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.     

Anatomical dissemination of circumsporozoite protein in wild Afrotropical Anopheles affects malaria sporozoite rate determination by ELISA

The head, thorax, wings, legs and abdomen of 320 wild-caught Anopheles gambiae Giles sensu lato and 115 An.funestus Giles were tested by an enzyme-linked immunosorbent assay (ELISA) for Plasmodium falciparum Welch to determine how anatomical dissemination of circumsporozoite (CS) protein could affect the estimation of malaria sporozoite rates by ELISA. Of fifty-three Anopheles with CS protein detected in any body part, positive reactions were observed for 58.5% of heads, 67.0% of thoraces, 39.6% of wings, 52.8% of legs and 60.4% of abdomens. Mean absorbance values (range 0-2.00) were highest in thorax samples (1.17), followed by heads (0.80), abdomens (0.67), wings (0.48) and legs (0.46). Circumsporozoite protein was present in the wings or legs, but not in the head or thorax, in 11.3% (6/53) of the infected Anopheles. The ELISA infection rate of 12.8% (41/320) for An.gambiae would have increased to 14.7% (47/320) by inclusion of six mosquitoes with CS protein in wings or legs alone. The slight overestimation of the proportion of infective mosquitoes due to disseminated CS protein would have little effect on estimates of relative infection rates by ELISA for field-collected Anopheles, with abdomens removed prior to testing. However, the widespread dissemination of CS protein indicates that sporozoite load estimates by ELISA, for mosquitoes without abdomens, may not provide adequate measurements of the numbers of sporozoites in the salivary glands. Operationally, careful processing of mosquito samples for the determination of infectivity rates by ELISA is necessary to prevent the mixing of wings or legs among samples representing individual mosquitoes.     

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.     

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.     

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.     

ELISA study of oocyst-sporozoite transition in malaria vectors

Intrinsic vector characteristics and environmental factors affect the sporogonic development of P. falciparum in Anopheles mosquitoes. We tested for the presence of the circumsporozoite protein, as a marker of the oocyst to sporozoite transition in naturally infected Anopheles gambiae s.l. and Anopheles funestus. Malaria vectors were collected in a village in the Sahel of Niger during the rainy and dry seasons. ELISA-CSP was carried out on abdomen and head/thorax portions from more than 2000 samples. No significant difference was found in the overall rates of infection of An. gambiae s.l. (4.13%) and An. funestus (3.58%). Given the differences in duration of the two parasite stages, P. falciparum CSP antigen prevalence was nearly as high in the abdomen as in the head/thorax, and did not differ significantly between An. gambiae s.l. and An. funestus. These preliminary results suggest that development from oocysts to salivary gland sporozoites is similar in the two vectors. However, these developmental indices varied as a function of the season in which samples were collected, particularly for An. gambiae s.l. This simple method may be useful for field studies assessing the effect of environmental and genetic factors on parasite survival.     

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.     

Anopheles gambiae eicosanoids modulate Plasmodium berghei survival from oocyst to salivary gland invasion

Eicosanoids affect the immunity of several pathogen/insect models, but their role on the Anopheles gambiae response to Plasmodium is still unknown. Plasmodium berghei-infected mosquitoes were injected with an eicosanoid biosynthesis inhibitor, indomethacin (IN), or a substrate, arachidonic acid (AA), at day 7 or day 12 post-infection (p.i.). Salivary gland invasion was evaluated by sporozoite counts at day 21 p.i. IN promoted infection upon sporozoite release from oocysts, but inhibited infection when sporozoites were still maturing within the oocysts, as observed by a reduction in the number of sporozoites reaching the salivary glands. AA treatment had the opposite effect. We show for the first time that An. gambiae can modulate parasite survival through eicosanoids by exerting an antagonistic or agonistic effect on the parasite, depending on its stage of development.     

Efficiency of salivary gland invasion by malaria sporozoites is controlled by rapid sporozoite destruction in the mosquito haemocoel

For successful transmission to the vertebrate host, malaria sporozoites must migrate from the mosquito midgut to the salivary glands. Here, using purified sporozoites inoculated into the mosquito haemocoel, we show that salivary gland invasion is inefficient and that sporozoites have a narrow window of opportunity for salivary gland invasion. Only 19% of sporozoites invade the salivary glands, all invasion occurs within 8h at a rate of approximately 200 sporozoites per hour, and sporozoites that fail to invade within this time rapidly die and are degraded. Then, using natural release of sporozoites from oocysts, we show that haemolymph flow through the dorsal vessel facilitates proper invasion. Most mosquitoes had low steady-state numbers of circulating sporozoites, which is remarkable given the thousands of sporozoites released per oocyst, and suggests that sporozoite degradation is a rapid immune process most efficient in regions of high haemolymph flow. Only 2% of Anopheles gambiae haemocytes phagocytized Plasmodium berghei sporozoites, a rate insufficient to explain the extent of sporozoite clearance. Greater than 95% of haemocytes phagocytized Escherichia coli or latex particles, indicating that their failure to sequester large numbers of sporozoites is not due to an inability to engage in phagocytosis. These results reveal the operation of an efficient sporozoite-killing and degradation machinery within the mosquito haemocoel, which drastically limits the numbers of infective sporozoites in the mosquito salivary glands.     

