Resistance of early midgut stages of natural Plasmodium falciparum parasites to high temperatures in experimentally infected Anopheles gambiae (Diptera: Culicidae)

We studied the effects of high temperature, 30 and 32 versus 27 C on early Plasmodium falciparum development in Anopheles gambiae experimentally infected with gametocytes from 30 volunteers with mean density of 264.1 gametocytes/microl blood (range: 16-1,536/microl). From several batches of mosquitoes, fed by membrane feeding, midguts of individual mosquitoes were dissected at 24 hr for ookinete enumeration and at 7 days to quantify oocysts. There were temperature-related differences in mean ookinete intensity per mosquito midgut, with 9.71 +/- 1.6 at 27 C, 9.85 +/- 2.32 at 30 C, and 3.89 +/- 0.81 at 32 C. The prevalence of oocyst infection decreased with an increase in temperatures from 15.9 to 8.5 to 6.4% at 27, 30, and 32 C, respectively. The average oocyst intensities for the infected mosquitoes increased with temperatures from 2.9 at 27 C to 3.5 at 30 C, and to 3.3 at 32 C. However, the success of infections was reduced at 30 and 32 C, and resulted in greater losses during consecutive inter-stage parasite development. The most significant impact of high temperatures occurred at the transition between macrogametocytes and ookinetes, whereas the transition between ookinetes and oocysts apparently was not affected. In contrast to other reports, exposure of mosquitoes infected with natural parasites to high temperatures did not eliminate preoocyst stages, as has been observed from laboratory studies using the NF-54 strain of P. falciparum. This observation of parasite resistance to high temperatures is consistent with the natural situation in tropical environments where perennial malaria transmission occurs during hot dry seasons.     

Population dynamics of sporogony for Plasmodium vivax parasites from western Thailand developing within three species of colonized Anopheles mosquitoes

Background

The population dynamics of Plasmodium sporogony within mosquitoes consists of an early phase where parasite abundance decreases during the transition from gametocyte to oocyst, an intermediate phase where parasite abundance remains static as oocysts, and a later phase where parasite abundance increases during the release of progeny sporozoites from oocysts. Sporogonic development is complete when sporozoites invade the mosquito salivary glands. The dynamics and efficiency of this developmental sequence were determined in laboratory strains of Anopheles dirus, Anopheles minimus and Anopheles sawadwongporni mosquitoes for Plasmodium vivax parasites circulating naturally in western Thailand.

Methods

Mosquitoes were fed blood from 20 symptomatic Thai adults via membrane feeders. Absolute densities were estimated for macrogametocytes, round stages (= female gametes/zygotes), ookinetes, oocysts, haemolymph sporozoites and salivary gland sporozoites. From these census data, five aspects of population dynamics were analysed; 1) changes in life-stage prevalence during early sporogony, 2) kinetics of life-stage formation, 3) efficiency of life-stage transitions, 4) density relationships between successive life-stages, and 5) parasite aggregation patterns.

Results

There was no difference among the three mosquito species tested in total losses incurred by P. vivax populations during early sporogony. Averaged across all infections, parasite populations incurred a 68-fold loss in abundance, with losses of ca. 19-fold, 2-fold and 2-fold at the first (= gametogenesis/fertilization), second (= round stage transformation), and third (= ookinete migration) life-stage transitions, respectively. However, total losses varied widely among infections, ranging from 6-fold to over 2,000-fold loss. Losses during gametogenesis/fertilization accounted for most of this variability, indicating that gametocytes originating from some volunteers were more fertile than those from other volunteers. Although reasons for such variability were not determined, gametocyte fertility was not correlated with blood haematocrit, asexual parasitaemia, gametocyte density or gametocyte sex ratio. Round stages and ookinetes were present in mosquito midguts for up to 48 hours and development was asynchronous. Parasite losses during fertilization and round stage differentiation were more influenced by factors intrinsic to the parasite and/or factors in the blood, whereas ookinete losses were more strongly influenced by mosquito factors. Oocysts released sporozoites on days 12 to 14, but even by day 22 many oocysts were still present on the midgut. The per capita production was estimated to be approximately 500 sporozoites per oocyst and approximately 75% of the sporozoites released into the haemocoel successfully invaded the salivary glands.

Conclusion

The major developmental bottleneck in early sporogony occurred during the transition from macrogametocyte to round stage. Sporozoite invasion into the salivary glands was very efficient. Information on the natural population dynamics of sporogony within malaria-endemic areas may benefit intervention strategies that target early sporogony (e.g., transmission blocking vaccines, transgenic mosquitoes).     

