Physiological aspects of multiple blood feeding in the malaria vector Anopheles tessellatus

The malaria vector Anopheles tessellatus is able to take several blood meals in a gonotrophic cycle. The fecundity is largely dependent on the first blood meal and is not generally increased by subsequent blood meals during a gonotrophic cycle. Larval rearing densities influenced adult body size. There is an inverse relationship between wing length and larval rearing densities. Smaller mosquitoes produced from larvae reared at higher densities had reduced body reserves of protein, lipid and carbohydrates. At emergence, ovarian development in An. tessellatus is in the previtellogenic stage and it remained at this stage until the intake of a blood meal. The number of ovarian follicles is related to wing length and, irrespective of adult body size, An. tessellatus developed oocytes to maturity with a single blood meal. This is attributed to the availability of metabolic reserves above the threshold level required for further development of oocytes. Mosquitoes that took more than one blood meal had largely digested their previous blood meal and had ongoing vitellogenesis. Blood meals subsequent to the first one apparently contribute mainly to increasing metabolic reserves. The stimulus for a second and third blood meal in An. tessellatus appears to be completion of the digestion of the previous blood meal. There was no evidence that multiple blood meals taken in the first gonotrophic cycle influenced fecundity significantly in the second cycle.     

Effects of post-ingestion and physical conditions on PCR amplification of host blood meal DNA in mosquitoes

The effects of post-ingestion and physical conditions under which killed mosquitoes are stored on the success of detecting blood meal DNA of Anopheles stephensi and Culex quinquefasiatus were investigated by polymerase chain reaction (PCR) amplification at the human mitochondrial DNA cytochrome b (cytB) gene. Host DNA extracted from the blood meal up to 33 h post-ingestion in both species acts as an efficient template for PCR amplification. However, more DNA concentration is needed for meals digested for a longer time, and successful PCR amplification from meals digested for 36 h,dropped to a faint band. There were no differences between PCR success rate for samples stored at +4 or -20 degrees C, but less successful products were observed in samples kept at 4 degrees C for the periods longer than 30 h digestion. The results of this study are important for conducting malaria epidemiological studies that provide information about the degree of contact between human hosts and mosquito vectors, impact of vector controls such as bed nets and repellents, and the transmission dynamics of human malaria and other vector-borne diseases.     

The role of the mosquito peritrophic membrane in bloodmeal digestion and infectivity of Plasmodium species.

Secretion and luminal formation of the peritrophic membrane (PM) were induced in female Anopheles stephensi and Aedes aegypti by feeding the mosquitoes on a warmed suspension of latex particles in Ringer's solution. The PM in A. stephensi was produced from apical secretion vesicles stored in the midgut epithelial cells and secreted into the lumen during feeding. In A. aegypti, the PM was formed de novo. When the latex feeding was followed 24 hr later by a meal of lyophilized pig blood, the 2 mosquito species exhibited very different modifications to their PM structure; in A. stephensi no PM was formed around the blood meal, whereas de novo synthesis of the PM in A. aegypti continued during the blood meal, with the resulting PM greatly thickened compared to the normal feeding. This artificial induction of PM formation was used as the basis to study the role of the PM in blood meal digestion and in infectivity of mosquitoes by the appropriate species of Plasmodium. The feeding of a latex suspension alone had no stimulatory effect on the 2 major midgut proteases, trypsin and aminopeptidase, in either species. After a blood meal alone, proteases rose to maximum activity at 30 hr and 24 hr after feeding in A. stephensi and A. aegypti, respectively. After double feeding, protease activities in both species were almost identical to those in blood-fed mosquitoes. Neither the absence of a PM (in A. stephensi) nor the presence of a thickened PM (in A. aegypti), therefore, has any effect on the ability of mosquitoes to digest a blood meal. Malaria infectivity, measured by oocyst counts, also was compared after normal and double feeding using infective blood meals. Infectivity of A. stephensi by Plasmodium berghei was unaffected by the presence or absence of the PM. The thickened PM produced by double feeding in A. aegypti caused a reduction of midgut infectivity by Plasmodium gallinaceum. These results suggest that the PM may act as a partial, but not an absolute, barrier to invasion of the midgut by the ookinete.     