Efficacy of permethrin-impregnated curtains for malaria vector control

Preliminary results obtained by the use of permethrin-impregnated curtains against the Afrotropical malaria vectors Anopheles gambiae Giles s.l. and An.funestus Giles are reported and discussed. Field trials were carried out in villages near Ouagadougou, Burkina Faso. Houses were provided with curtains made from 100% cotton netting, impregnated with permethrin at the dose of 1 g a.i./m2, to cover the doorway, the window(s) and the space under the eaves. Entomological data collected during the period 1985-86 showed residual permethrin activity for about a year, and almost complete prevention of indoor-resting mosquitoes. Increased exit-rate and mortality-rate of house-entering malaria vectors were also obtained. Utilization of this malaria vector control method in primary health care programmes is advocated.     

A mosquito-specific protein family includes candidate receptors for malaria sporozoite invasion of salivary glands

We describe a previously unrecognized protein family from Aedes and Anopheles mosquitoes, here named SGS proteins. There are no SGS homologues in Drosophila or other eukaryotes, but SGS presence in two mosquito genera suggests that the protein family is widespread among mosquitoes. Ae. aegypti aaSGS1 mRNA and protein are salivary gland specific, and protein is localized in the basal lamina covering the anatomical regions that are preferentially invaded by malaria sporozoites. Anti-aaSGS1 antibodies inhibited sporozoite invasion into the salivary glands in vivo, confirming aaSGS1 as a candidate sporozoite receptor. By homology to aaSGS1 we identified the complete complement of four SGS genes in An. gambiae, which were not recognized in the genome annotation. Two An. gambiae SGS genes display salivary gland specific expression like aaSGS1. Bioinformatic analysis predicts that SGS proteins possess heparin-binding domains, and have among the highest density of tyrosine sulphation sites of all An. gambiae proteins. The major sporozoite surface proteins (CS and TRAP) also bind heparin, and interact with sulphoconjugates during liver cell invasion. Thus, we speculate that sporozoite invasion of mosquito salivary glands and subsequently the vertebrate liver may share similar mechanisms based on sulphation. Phylogenomic analysis suggests that an SGS ancestor was involved in a lateral gene transfer.     

Simultaneous identification of species and molecular forms of the Anopheles gambiae complex by PCR-RFLP

For differential identification of sibling species in the Anopheles gambiae Giles complex (Diptera: Culicidae), including simultaneous separation of M and S molecular forms within An. gambiae Giles sensu stricto, we describe a PCR-RFLP method. This procedure is more efficient, faster and cheaper than those used before, so is recommended for large-scale processing of field-collected larval and adult specimens to be identified in malaria vector studies.     

Use of a male-specific DNA probe to distinguish female mosquitoes of the Anopheles gambiae species complex

Abstract. A method has been developed to distinguish between mated female individuals of the sibling species Anopheles gambiae Giles sensu stricto and Anopheles arabiensis Patton. The DNA probe pAnal, reported by Gale & Crampton (1987a) to be useful for the specific identification of An.arabiensis males, is here shown to be sufficiently sensitive to deduce the species identity of inseminated females from the identity of the sperm contained within the spermatheca.     

The parasite invasion marker SRPN6 reduces sporozoite numbers in salivary glands of Anopheles gambiae

For malaria transmission to occur, Plasmodium sporozoites must infect the salivary glands of their mosquito vectors. This study reports that Anopheles gambiae SRPN6 participates in a local salivary gland epithelial response against the rodent malaria parasite, Plasmodium berghei . We showed previously that SRPN6, an immune inducible midgut invasion marker, influences ookinete development. Here we report that SRPN6 is also specifically induced in salivary glands with the onset of sporozoite invasion. The protein is located in the basal region of epithelial cells in proximity to invading sporozoites. Knockdown of SRPN6 during the late phase of sporogony by RNAi has no effect on oocyst rupture but significantly increases the number of sporozoites present in salivary glands. Despite several differences between the passage of Plasmodium through the midgut and the salivary glands, this study identifies a striking overlap in the molecular responses of these two epithelia to parasite invasion.     

