Activation of vitellogenin synthesis in the mosquito Aedes aegypti by ecdysone

Injected β-ecdysone was found to induce the synthesis of yolk protein (vitellogenin) in adult female Aedes aegypti without a blood meal. After injection of 5 μg ecdysone per mosquito, vitellogenin constituted 80 per cent of the total protein secreted by explanted fat body, a proportion comparable to that produced by fat body from blood-fed females. Moreover, the time course of induction of vitellogenin synthesis in ecdysone-injected mosquitoes was similar to that triggered by a blood meal. Response to ecdysone is dosedependent: 0·5 μg per female was required to stimulate synthesis to 50 per cent of the level found 18 hr after a blood meal. Ecdysone was effective in decapitated or ovariectomized mosquitoes, and also when applied directly to fat body preparations in vitro. Thus it appears that ecdysone acts directly on the fat body to induce specific protein synthesis, as does the vitellogenin stimulating hormone (VSH) from the ovary of blood-fed mosquitoes. These results suggest that ecdysone can replace VSH in inducing vitellogenin synthesis in the unfed mosquito.     

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.     

Development of responsiveness to hormones after a blood meal in the mosquito Aedes aegypti

The effects of exogenous hormones on oocyte development in isolated abdomens from blood-fed female Aedes aegypti were examined. Abdomens were prepared immediately after a blood meal. Single applications of hormones were administered immediately after ligation or 18 hr after the blood meal. Double applications were done at both times. Oocyte development was assayed by measuring the amount of yolk in oocytes 66 hr after the blood meal. Topical application of maximum doses of methoprene immediately after ligation caused oocytes to mature in 60% of the abdomens; a half-maximum response was obtained with 300 pg. Injection of 700 ng of 20-hydroxyecdysone (20-HE) was necessary to cause an equivalent response. Delaying the injection of 20-HE until 18 hr after feeding reduced the amount necessary to obtain a half-maximum response to 150 ng. Treating the abdomens twice dramatically reduced the amount of 20-HE needed for the second dose: pretreatment of abdomens immediately after ligation with 50 pg of 20-HE reduced the amount of 20-HE needed in the second injection to 30 ng. Pretreatment with a topical application of 50 pg of methoprene had a similar effect. These data indicate that the sensitivity of the mosquito to exogenous hormones changes after a blood meal, and that either 20-HE or methoprene can promote a further increase in sensitivity.     

Parasitic pattern of rodent Plasmodium in blood from mouse tail and from Anopheles blood meal

During the course of the infection of mice by rodent malaria parasites (P. yoelii yoelii, P. vinckei petteri, and P. chabaudi chabaudi) the parasitic pattern in the blood smears differed according to the site of sampling: mosquito blood meal (usually actively sucked from capillaries) or mouse tail (issued from larger vessels). There were fewer old trophozoites and mature schizonts, and more immature schizonts, in the mosquito blood meal than in the tail blood of mice. It is suggested that the erythrocyte containing a schizont becomes less rigid when merozoites formation begins. The greater abundance in the mosquito blood meal of the gametocytes infective stage (0-I for P. yoelii, II for P. vinckei), previously shown, could be due to a particular flexibility of this stage, a character progressively selected.     

Localization of the mosquito insulin receptor homolog (MIR) in reproducing yellow fever mosquitoes (Aedes aegypti)

The female mosquito takes a blood meal to produce a batch of eggs. Initiation of egg maturation and growth of oocytes is governed by several endocrine factors. Peptide factors from the brain are involved in this process and some are also responsible for the induction of ecdysone secretion. The latter appears to be required to maintain a high rate of vitellogenin synthesis. By analogy with the known functions of insulin-like molecules (e.g. bombyxins) which in insects activate the secretion of ecdysteroids, we have postulated that there is an insulin receptor homolog responsible for activation of endysone secretion in the ovary. We have recently cloned the mosquito homolog (MIR) and are now investigating its spatial and temporal distribution. Here, we have localized the insulin receptor (MIR) both at the mRNA and protein level using in situ-hybridization and immunocytochemistry. The receptor is expressed before a blood meal mainly in the nurse cells of ovaries. After a meal, follicle and nurse cells contain mRNA coding for the receptor. The intensity of expression rises in the follicle cells until they degenerate during choriogenesis. Immunocytochemical localization confirms the in situ data: the protein is present before and after a meal. Both methods confirm our previous findings by Northern blot analysis, in which the ovary was found to be the main source of the receptor mRNA. The dynamics of receptor mRNA are related to the dynamics of ecdysone secretion and its action on physiological processes.     