Plasmodium falciparum produce lower infection intensities in local versus foreign Anopheles gambiae populations

Both Plasmodium falciparum and Anopheles gambiae show great diversity in Africa, in their own genetic makeup and population dynamics. The genetics of the individual mosquito and parasite are known to play a role in determining the outcome of infection in the vector, but whether differences in infection phenotype vary between populations remains to be investigated. Here we established two A. gambiae s.s. M molecular form colonies from Cameroon and Burkina Faso, representing a local and a foreign population for each of the geographical sites. Experimental infections of both colonies were conducted in Cameroon and Burkina Faso using local wild P. falciparum, giving a sympatric and allopatric vector-parasite combination in each site. Infection phenotype was determined in terms of oocyst prevalence and intensity for at least nine infections for each vector-parasite combination. Sympatric infections were found to produce 25% fewer oocysts per midgut than allopatric infections, while prevalence was not affected by local/foreign interactions. The reduction in oocyst numbers in sympatric couples may be the result of evolutionary processes where the mosquito populations have locally adapted to their parasite populations. Future research on vector-parasite interactions must take into account the geographic scale of adaptation revealed here by conducting experiments in natural sympatric populations to give epidemiologically meaningful results.     

Rodent Plasmodium: population dynamics of early sporogony within Anopheles stephensi mosquitoes

Early sporogony of Plasmodium parasites involves 2 major developmental transitions within the insect vector, i.e., gametocyte-to-ookinete and ookinete-to-oocyst. This study compared the population dynamics of early sporogony among murine rodent Plasmodium (Plasmodium berghei, Plasmodium chabaudi, Plasmodium vinckei, and Plasmodium yoelii) developing within Anopheles stephensi mosquitoes. Estimates of absolute densities were determined for gametocytes, ookinetes, and oocysts for 108 experimental infections. Total losses throughout early sporogony were greatest in P. vinckei (ca. 250,000-fold loss), followed by P. yoelii (ca. 70,000-fold loss), P. berghei (ca. 45,000-fold loss), and P. chabaudi (ca. 15,000-fold loss). The gametocyte-to-ookinete transition represented the most severe population bottleneck. Numerical losses during this transition (ca. 3,000- to 30,000-fold, depending on species) were orders of magnitude greater than losses incurred during the ookinete-to-oocyst transition (3- to 14-fold). There were no significant correlations between gametocyte and ookinete densities. Significant correlations between ookinete and oocyst densities existed for P. berghei, P. chabaudi, and P. yoelii (but not for P. vinckei), and were best described by nonlinear functions (P. berghei = sigmoid, P. chabaudi = hyperbolic, P. yoelii = sigmoid), indicating that conversion of ookinetes to oocysts in these species is density dependent. The upper theoretical limit for oocyst density on the mosquito midgut for P. chabaudi and P. yoelii (ca. 300 oocysts per midgut) was higher than for P. berghei (ca. 30 oocysts per midgut). This study provides basic information about population processes that occur during the early sporogonic development of some common laboratory model systems of malaria.     

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.     

Anopheles gambiae SRPN2 facilitates midgut invasion by the malaria parasite Plasmodium berghei

We report on a phylogenetic and functional analysis of genes encoding three mosquito serpins (SRPN1, SRPN2 and SRPN3), which resemble known inhibitors of prophenoloxidase-activating enzymes in other insects. Following RNA interference induction by double-stranded RNA injection, knockdown of SRPN2 in adult Anopheles gambiae produced a notable phenotype: the appearance of melanotic pseudotumours, which increased in size and number with time, indicating spontaneous melanization and association with an observed lifespan reduction. Furthermore, knockdown of SRPN2 strongly interfered with the invasion of A. gambiae midguts by the rodent malaria parasite Plasmodium berghei. It did not affect ookinete formation, but markedly reduced oocyst numbers, by 97%, as a result of increased ookinete lysis and melanization.     

Engineered resistance to Plasmodium falciparum development in transgenic Anopheles stephensi

Transposon-mediated transformation was used to produce Anopheles stephensi that express single-chain antibodies (scFvs) designed to target the human malaria parasite, Plasmodium falciparum. The scFvs, m1C3, m4B7, and m2A10, are derived from mouse monoclonal antibodies that inhibit either ookinete invasion of the midgut or sporozoite invasion of salivary glands. The scFvs that target the parasite surface, m4B7 and m2A10, were fused to an Anopheles gambiae antimicrobial peptide, Cecropin A. Previously-characterized Anopheles cis-acting DNA regulatory elements were included in the transgenes to coordinate scFv production with parasite development. Gene amplification and immunoblot analyses showed promoter-specific increases in transgene expression in blood-fed females. Transgenic mosquito lines expressing each of the scFv genes had significantly lower infection levels than controls when challenged with P. falciparum.     