Population dynamics of Plasmodium falciparum sporogony in laboratory-infected Anopheles gambiae.

The population dynamics of cultured Plasmodium falciparum parasites was examined during their sporogonic development in Anopheles gambiae mosquitoes. Estimates of absolute densities were determined for each life stage, and life tables were constructed for each of 38 experimental infections. Macrogametocyte and ookinete mortalities contributed equally to the overall mortality. On average, there was a 40-fold decrease in parasite numbers in the transition from the macrogametocyte to the ookinete stage, a 69-fold decrease in the transition from ookinete to oocyst stages, and a total net decrease in parasite numbers from macrogametocyte to oocyst stage of 2,754-fold (i.e., multiplicative). There was no relationship between macrogametocyte and ookinete densities due to the inherent variability in fertility among different gametocyte cultures. There was a curvilinear relationship (r2 = 0.66) between ookinete and oocyst densities. Above a threshold of about 30 ookinetes/mosquito, the oocyst yield per ookinete became increasingly greater with increasing ookinete density. There was a linear relationship (r2 = 0.73) between oocyst and sporozoite densities, with an average of 663 salivary gland sporozoites produced per oocyst. Sporozoite production per oocyst was not affected by oocyst density and virtually all oocyst infections resulted in sporozoite infections of the salivery glands. This quantitative study indicates that the sporogony of cultured P. falciparum in laboratory-infected A. gambiae is an inefficient process and that the ookinete is the key transitional stage affecting the probability of vector infectivity.     

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.     

Extent of digestion affects the success of amplifying human DNA from blood meals of Anopheles gambiae (Diptera: Culicidae)

The success of distinguishing blood meal sources of Anopheles gambiae Giles through deoxyribonucleic acid (DNA) profiling was investigated by polymerase chain reaction (PCR) amplification at the TC-11 and VWA human short tandem repeats (STR) loci. Blood meal size and locus had no significant effect on the success of amplifying human DNA from blood meals digested for 0, 8, 16, 24 and 32 h (P = 0.85 and 0.26 respectively). However, logistic regression found a significant negative relationship between time since ingestion and the success probability of obtaining positive PCR products among meals digested for between 8 and 32 h (P = 0.001). Approximately 80% of fresh blood meals were successfully profiled. After 8 h, the proportion of blood meals that could be successfully profiled decreased slowly with time after ingestion, dropping to below 50% after approximately 15 h. There was no significant difference in the success of amplifying human DNA from blood meals of mosquitoes killed at time 0 and 8 h after ingestion (P = 0.272).     

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.     

Unbiased Characterization of Anopheles Mosquito Blood Meals by Targeted High-Throughput Sequencing

Understanding mosquito host choice is important for assessing vector competence or identifying disease reservoirs. Unfortunately, the availability of an unbiased method for comprehensively evaluating the composition of insect blood meals is very limited, as most current molecular assays only test for the presence of a few pre-selected species. These approaches also have limited ability to identify the presence of multiple mammalian hosts in a single blood meal. Here, we describe a novel high-throughput sequencing method that enables analysis of 96 mosquitoes simultaneously and provides a comprehensive and quantitative perspective on the composition of each blood meal. We validated in silico that universal primers targeting the mammalian mitochondrial 16S ribosomal RNA genes (16S rRNA) should amplify more than 95% of the mammalian 16S rRNA sequences present in the NCBI nucleotide database. We applied this method to 442 female Anopheles punctulatus s. l. mosquitoes collected in Papua New Guinea (PNG). While human (52.9%), dog (15.8%) and pig (29.2%) were the most common hosts identified in our study, we also detected DNA from mice, one marsupial species and two bat species. Our analyses also revealed that 16.3% of the mosquitoes fed on more than one host. Analysis of the human mitochondrial hypervariable region I in 102 human blood meals showed that 5 (4.9%) of the mosquitoes unambiguously fed on more than one person. Overall, analysis of PNG mosquitoes illustrates the potential of this approach to identify unsuspected hosts and characterize mixed blood meals, and shows how this approach can be adapted to evaluate inter-individual variations among human blood meals. Furthermore, this approach can be applied to any disease-transmitting arthropod and can be easily customized to investigate non-mammalian host sources.     

