mechanism of ligand binding and release in the odorant binding protein 20 from the malaria mosquito Anopheles gambiae

Anopheles gambiae mosquitoes that transmit malaria are attracted to humans by the odor molecules that emanate from skin and sweat. Odorant binding proteins (OBPs) are the first component of the olfactory apparatus to interact with odorant molecules, and so present potential targets for preventing transmission of malaria by disrupting the normal olfactory responses of the insect. AgamOBP20 is one of a limited subset of OBPs that it is preferentially expressed in female mosquitoes and its expression is regulated by blood feeding and by the day/night light cycles that correlate with blood‐feeding behavior. Analysis of AgamOBP20 in solution reveals that the apo‐protein exhibits significant conformational heterogeneity but the binding of odorant molecules results in a significant conformational change, which is accompanied by a reduction in the conformational flexibility present in the protein. Crystal structures of the free and bound states reveal a novel pathway for entrance and exit of odorant molecules into the central‐binding pocket, and that the conformational changes associated with ligand binding are a result of rigid body domain motions in α‐helices 1, 4, and 5, which act as lids to the binding pocket. These structures provide new insights into the specific residues involved in the conformational adaptation to different odorants and have important implications in the selection and development of reagents targeted at disrupting normal OBP function.     

Role of species composition in malaria transmission by the Anopheles funestus group (Diptera: Culicidae) in Ghana

Malaria remains a public health problem in Ghana, with Anopheles gambiae and Anopheles funestus as the predominant vectors. While much information exists on the species composition of An. gambiae, very little exists for An. funestus. This study was carried out to determine the species composition of An. funestus Giles populations from three ecological areas in Ghana and investigate their role in malaria transmission. Mosquitoes were collected using human landing and pyrethrum spray methods. A total of 10,254 Anopheles individuals were collected, out of which An. funestus constituted 53.6% (5,496). An. funestus sensu stricto (s.s.) and Anopheles lessoni were identified as the only members of the An. funestus group in all three ecological areas. All 62 sporozoite positive specimens that were identified as An. funestus s.s. were highly anthropophilic with a human blood index in the range of 80–96%, whereas more than 83% of the An. leesoni had fed on either bovine, goat, or sheep. Malaria transmission was higher in the Sahel savannah area than the rest of the ecological zones, with An. funestus s.s. being implicated as a vector of malaria in all ecological zones. Anopheles leesoni occurred in all the ecological areas but played no role in malaria transmission. The study established the importance of An. funestus s.s. in malaria transmission in Ghana.     

Anopheles arabiensis and An. funestus are equally important vectors of malaria in Matola coastal suburb of Maputo, southern Mozambique

Transmission characteristics of malaria were studied in Matola, a coastal suburb of Maputo, the capital City, in southern Mozambique, from November 1994 to April 1996. The local climate alternates between cool dry season (May-October) and hot rainy season (November-April) with mean annual rainfall 650-850 mm. Saltmarsh and freshwater pools provide mosquito breeding sites in Matola. Malaria prevalence reached approximately 60% among people living nearest to the main breeding sites of the vectors. Plasmodium falciparum caused 97% of malaria cases, others being P. malariae and P. ovale. Potential malaria vector mosquitoes (Diptera: Culicidae) collected at Matola during daytime indoor-resting (n = 1021) and on human bait at night (n = 5893) comprised 12% Anopheles coustani Laveran (93% biting outdoors), 46% An. funestus Giles (68% biting indoors) and 42% An. gambiae Giles sensu lato (60% biting outdoors). All 215 specimens of An. gambiae s.l. identified genetically were An. arabiensis Patton. Anopheles funestus populations remained stable throughout the year, whereas densities of the An. gambiae complex fluctuated considerably, with An. arabiensis peaking during the rainy season. No concomitant rise in malaria incidence was observed. Human landing indices of An. funestus and An. arabiensis averaged 1.8 and 3.8 per man-night, respectively. Overall Plasmodium sporozoite rates were 2.42+/-1.24% in 2181 An. funestus and 1.11+/-1.25% in 1689 An. arabiensis dissected and examined microscopically. Mean daily survival rates were 0.79 for both vector species. Estimated infective bites/person/year were 15 An. funestus and 12 An. arabiensis. Biting rates were greatest at 2100-24.00 hours for An. funestus (68% endophagic) and 21.00-03.00 hours for An. arabiensis (40% endophagic). The entomological inoculation rate (EIR) declined sharply over very short distances (50% per 90m) away from breeding-sites of the vectors. Consequently, P. falciparum prevalence among Matola residents was halved 350 m within the town. Implications for the protective effectiveness of a 'cordon sanitaire' by residual house-spraying and/or the use of insecticide-treated bednets are discussed.     

Impact of permethrin-treated bednets on malaria transmission by the Anopheles gambiae complex in The Gambia

Malaria vector mosquitoes belonging to the Anopheles gambiae complex were studied in four hamlets in The Gambia. All inhabitants were given bednets treated either with a placebo (milk) in two hamlets or with the pyrethroid insecticide permethrin (500 mg/m2) in two other hamlets. Malaria transmission occurred mainly during a few weeks of the rainy season, in September and October 1987. The indoor resting densities of mosquitoes in permethrin-treated hamlets were reduced, and we estimated over 90% reduction in biting on man by An. gambiae Giles sensu stricto in these hamlets. No mosquitoes were found under permethrin-treated bednets compared with eighty-one recovered from placebo-treated bednets. Mosquitoes exited more readily from rooms where permethrin-treated bednets were used than from rooms with placebo-treated nets. The annual mean probability that a child would receive an infective bite was estimated to be 0.09 in hamlets with insecticide-treated bednets, compared with 1.9 where placebo-treated bednets were used. Permethrin-treated bednets are therefore recommended as a means of effectively reducing the risk of exposure to malaria transmission, particularly in areas of low seasonal transmission.     

