The effects of parabiotic twinning of susceptible and refractory mosquitoes on the development of Plasmodium gallinaceum

Two species of mosquitoes were joined parabiotically with glass capillaries so as to share common hemolymph. In experiments designed to determine optimum physical factors was found that capillaries of 2.5 mm in length, 100 microns OD and with pointed ends were tolerated best by mosquitoes and permitted optimum hemolymph transfer. Maximum survival of mosquitoes was noted when capillaries were inserted in the post mesospiracular membranous area, in the largest mosquito first and allowed to fill with hemolymph prior to inserting in the second mosquito. Mosquitoes having blood meals prior to twinning retained capillaries best. Use of CO2 anesthetization and a 30-min holding period while anesthetized contributed to greater survival and union of the mosquitoes. In the principal experiments, designed to study the nature of innate immunity of Culex pipiens to Plasmodium gallinaceum, 243 of 2,126 parabiotic twins of C. pipiens and infected Aedes aegypti survived to be evaluated. None of the C. pipiens became infected and only four A. aegypti remained infected. The controls were 93 to 95% infected. It was concluded that the refractory species possessed substances that were toxic to the parasites and prevented parasite development in both species. If there was a lack of essential substances (that could not be transferred) in the refractory C. pipiens they could have been provided by the highly susceptible A. aegypti and both species would have become infected. Innate immunity is therefore antiblastic not atreptic.     

Wolbachia strain wAlbB enhances infection by the rodent malaria parasite Plasmodium berghei in Anopheles gambiae mosquitoes

Wolbachia, a common bacterial endosymbiont of insects, has been shown to protect its hosts against a wide range of pathogens. However, not all strains exert a protective effect on their host. Here we assess the effects of two divergent Wolbachia strains, wAlbB from Aedes albopictus and wMelPop from Drosophila melanogaster, on the vector competence of Anopheles gambiae challenged with Plasmodium berghei. We show that the wAlbB strain significantly increases P. berghei oocyst levels in the mosquito midgut while wMelPop modestly suppresses oocyst levels. The wAlbB strain is avirulent to mosquitoes while wMelPop is moderately virulent to mosquitoes pre-blood meal and highly virulent after mosquitoes have fed on mice. These various effects on P. berghei levels suggest that Wolbachia strains differ in their interactions with the host and/or pathogen, and these differences could be used to dissect the molecular mechanisms that cause interference of pathogen development in mosquitoes.     

Analysis of the sporogonic development of Plasmodium falciparum and Plasmodium berghei in anopheline mosquitoes

Basic knowledge of the sporogonic development of malarial parasites is crucial when evaluating the sporontocidal activity of antimalarial drugs or when determining why certain vectors are refractory to a particular parasite while others are competent vectors. We have developed a model which we have used to i) assess the sporogonic development of Plasmodium berghei ANKA in Anopheles stephensi and A. freeborni mosquitoes and ii) determine the effect of chloroquine on the sporogony of P. falciparum NF-54 in A. stephensi. Criteria used to assay sporogonic development include: i) number of oocysts present, ii) percentage of mosquitoes with oocysts, iii) time of release of sporozoites from the oocysts into the hemolymph, iv) time and degree of sporozoite invasion of salivary glands, and v) transmission (P. berghei) into vertebrate hosts. Parasite development in the mosquito is evaluated every other day, commencing on ca. day 7 post-feed (PF) and continuing until ca. day 22 PF. These detailed observations allow us to delineate the chronology of sporogonic development.     

Reproductive physiology of Anopheles gambiae and Anopheles atroparvus.

When exposed to a human host, Anopheles gambiae started probing 4 h post-eclosion, but 95% successfully blood-fed by 16-20 h with maximal blood volumes of 5- 10 microl per female. When fed sugar, the 95% feeding was not observed until 36-40 h post-eclosion; sugar meals appeared to interfere with blood meals. Similarly in An. atroparvus, maximum volumes were 10 microl when starved but only 6 microl when fed sugar. This species did not bite before 2 d, and 95% biting was by 4 d. Given single blood meals to water-kept An. gambiae, a threshold body size for oogenesis was detected. With wing lengths below 2.8 mm, eggs never matured, but when sugar-fed, females of all sizes matured eggs including the synthesis of maternal deposits. Although sugar feeding interfered with blood feeding, more lipid was transferred to the yolk. In water-kept An. atroparvus only 5% of the females produced eggs. When sugar-fed for 4 d, all females matured eggs, so in this species sugar feeding appeared to be essential for oogenesis. An. gambiae always took multiple blood meals, tested at any time after the first ones, leading to 120 mature eggs/female. Yolk composition was 3.9 mcal protein and 3.8 mcal lipid/oocyte when kept on water, but 2.8 meal protein and 4.3 mcal lipid/oocyte with intermittent sugar meals, thus marking a surprising flexibility in synthesis of yolk protein and lipid that strongly depends on additional carbohydrates sources. Only 80% of water-fed An. atroparvus re-fed 2 d after a first blood meal with small females taking three blood meals but they still showed reduced fecundity. Only the large water-fed females matured eggs, with blood volumes higher than 9-12 microl. When fed sugar, the blood meal input was reduced, but oogenesis was possible, whereas water-fed females required three blood meals to reach the caloric level comparable to pre-feeding sugar-fed females. Water-fedAn. gambiae could survive on daily blood meals alone, but survival was further extended by intermittent sugar meals. When offered a blood donor daily, there was a behavioral difference. Females maintained alone showed a more or less regular 3 d feeding and oviposition activity, while females kept in groups fed daily followed a daily oviposition pattern, suggesting gonotrophic discordance.     