Evaluation of procedures to determine absolute density of Plasmodium vivax ookinetes

The ookinete is the key determinant of infection within the mosquito vector, yet there are few population studies of ookinetes in nature. This investigation compared different techniques used to estimate ookinete densities in mosquitoes. Laboratory-reared Anopheles dirus mosquitoes were fed on gametocytemic blood drawn from 7 Plasmodium vivax patients at a malaria clinic in Mae Sot, Thailand. At 20-26 hr, bloodmeals were excised. Three techniques were evaluated, i.e., hemacytometer counts under phase-contrast microscope, Giemsa staining of bloodmeal smears, and immunofluorescent staining with a monoclonal antibody specific against the 25-kDa antigen expressed on the surface of P. vivax zygotes and ookinetes. Additional mosquitoes were dissected at day 10 for oocysts. The hemacytometer method was the simplest and quickest method but lacked precision at low ookinete densities. Immunofluorescent staining was the most sensitive, accurate, and the only method that enabled unequivocal detection of zygotes. Bloodmeals contained a mixture of zygotes, retorts, and mature ookinetes, indicating that postzygotic development of P. vivax in A. dirus was asynchronous. The conversion efficiency of zygotes/ookinetes to oocysts varied among patients and was independent of zygote-ookinete density, suggesting that variations in host blood composition, e.g., antibodies, drugs, etc., may influence the success of zygote-ookinete development.     

Fecundity, parity, and adult feeding relationships among Nyssorhynchus malaria vectors from Venezuela.

Relative to their pre-engorgement weights, nulliparous Anopheles nuneztovari consumed significantly smaller blood meals than A. marajoara, A. triannulatus or A. aquasalis. When females were deprived of sugar before blood feeding, only one-quarter of A. nuneztovari, but more than two-thirds of A. marajoara, A. triannulatus and A. aquasalis matured eggs. Sugar feeding before blood, or two successive blood meals by sugar-deprived females, increased the proportion of nulliparous A. nuneztovari which developed eggs, but not significantly so. Nearly all individuals of nulliparous, sugar-fed A. marajoara, A. triannulatus and A. aquasalis matured eggs after one blood feeding. Among A. nuneztovari, A. marajoara and A. aquasalis that matured some eggs in the laboratory, there were no positive correlations between the number of eggs developed and relative blood meal size. However, blood meals larger than the mean size significantly increased the chance that A. nuneztovari would develop some eggs. Mean fecundities of gravid A. nuneztovari and A. marajoara reared in the laboratory were significantly lower than those of the same species captured at human bait in nature. Post-engorgement access to sugar by A. nuneztovari (captured at human bait) did not influence fecundity, but significantly enhanced survivorship and the proportion of individuals which retained eggs. Release-recapture experiments revealed that relatively small blood meals are typical of A. nuneztovari only during the first gonotrophic cycle. We suggest that multiple blood feeding, seemingly necessary for most A. nuneztovari to develop a first clutch of eggs, may increase the probability of infection with Plasmodium vivax where this mosquito species is a primary vector.     

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).     

Ovarian control of blood meal retention the the mosquito Anopheles freeborni

Female Anopheles freeborni begin to defaecate the haem containing residue of a blood meal about the time their ova mature, 40–45 hr after feeding. Ovariectomized females begin to defaecate 15 hr prematurely, while ovariectomized females treated with 20-hydroxyecdysone retain blood meals longer, proportional to dose, than do those untreated. It is concluded that the production of ecdysteroids by the ovary initiates a mechanism for retention of the blood meal in the mosquito.     