Egg development in the mosquito Anopheles albimanus

Summary

In the mosquito, Anopheles albimanus, previtellogenic egg development was completed by 48 h after emergence, and vitellogenic growth was completed by 36 h after a blood meal. Ecdysteroid levels reached a peak of 800 pg/female by 18 h, while vitellin levels rose to their maximum 36–48 h after a blood meal. Most of the ecdysteroids present in the female before 36 h behaved as ‘free’ hormone, while after 42 h the ecdysteroids were ‘conjugated’. Injection of 20-hydroxyecdysone into non-blood-fed females induced degeneration of the resting stage oocytes, but vitellogenin synthesis was detectable by autoradiography. Injection of 5 μ of 20-hy-hroxyecdysone into blood-fed decapitated females induced almost precisely normal levels of vitellin. Detailed analysis of the effect of decapitating blood-fed females suggested that the release of factors from the head (e.g., egg development neurosecretory hormone) occurs as an all-or-none phenomenon, and probably occurs twice.     

Dynamic gut microbiome across life history of the malaria mosquito Anopheles gambiae in Kenya

The mosquito gut represents an ecosystem that accommodates a complex, intimately associated microbiome. It is increasingly clear that the gut microbiome influences a wide variety of host traits, such as fitness and immunity. Understanding the microbial community structure and its dynamics across mosquito life is a prerequisite for comprehending the symbiotic relationship between the mosquito and its gut microbial residents. Here we characterized gut bacterial communities across larvae, pupae and adults of Anopheles gambiae reared in semi-natural habitats in Kenya by pyrosequencing bacterial 16S rRNA fragments. Immatures and adults showed distinctive gut community structures. Photosynthetic Cyanobacteria were predominant in the larval and pupal guts while Proteobacteria and Bacteroidetes dominated the adult guts, with core taxa of Enterobacteriaceae and Flavobacteriaceae. At the adult stage, diet regime (sugar meal and blood meal) significantly affects the microbial structure. Intriguingly, blood meals drastically reduced the community diversity and favored enteric bacteria. Comparative genomic analysis revealed that the enriched enteric bacteria possess large genetic redox capacity of coping with oxidative and nitrosative stresses that are associated with the catabolism of blood meal, suggesting a beneficial role in maintaining gut redox homeostasis. Interestingly, gut community structure was similar in the adult stage between the field and laboratory mosquitoes, indicating that mosquito gut is a selective eco-environment for its microbiome. This comprehensive gut metatgenomic profile suggests a concerted symbiotic genetic association between gut inhabitants and host.     

Formation of lipid reserves in fat body and eggs of the yellow fever mosquito, Aedes aegypti

We examined the accumulation of lipids in adult females of the mosquito, Aedes aegypti. Females emerged with about 100 μg lipid in the fat body. With access to sugar water lipids increased over seven days to 300 μg. After a blood meal on day five, sugar-fed females accumulated 120-140 μg of lipids in their ovaries within 2 days. At the same time the lipid content of the fat body decreased by 100 μg, indicating transfer of lipids from fat body to oocytes. Experiments in which fat body lipids were prelabelled support this conclusion. Label was transferred to oocytes: in mature oocytes the specific radioactivity of lipids was 80% of the specific radioactivity of prelabeled fat body lipids. Components of blood meals are also used to synthesize oocyte lipids. Fat bodies of females starved for four days had only 27 μg of lipids left. When these females were given a blood meal, they matured oocytes, although the number of ooyctes was reduced and ovaries contained only half the amount of lipids found in ovaries of females which had first fed on sugar water. Fat body lipids of these females had only slightly increased to 36 μg. This demonstrates that female Ae. aegypti use sugar to synthesize lipids, but they can also use components of blood for this purpose.     

Initiation and termination of host-seeking inhibition in Aedes aegypti during oöcyte maturation