Molecular interactions between Anopheles stephensi midgut cells and Plasmodium berghei: the time bomb theory of ookinete invasion of mosquitoes

We present a detailed analysis of the interactions between Anopheles stephensi midgut epithelial cells and Plasmodium berghei ookinetes during invasion of the mosquito by the parasite. In this mosquito, P. berghei ookinetes invade polarized columnar epithelial cells with microvilli, which do not express high levels of vesicular ATPase. The invaded cells are damaged, protrude towards the midgut lumen and suffer other characteristic changes, including induction of nitric oxide synthase (NOS) expression, a substantial loss of microvilli and genomic DNA fragmentation. Our results indicate that the parasite inflicts extensive damage leading to subsequent death of the invaded cell. Ookinetes were found to be remarkably plastic, to secrete a subtilisin-like serine protease and the GPI-anchored surface protein Pbs21 into the cytoplasm of invaded cells, and to be capable of extensive lateral movement between cells. The epithelial damage inflicted is repaired efficiently by an actin purse-string-mediated restitution mechanism, which allows the epithelium to 'bud off' the damaged cells without losing its integrity. A new model, the time bomb theory of ookinete invasion, is proposed and its implications are discussed.     

Dipsticks for rapid detection of Plasmodium in vectoring Anopheles mosquitoes

Malaria remains the most serious vector-borne disease, affecting some 300-500 million people annually, transmitted by many species of Anopheles mosquitoes (Diptera: Culicidae). Monoclonal antibodies developed against specific circumsporozoite (CS) proteins of the main malaria parasites Plasmodium falciparum and P. vivax have been used previously for enzyme-linked immunosorbent assays (ELISA), widely employed for detection of malaria sporozoites in vector Anopheles for local risk assessment, epidemiological studies and targeting vector control. However, ELISA procedures are relatively slow and impractical for field use. To circumvent this, we developed rapid wicking assays that identify the presence or absence of specific peptide epitopes of CS protein of the most important P. falciparum and two strains (variants 210 and 247) of the more widespread P. vivax. The resulting assay is a rapid, one-step procedure using a 'dipstick' wicking test strip. In laboratory assessment, dipsticks identified 1 ng/ mL of any of these three CS protein antigens, with sensitivity nearly equal to the CS standard ELISA. We have developed and are evaluating a combined panel assay that will be both qualitative and quantitative. This quick and easy dipstick test (VecTest Malaria) offers practical advantages for field workers needing to make rapid surveys of malaria vectors.     

Radiation-induced inversions and reciprocal translocations in Anopheles atroparvus

Inversions and reciprocal translocations were induced in Anopheles atroparvus by irradiation of males with X-rays. 22 aberrations were produced in stocks and were identified as follows: 6 paracentric, 6 pericentric inversions and 10 reciprocal translocations (9 autosomal and 1 sex-linked). Partial sterility in the offspring of this stock is demonstrated. The practical significance of constructing stocks with inversions and translocations for genetic control of pest insects is considered.     

Probing behaviour and sporozoite delivery by Anopheles stephensi infected with Plasmodium berghei

We observed that Plasmodium berghei sporozoite-infected Anopheles stephensi was not impaired in its ability to locate blood on a host. When probing rats, infected mosquitoes took as long as non-infected mosquitoes to locate blood. Contrary to previous suggestions, infective mosquitoes delivered sporozoites into mineral oil even after extensively probing a vertebrate host. We observed that, in mosquitoes having probed a host, both the mean number of sporozoites ejected over 3 min into oil (35.9 v. 31.7 sporozoites) and the proportion of mosquitoes delivering sporozoites (60% v. 50%) were similar to mosquitoes not having probed. We then developed a model of sporozoite delivery, taking into account observations that sporozoites are clumped in the lumen of the glands as well as upon delivery, and that output is uneven and inconsistent. We conclude that clumping optimizes transmission, if a threshold of infection exists and the mean number of sporozoites per clump is greater than the threshold.     