Plasmodium vivax: impaired escape of Vk210 phenotype ookinetes from the midgut blood bolus of Anopheles pseudopunctipennis

The site in the midguts of Anopheles pseudopunctipennis where the development of Plasmodium vivax circumsporozoite protein Vk210 phenotype is blocked was investigated, and compared to its development in An. albimanus. Ookinete development was similar in time and numbers within the blood meal bolus of both mosquito species. But, compared to An. pseudopunctipennis, a higher proportion of An. albimanus were infected (P=0.0001) with higher ookinete (P=0.0001) and oocyst numbers (P=0.0001) on their internal and external midgut surfaces, respectively. Ookinetes were located in the peritrophic matrix (PM), but neither inside epithelial cells nor on the haemocoelic midgut surface by transmission electron microscopy in 24h p.i.-An. pseudopunctipennis mosquito samples. In contrast, no parasites were detected in the PM of An. albimanus at this time point. These results suggest that P. vivax Vk210 ookinetes cannot escape from and are destroyed within the midgut lumen of An. pseudopunctipennis.     

Infection of the malaria mosquito Anopheles gambiae with the entomopathogenic fungus Metarhizium anisopliae reduces blood feeding and fecundity

The entomopathogenic fungus Metarhizium anisopliae is being considered as a biocontrol agent against adult African malaria vectors. In addition to causing significant mortality, this pathogen is known to cause reductions in feeding and fecundity in a range of insects. In the present study we investigated whether infection with M. anisopliae affected blood feeding and fecundity of adult female malaria vectors Anopheles gambiae Giles sensu stricto. Mosquitoes were contaminated with either a low or a moderately high dose of oil-formulated conidia of M. anisopliae, and offered a single human blood meal 48, 72, or 96 h later to assess feeding propensity and individual blood meal size. In a second experiment, individual fungus-infected females were offered a blood meal every third day (to a total of 8 gonotrophic cycles), and allowed to oviposit after each cycle in order to quantify feeding propensity and fecundity. Infected females took smaller blood meals and displayed reduced feeding propensity. It was found that mosquitoes, inoculated with a moderately high dose of fungal conidia, exhibited reduced appetite related to increasing fungal growth. Of the fungus-infected females, the proportion of mosquitoes taking the second blood meal was reduced with 51%. This was further reduced to 35.3% by the 4th blood meal. During 8 feeding opportunities, the average number of blood meals taken by uninfected females was 4.39, against 3.40 (low dose), and 2.07 (high dose) blood meals for the fungus-infected females. Moreover, infected females produced fewer eggs per gonotrophic cycle and had a lower life-time fecundity. Epidemiological models show that both blood feeding and fecundity are among the most important factors affecting the likelihood of a mosquito transmitting malaria, which suggests that this fungus may have potential as biocontrol agent for vector-borne disease control.     

Multiple blood meals in Anopheles darlingi (Diptera: Culicidae)

Anopheles darlingi is an important vector of human malaria in the Amazon. Adult females of this mosquito species require a blood meal to develop eggs, preferring humans to other blood sources. Although gonotrophic concordance has been described as the norm for An. darlingi, here we report An. darlingi female mosquitoes taking two or more blood meals within their first gonotrophic cycle. Only half of field‐captured adult females fed one blood meal developed follicles to Christophers' stage V. This outcome is dependent on larval nutrition, as 88% of laboratory‐raised well‐nourished females completed the first gonotrophic cycle with only one blood meal, while less nourished females needed additional blood meals. Half of the field‐captured blood‐seeking An. darlingi females had follicles in intermediate (IIIa and IIIb) and final (V) stages of the gonotrophic cycle, supporting the conclusion that An. darlingi blood feed more than once during a gonotrophic cycle. Additionally, we observed females attempting to blood feed a second time during the same day. Additional studies of An. darlingi biting behavior are necessary to accurately estimate Plasmodium sp. entomologic inoculation rates throughout the An. darlingi vast geographical distribution.     

Hemolymph of Anopheles stephensi from noninfected and Plasmodium berghei-infected mosquitoes. 3. Carbohydrates.

Determinations were made of carbohydrates in hemolymph collected from adult female mosquitoes (Anopheles stephensi). First the hemolymph was fractionated by extraction and precipitation procedures, after which qualitative and quantitative determinations of carbohydrates were made by thin layer chromatography. The most abundant sugars found in the hemolymph were glucose and trehalose, though maltose, glucuronic acid, and inositol could be found after the mosquitoes took blood meals. After the mosquitoes ingested a noninfected blood meal, their hemolymph sugar levels rose almost 4-fold. There was less of an increase following a blood meal infected with the rodent malaria parasite, Plasmodium berghei. Depletion of sugars in the hemolymph of infected mosquitoes may result from direct utilization of sugar by the malaria parasite developing within the mosquito.     