Progression of Plasmodium berghei through Anopheles stephensi is density-dependent

It is well documented that the density of Plasmodium in its vertebrate host modulates the physiological response induced; this in turn regulates parasite survival and transmission. It is less clear that parasite density in the mosquito regulates survival and transmission of this important pathogen. Numerous studies have described conversion rates of Plasmodium from one life stage to the next within the mosquito, yet few have considered that these rates might vary with parasite density. Here we establish infections with defined numbers of the rodent malaria parasite Plasmodium berghei to examine how parasite density at each stage of development (gametocytes; ookinetes; oocysts and sporozoites) influences development to the ensuing stage in Anopheles stephensi, and thus the delivery of infectious sporozoites to the vertebrate host. We show that every developmental transition exhibits strong density dependence, with numbers of the ensuing stages saturating at high density. We further show that when fed ookinetes at very low densities, oocyst development is facilitated by increasing ookinete number (i.e., the efficiency of ookinete-oocyst transformation follows a sigmoid relationship). We discuss how observations on this model system generate important hypotheses for the understanding of malaria biology, and how these might guide the rational analysis of interventions against the transmission of the malaria parasites of humans by their diverse vector species.     

Plasmodium yoelii: axenic development of the parasite mosquito stages

Study of the parasite mosquito stages of Plasmodium and its use in the production of sporozoite vaccines against malaria has been hampered by the technical difficulties of in vitro development. Here, we show the complete axenic development of the parasite mosquito stages of Plasmodium yoelii. While we demonstrate that matrigel is not required for parasite development, soluble factors produced and secreted by Drosophila melanogaster S2 cells appear to be crucial for the ookinete to oocyst transition. Parasites cultured axenically are both morphologically and biologically similar to mosquito-derived ookinetes, oocysts, and sporozoites. Axenically derived sporozoites were capable of producing an infection in mice as determined by RT-PCR; however, the parasitemia was significantly much less than that produced by mosquito-derived sporozoites. Our cell free system for development of the mosquito stages of P. yoelii provides a simplified approach to generate sporozoites that may be for biological assays and genetic manipulations.     

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.     

Anopheles and Plasmodium: from laboratory models to natural systems in the field

Parasites that cause malaria must complete a complex life cycle in Anopheles vector mosquitoes in order to be transmitted from human to human. Previous gene-silencing studies have shown the influence of mosquito immunity in controlling the development of Plasmodium. Thus, parasite survival to the oocyst stage increased when the parasite antagonist gene LRIM1 (leucine-rich repeat immune protein 1) of the mosquito was silenced, but decreased when the C-type lectin agonist gene CTL4 or CTLMA2 (CTL mannose binding 2) was silenced. However, such effects were shown for infections of the human mosquito vector Anopheles gambiae with the rodent parasite Plasmodium berghei. Here, we report the first results of A. gambiae gene silencing on infection by sympatric field isolates of the principal human pathogen P. falciparum. In contrast with the results obtained with the rodent parasite, silencing of the same three genes had no effect on human parasite development. These results highlight the importance of following up discoveries in laboratory model systems with studies on natural parasite-mosquito interactions.     

Plasmodium gallinaceum: ookinete formation and proteolytic enzyme dynamics in highly refractory Aedes aegypti populations

Despite significant progress in the identification of the genetic basis of the refractory phenotype, little is known about the physiological mechanism of refractoriness. This study therefore examined the physiological basis of mosquito refractoriness in the Aedes aegypti/P. gallinaceum system, in which a selected refractory strain does not permit Plasmodium oocyst formation. We examined the kinetics of two major proteolytic enzymes involved in blood meal digestion and the dynamics of ookinete formation for two refractory populations (strains Moyo-R and Formosus) and one susceptible population (strain Red). Healthy ookinetes were observed in both the susceptible and the refractory populations, although the susceptible population generally exhibited higher enzymatic activity for trypsin and aminopeptidase than the refractory populations. Parasite numbers in the susceptible Red population showed a 4- to 7-fold decrease in abundance during the transition from the ookinete stage to the oocyst stage, far less than the refractory populations (30- to 92-fold reduction). Due to its smaller body size, Moyo-R individuals generally ingest a smaller blood meal and thus intake fewer gametocytes than Red individuals. Thus, the possibility that refractoriness in the Moyo-R population results from fewer gametocytes being ingested is examined. We found that the Red population remained highly susceptible and the Moyo-R population stayed refractory when those individuals with similar blood meal size were compared. We conclude that failure of oocyst development in the refractory mosquitoes is not due to ookinete damage by proteolytic enzymes or to fewer gametocytes being ingested, but rather is due to a midgut barrier or to some other mechanism.     