Plasmodium berghei: mechanisms and sites of resistance to sporogonic development in different mosquitoes

An experimental study of the mechanisms and patterns of resistance to Plasmodium berghei in different mosquito species revealed a diversity of factors which prevent or inhibit sporogonic development in its different phases and in different sites in the mosquito vector. The experiments showed that Culex salinarius was a totally resistant species in which exflagellation and ookinete formation are prevented. In Aedes aegypti, ookinetes in small or moderate numbers are formed but penetration of the mosquito midgut wall is blocked and oocysts are not formed. In the three experimental vectors, Anopheles quadrimaculatus, Anopheles aztecus, and Anopheles stephensi grades of enhanced susceptibility are recognized. They are expressed in lesser numbers of abnormal and degenerative oocysts, in higher numbers of sporozoites in the salivary gland, and greater viability and invasiveness of these sporozoites. In Anopheles dureni, the natural vector of rodent malaria, one observes both in nature and under experimental conditions the highest adaptation and most pronounced grade of susceptibility to P. berghei.     

Pathology of Anopheles stephensi after infection with Plasmodium berghei berghei. I. Mortality rate.

The mortality of P. berghei-infected Anopheles stephensi females can be about 30% higher during the first three days than in normal blood-fed mosquitoes. As expected the mortality is higher after feeding on highly infected mice but also depends on the date of feeding and the temperature. Infected mosquitoes kept at 25 degrees C die more often than those kept at 21 degrees C. On the other hand sporozoite production needs the low temperature of 21 degrees C. So the sporozoite production rate falls with increasing temperature, and the mortality rate increases.     

Defined medium supporting development of cleansed Plasmodium berghei ookinetes in Anopheles stephensi.

Hamsters blood infected with Plasmodium berghei was cultured in vitro for the development of ookinetes. The ookinetes were separated from blood components, suspended in various defined media and fed to Anopheles stephensi through a membrane. The development of the oocysts and infective sporozoites was recorded. Mosquitoes infected with ookinetes suspended in L15 formulated into L15-B, L15-D (a medium specially modified for this purpose), IPL-41 or 199 media with no proteins added, developed at least as many oocysts as the control mosquitoes fed ookinetes suspended in blood. Ookinetes suspended in the L15-B medium yielded more oocysts than after feeding ookinetes suspended in L15-B with 5% casein. Sporozoites from mosquitoes maintained on blood, L15-B, L15-D, or L15-B with 5% casein were shown to be infective to hamsters. Mosquitoes fed ookinetes suspended in sucrose solutions showed very few oocysts, but the yield was increased when a blood meal was given 2-4 days after the infective meal. Some of the oocysts which had developed from the ookinetes suspended in artificial media were found to have degenerated. The described system could be potentially useful for a study of the interaction between the vector physiology and the parasite. The possible use of the system to learn which media should be developed in the future for in vitro cultivation of oocysts is discussed.     

Energy metabolism affects susceptibility of Anopheles gambiae mosquitoes to Plasmodium infection

Previous studies showed that Anopheles gambiae L3-5 females, which are refractory (R) to Plasmodium infection, express higher levels of genes involved in redox-metabolism and mitochondrial respiration than susceptible (S) G3 females. Our studies revealed that R females have reduced longevity, faster utilization of lipid reserves, impaired mitochondrial state-3 respiration, increased rate of mitochondrial electron leak and higher expression levels of several glycolytic enzyme genes. Furthermore, when state-3 respiration was reduced in S females by silencing expression of the adenine nucleotide translocator (ANT), hydrogen peroxide generation was higher and the mRNA levels of lactate dehydrogenase increased in the midgut, while the prevalence and intensity of Plasmodium berghei infection were significantly reduced. We conclude that there are broad metabolic differences between R and S An. gambiae mosquitoes that influence their susceptibility to Plasmodium infection.     

Rapid, large-scale isolation of Plasmodium berghei sporozoites from infected mosquitoes.

The discontinuous gradient technique for recovery of malarial sporozoites from mosquitoes (Beaudoin et al., 1977) has been modified to speed up recovery and prevent sensitization of mice by components of the gradient which contaminate the sporozoites used as antigen. Mouse serum was substituted for BSA in the gradient because the latter produced hypersensitivity. Best results were obtained with gradients consisting of Medium 199, Renografin and mouse serum. Heavy and light solution of gradient components are layered in a centrifuge tube. Centrifugation of comminuted, infected mosquitoes applied to the top of the discontinuous gradient concentrates sporozoites at the interface. Sporozoites recovered from the gradient were infective, immunogenic, and relatively free of mosquito tissue. This improved method enables recovery of 100,000 sporozoites from each Anopheles stephensi infected with the ANKA strain of Plasmodium berghei. As many as 2,800 mosquitoes have been processed in 2 hr without a significant decrease in yield.