Evidence for late Pleistocene population expansion of the malarial mosquitoes, Anopheles arabiensis and Anopheles gambiae in Nigeria

Anopheles gambiae Giles s.s. and Anopheles arabiensis Patton (Diptera: Culicidae) are major vectors of malaria in Nigeria. We used 1115 bp of the mitochondrial COI gene to assess their population genetic structures based on samples from across Nigeria (n = 199). The mtDNA neighbour-joining tree, based on F(ST) estimates, separated An. gambiae M and S forms, except that samples of An. gambiae M from Calabar clustered with all the An. gambiae S form. Anopheles arabiensis and An. gambiae could be combined into a single star-shaped, parsimonious haplotype network, and shared three haplotypes. Haplotype diversity values were high in An. arabiensis and An. gambiae S, and intermediate in An. gambiae M; all nucleotide diversities were relatively low. Taken together, patterns of haplotype diversity, the star-like genealogy of haplotypes, five of seven significant neutrality tests, and the violation of the isolation-by-distance model indicate population expansion in An. arabiensis and An. gambiae S, but the signal was weak in An. gambiae M. Selection is supported as an important factor shaping genetic structure in An. gambiae in Nigeria. There were two geographical subdivisions in An. arabiensis: one included all southern localities and all but two central localities; the other included all northern and two central localities. Re-analysing an earlier microsatellite dataset of An. arabiensis using a Bayesian method determined that there were two distinctive clusters, northern and southern, that were fairly congruent with the mtDNA subdivisions. There was a trend towards decreasing genetic diversity in An. arabiensis from the northern savannah to the southern rainforest that corroborated previous data from microsatellites and polytene chromosomes.     

Malaria transmission dynamics in central Côte d'Ivoire: the influence of changing patterns of irrigated rice agriculture

The dynamics of malaria transmission was studied comparatively in the villages of Zatta and Tiemelekro, central Cote d'Ivoire, from February 2002 to August 2003. Prominent agroecosystems in these villages are irrigated rice growing and vegetable farming, respectively. Mosquitoes (Diptera: Culicidae) were collected on human bait at night and by pyrethrum knock-down spray sheet collections at four randomly selected sentinel sites in each village. In 2002, for a total of 96 man-nights per village, 7716 mosquitoes were collected in Zatta and 3308 in Tiemelekro. In 2003, with half the sampling effort, 859 and 2056 mosquitoes were collected in Zatta and Tiemelekro, respectively. Anopheles gambiae Giles s.l. was the predominant mosquito and the key malaria vector throughout, followed by An. funestus Giles. Anthropophily among adult female Anopheles exceeded 95% in both villages. Comparison between years revealed that the biting rate of An. gambiae s.l. in Zatta decreased several-fold from 49.3 bites per person per night (b/p/n) in 2002 to 7.9 b/p/n in 2003 (likelihood ratio test (LRT) = 1072.66; P < 0.001). Although the biting rate remained fairly constant in Tiemelekro, the difference between years was significant (16.1 vs. 18.2 b/p/n; LRT = 148.06; P < 0.001). These observations were paralleled by a marked decrease in the infective rate of An. gambiae s.l. in Zatta (4.6-1.2%), and an increase in Tiemelekro (3.1-7.6%). Meanwhile, the entomological inoculation rate of An. gambiae s.l. decreased 21-fold in Zatta, from 789 to 38 infective bites per person per year (ib/p/y), whereas it remained high in Tiemelekro (233 vs. 342 ib/p/y). The interruption of irrigated rice growing in Zatta in 2003, consequential to a farmers' conflict over land, might be the underlying cause for the significant reduction in malaria transmission, whereas more stable conditions occurred in Tiemelekro.     

A role for heparan sulfate proteoglycans in Plasmodium falciparum sporozoite invasion of anopheline mosquito salivary glands

HS (heparan sulfate) has been shown to be an important mediator of Plasmodium sporozoite homing and invasion of the liver, but the role of this glycosaminoglycan in mosquito vector host-sporozoite interactions is unknown. We have biochemically characterized the function of AgOXT1 (Anopheles gambiae peptide-O-xylosyltransferase 1) and confirmed that AgOXT1 can modify peptides representing model HS and chondroitin sulfate proteoglycans in vitro. Moreover, we also demonstrated that the mosquito salivary gland basal lamina proteoglycans are modified by HS. We used RNA interference-mediated knockdown of HS biosynthesis in A. gambiae salivary glands to determine whether Plasmodium falciparum sporozoites that are released from mosquito midgut oocysts use salivary gland HS as a receptor for tissue invasion. Our results suggest that salivary gland basal lamina HS glycosaminoglycans only partially mediate midgut sporozoite invasion of this tissue, and that in the absence of HS, the presence of other surface co-receptors is sufficient to facilitate parasite entry.     

Engorgement response of anopheline mosquitoes to blood fractions and artificial solutions

ABSTRACT. Anopheles stephensi Liston, Anopheles freeborni Aitken, Anopheles gambiae Giles and Anopheles dirus (Peyton & Harrison) fed equally well on whole blood, red blood cells, platelet-rich plasma and platelet-poor plasma. Similar feeding ability on 0.15 M NaCl containing 10^-2 M NaHCO₃ was shown by the first three species, but An. dirus required an addition of albumin. The need for ATP as a phagostimulant could not be demonstrated in any of these species.