Malaria infection potential of anopheline mosquitoes sampled by light trapping indoors in coastal Tanzanian villages

Abstract. Anopheline mosquito populations were studied during 1992 in seven villages south of Bagamoyo, coastal Tanzania, prior to malaria control intervention using insecticide treated bednets. To collect mosquitoes, CDC light traps were used in ten houses per village fortnightly for 12 months. Anopheles females were identified and checked by ELISA for the presence of malaria sporozoite antigen and source of bloodmeal. An. funestus peaked in June-July after the long rains. Three members of the An. gambiae complex had different seasonality: An. arabiensis, An. gambiae and small numbers of An. merus were collected.
In most villages transmission was extremely high and perennial with the entomological inoculation rate reaching three to eleven infective bites per person per night in July and persisting at around 0.1 and 1 for most of the remainder of the year. Sporozoite infection rates within the An. gambiae complex ranged from 2% to 25%, with the peaks in January and July following the two rainy periods. An. funestus showed a similar pattern. The light traps were reliable, simple to operate, and proved to be satisfactory to study the mosquito vector population.     

Feeding and survival of the malaria vector Anopheles gambiae on plants growing in Kenya

The propensity of the malaria vector mosquito Anopheles gambiae Giles (Diptera: Culicidae) to ingest sugars from various plants, and subsequent survival rates, were assessed with laboratory-reared males and females offered eight species of plants commonly cultivated and/or growing wild in western Kenya. In cages (no-choice bioassay), mosquitoes given the opportunity to feed on castorbean (Ricinus communis L.) had the longest survival times (mean and median survival time of 6.99 +/- 0.23 and 5.67 +/- 0.17 days, respectively), comparable to mosquitoes given 6% glucose (mean and median survival time of 8.70 +/- 0.23 and 6.67 +/- 0.33 days, respectively). Survival rates of An. gambiae were low on the other plants, comparable to mosquitoes given only water. Three plants: sweet potato (Ipomoea batatas L.), wild sage (Lantana camara L.) and castorbean provided levels of sugar ingestion by both sexes of An. gambiae detectable using the cold anthrone method, showing a positive correlation between median survival and sugar consumption (Spearman rank correlation coefficient = 0.905, P < 0.0001). Equal numbers of males and females were released in an enclosed semi-field screenhouse system containing a range of local plants, but no host for blood, and allowed to feed ad libitum: 6.7 +/- 0.5% (11/64) of those recaptured were found to contain detectable fructose (all females). Common plants are clearly a viable source of nutrition for adult female An. gambiae, as well as males, and may constitute and important resource for this important malaria vector.     

Impact of permethrin-impregnated mosquito nets compared with DDT house-spraying against malaria transmission by Anopheles farauti and An.punctulatus in the Solomon Islands

Abstract. In villages of northern Guadalcanal in the Solomon Islands, where the predominant malaria vector is An.farauti No. 1 and An. puctulatus is also involved, malaria transmission rates were compared for three zones: (1) non-intervention: 438 people in seventeen villages; (2) residual DDT house-spraying two cycles per year: 644 people in thirty villages; (3) bednets impregnated with permethrin 0.5 g/m² twice per year, used by 580 people in sixteen villages. Regular DDT spraying in zones 1 and 3 had been withdrawn 18 months previously. Malariological blood smear surveys of children aged 1-9 years in August 1986 to January 1987 showed a mean-baseline malaria parasite rate of 38% (32/84). By February 19 88 , 18 months after introduction of impregnated bednets, the Plasmodium falciparum infection rate in children was lowest in the zone using impregnated bednets (21% of 29), intermediate in the untreated zone (29% of 34) and highest in the DDT zone (46% of 53), but these differences were not statistically significant. P.vivax infection rates were 9–14%. Using ELISA tests for malaria circumsporozoite antigen in the vectors, overall positivity rates were 0.7% of 49 ,902 An.farauti and 2.54% of 118 An.punctulatus, comprising 228 P.falciparum and 124 P. vivax infections. In the study zones, vector positivity rates were 0.93% of 31 ,615 An.farauti in the untreated zone; 0.32% of 16, 883 An.farauti in the DDT zone; 0.07% of 1404 An.farauti and 2.54% of 118 An.puctulatus in the impregnated bednet zone. There was no significant correlation between malaria parasite rates in the vectors and the children. Entomological inoculation rates were consistently highest in the untreated zone (1.6–2.8 infective bites/night), intermediate in the DDT zone (0.8– 1.1/night) and significantly lowest in the bednet zone (0.03-0.23/night). Geometric mean densities of P.falciparum sporozoites were also significantly higher in the DDT zone (50% > 10,000 sporozoites/mosquito compared with 20% in untreated zone). The highest individual infection density was an estimated 52,080 sporozoites of P.falciparum in a specimen of An.punctulatus from the bednet zone. P.vivax sporozoite densities were not significantly different between zones, and both species of vector had similar mean sporozoite loads for both species of malaria. It is concluded that permethrin-impregnated mosquito nets exerted significantly more impact on vector infectivity and the inoculation rate than resulted from DDT spraying. Even so, the inoculation rate for people in the bednet zone remained at one infective bite every 4–32 days, an insufficient reduction to control malaria without additional countermeasures. Ineffectiveness of house-spraying and the limited impact of impregnated bednets are attributed to exophily and other behavioural aspects of An. farauti.