离体培养鼠疟原虫动合子形成过程的电子显微镜观察

本文报道了用透射电子显微镜观察离体培养的鼠疟原虫配子体到动合子的发育过程。 鼠疟原虫配子的发生是由嗜锇小体趋向配子体表面开始。雌配子体从红细胞中逸出后,嗜锇小体消失。雄配子体微管形成和鞭毛轴丝集合是从红细胞中逸出前出现的。合子转变为动合子由致密内膜及膜下微管形成时开始,继之形成顶端复合物,随着突起增大,表膜复合物逐渐向后延伸,最后包绕整个虫体,即完成动合子的发育。疟原虫生活史第一次核分裂可能发生在动合子形成期间。本文证实了离体培养的动合子与蚊体内发育的动合子结构相同。     

Transformation of sporozoites of Plasmodium berghei into exoerythrocytic forms in the liver of its mammalian host

Summary Intrahepatocytic transformation in vivo of the rodent malaria sporozoite of Plasmodium berghei, into the young trophic exoerythrocytic tissue stage was studied by immunofluorescence, light- and electron microscopy. The first 20 h of intracellular life were involved entirely in dedifferentiation with limited proliferation of organelles. From about 20 h onwards nuclear division commenced, rough endoplasmic reticulum became markedly expanded, and mitochondria increased in numbers. However, remains of the sporozoite pellicle (i.e., inner membranes and subpellicular microtubules) persisted for at least 28 h, which correlates with the persisting reaction of young exoerythrocytic forms with antisporozoite antibodies. In general, the basic mechanism of transformation resembles that of the ookinete into oocyst and that of the merozoite into erythrocytic trophozoite.     

Artemisinin-based combination therapy does not measurably reduce human infectiousness to vectors in a setting of intense malaria transmission

ABSTRACT: BACKGROUND: Artemisinin-based combination therapy (ACT) for treating malaria has activity against immature gametocytes. In theory, this property may complement the effect of terminating otherwise lengthy malaria infections and reducing the parasite reservoir in the human population that can infect vector mosquitoes. However, this has never been verified at a population level in a setting with intense transmission, where chronically infectious asymptomatic carriers are common and cured patients are rapidly and repeatedly re-infected. METHODS: From 2001 to 2004, malaria vector densities were monitored using light traps in three Tanzanian districts. Mosquitoes were dissected to determine parous and oocyst rates. Plasmodium falciparum sporozoite rates were determined by ELISA. Sulphadoxinepyrimethamine (SP) monotherapy was used for treatment of uncomplicated malaria in the contiguous districts of Kilombero and Ulanga throughout this period. In Rufiji district, the standard drug was changed to artesunate co-administered with SP (AS + SP) in March 2003. The effects of this change in case management on malaria parasite infection in the vectors were analysed. RESULTS: Plasmodium falciparum entomological inoculation rates exceeded 300 infective bites per person per year at both sites over the whole period. The introduction of AS + SP in Rufiji was associated with increased oocyst prevalence (OR [95%CI] = 3.9 [2.9-5.3], p < 0.001), but had no consistent effect on sporozoite prevalence (OR [95%CI] = 0.9 [0.7-1.2], p = 0.5). The estimated infectiousness of the human population in Rufiji was very low prior to the change in drug policy. Emergence rates and parous rates of the vectors varied substantially throughout the study period, which affected estimates of infectiousness. The latter consequently cannot be explained by the change in drug policy. CONCLUSIONS: In high perennial transmission settings, only a small proportion of infections in humans are symptomatic or treated, so case management with ACT may have little impact on overall infectiousness of the human population. Variations in infection levels in vectors largely depend on the age distribution of the mosquito population. Benefits of ACT in suppressing transmission are more likely to be evident where transmission is already low or effective vector control is widely implemented.     

Immunogold localization of circumsporozoite protein of the malaria parasite Plasmodium falciparum during sporogony in Anopheles stephensi midguts

The occurrence of the circumsporozoite (CS) proteins of Plasmodium falciparum sporozoites was monitored during sporogonic development in Anopheles stephensi mosquitoes. Using a monoclonal anti-CS protein antibody (3Sp2) and immunogold labeling on ultrathin cryosections it was found that CS protein is synthesized in immature oocysts from day 6 onwards when there are not yet signs of sporozoite formation. The CS protein is rapidly incorporated in the oocyst plasmalemma, which subsequently invaginates into the parasite. In the oocyst only the external sporozoite membrane contains CS protein. The inner pellicle membranes, rhoptries and micronemes do not react with monoclonal antibody (MoAb) 3Sp2.