Ultrastructure of the encapsulation of Plasmodium cynomolgi (B strain) on the midgut of a refractory strain of Anopheles gambiae

Using transmission electron microscopy, we investigated the encapsulation of the simian malaria parasite, Plasmodium cynomolgi, in a refractory strain of the mosquito, Anopheles gambiae. After the ookinete penetrates the mosquito midgut epithelium and lodges between the basal membrane and the basal lamina, an electron-dense, melanin-like substance begins to coalesce around the parasite. Completely encapsulated parasites were found as early as 16 hr after the blood meal. Granules of the melanin-like substance often appeared to condense onto the parasite from the fluid in the extracellular spaces of the basal membrane labyrinth. Melanin granules also appeared to condense from the hemolymph onto the basal lamina underlying the parasite. In addition, groups of tubules, vesicles, and membranous whorls often were found in midgut cells that were located next to or were enclosing parasites. These structures were unusually electron-dense, and may have been associated with melanization. Hemocytes rarely were observed near completed capsules and neither hemocytes nor their remnants were components of the capsules. During later stages of encapsulation, parasites appeared abnormal and often were infiltrated with melanin. Although late-stage capsules were usually located basally, completed capsules enclosed by membranes were occasionally observed near the apical border of the midgut. Other capsules associated with cellular debris, were found in the lumen of the midgut from 1 to 6 days after the blood meal.     

Reactive oxygen species modulate Anopheles gambiae immunity against bacteria and Plasmodium

The involvement of reactive oxygen species (ROS) in mosquito immunity against bacteria and Plasmodium was investigated in the malaria vector Anopheles gambiae. Strains of An. gambiae with higher systemic levels of ROS survive a bacterial challenge better, whereas reduction of ROS by dietary administration of antioxidants significantly decreases survival, indicating that ROS are required to mount effective antibacterial responses. Expression of several ROS detoxification enzymes increases in the midgut and fat body after a blood meal. Furthermore, expression of several of these enzymes increases to even higher levels when mosquitoes are fed a Plasmodium berghei-infected meal, indicating that the oxidative stress after a blood meal is exacerbated by Plasmodium infection. Paradoxically, a complete lack of induction of catalase mRNA and lower catalase activity were observed in P. berghei-infected midguts. This suppression of midgut catalase expression is a specific response to ookinete midgut invasion and is expected to lead to higher local levels of hydrogen peroxide. Further reduction of catalase expression by double-stranded RNA-mediated gene silencing promoted parasite clearance by a lytic mechanism and reduced infection significantly. High mosquito mortality is often observed after P. berghei infection. Death appears to result in part from excess production of ROS, as mortality can be decreased by oral administration of uric acid, a strong antioxidant. We conclude that ROS modulate An. gambiae immunity and that the mosquito response to P. berghei involves a local reduction of detoxification of hydrogen peroxide in the midgut that contributes to limit Plasmodium infection through a lytic mechanism.     

Diuresis after a bloodmeal in female Anopheles freeborni

Female Anopheles freeborni discharge urine rapidly and copiously for a brief time after taking a meal of blood. This diuresis begins immediately upon cessation of feeding and continues for about 30 min at a constant rate. A decline in this rate follows and diuresis is completed by 50 min after feeding. This time-course of diuresis is independent of the size of the meal; diuresis after large meals occures at a higher rate, not over a longer time, than diuresis after small meals. Heat-stable and saline-soluble substances that induce rapid excretion by isolated Malpighian tubules can be extracted from the head and thoracic nervous system, suggesting the presence of a neurosecretory diuretic hormone similar to that found in other blood-sucking insects. Decapitation or section of the ventral nerve cord abolishes the rapid phase of diuresis after a bloodmeal. Therefore, in analogy to the situation in the tsetse fly, the head is required either as the source of a diuretic hormone or as link in the pathway that stimulates its release.     

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.