The inhibition of host-seeking behaviour that accompanies vitellogenesis in the mosquito, Aedes aegypti, was examined by the removal and implantation of ovaries. Mosquitoes ovariectomized before a blood meal and between 1 and 6 hr after a blood meal responded to a host at 48 hr after a blood meal. However, when ovariectomy was delayed until 10 hr after the meal or later, most mosquitoes did not respond to the host. When a partial ovary was present for only the first 12 hr after a meal, there was no host-seeking inhibition at 48 hr, and only 58% of females with one complete ovary present during this time interval responded. Howver, these same amounts of ovarian tissue inhibited host-seeking when they remained for 48 hr after a meal. Vitellogenic ovaries from donors blood-fed 8–24 hr before, implanted into sugar-fed recipients, did not affect the host-seeking behaviour of these recipients. Ovaries removed and reimplanted before the blood meal inhibited host-seeking at 72 hr after the blood meal only in the absence of oviposition from intact ovaries. It is concluded that 2 humoral factors are involved in the promotion of host-seeking inhibition: the first factor is produced by the ovaries, and after reaching a critical threshold in the haemolymph, stimulates the release of a second factor that acts directly to inhibit mosquito behaviour. An ovary which retains 2 or fewer eggs after oviposition terminates the inhibition via nervous pathways. The role of 20-hydroxyecdysone in the behavioural inhibition is discussed.     

Juvenile hormone regulation of the second biting cycle in Culex pipiens

Juvenile hormone regulation of the second biting cycle was studied in Culex pipiens by allatectomizing mosquitoes at daily intervals after the first blood meal. Mosquitoes oviposited and were tested for biting 2 days later. Allatectomy on days 1 through 4 prevented biting in a high percentage of females, indicating that juvenile hormone was required for a second biting cycle. When allatectomy was delayed 6 or more days after the first blood meal, the mosquitoes took a second blood meal after oviposition even though eggs were retained at the time of allatectomy. Thus, egg retention did not prevent mosquitoes from releasing juvenile hormone for a second biting cycle. Unoperated mosquitoes also bit when they were forced to retain eggs after the first blood meal. However, these mosquitoes only fed on blood if they were confined on or near the host; they would not feed in cages where host seeking was required to locate the host. Based on these findings, it appeared that oviposition was necessary to initiate host-seeking behaviour, whereas juvenile hormone release initiated biting. These results, in conjunction with earlier work demonstrating juvenile hormone induction of the first biting cycle, indicated that alternating periods of juvenile hormone production and oviposition regulate the cyclic patterns of biting and host-seeking behaviour throughout the life of the mosquito.     

Ecdysteroid titer and oocyte growth in the northern house mosquito, Culex pipiens L

In an anautogenous strain of the northern house mosquito, Culex pipiens, the ovaries reached the resting stage (follicle length = 90 microns) three days after adult emergence. Follicle length increased from 90 to 550 microns between 0 and 60 hr after a blood meal. Total ecdysteroids reached a peak at 400 fmol/insect at 36 hr after a blood meal then declined rapidly. The ratio of 20-hydroxyecdysone to ecdysone increased in conjunction with the total ecdysteroid level. Oocyte growth beyond the resting stage and initiation of vitellogenesis was dependent on a head factor which was released within 4-8 min of the start of the blood meal.     

Control of follicular epithelium development and vitelline envelope formation in the mosquito; role of juvenile hormone and 20-hydroxyecdysone

Using microsurgical manipulations, hormone applications, and transmission electron microscopy we have investigated the regulation of differentiation of the follicular epithelium and formation of the vitelline envelope (VE) in primary follicles in the ovary of the mosquito, Aedes aegypti. During the first 3 days after eclosion, the primary follicle grows, and cells of the follicular epithelium differentiate, their content of mitochondria, rough endoplasmic reticulum, and Golgi complexes increases significantly. Growth and differentiation of the follicular epithelium appear to be under the control of juvenile hormone (JH), because they are blocked by removal of corpora allata in newly closed adult females and can be restored by either implantation of corpora allata or application of JH III. In insects, including mosquitoes, VE is the first layer of the eggshell to be deposited. It is formed from the secretory products of the follicle cells and its deposition coincides with yolk accumulation by developing oocytes. Only follicle cells adjacent to the oocyte deposit VE. In decapitated females, given a blood meal by enema and injected with picogram doses of 20-hydroxyecdysone (20-HE), follicle cells synthesize the VE precursors and deposit morphologically normal VE, in contrast to saline injected controls which deposit no VE. We conclude that 20-HE, as well as factors originating from the blood meal and the oocyte, are required for the normal formation of VE in the mosquito follicles.     

A method for estimating blood meal volume in Aedes aegypti using a radioisotope

A study was conducted to determine the feasibility of measuring blood meal volume in Aedes aegypti using a radioisotope blood label in lieu of weight differentials. This mosquito species defaecated numerous droplets of a clear fluid beginning while blood feeding was in progress and continuing for up to 3 hr post-feeding. Excretion of this clear fluid occurred regardless of the age or prior nutritional conditioning of the mosquito. When mosquitoes were fed on blood labeled with Ce-144, this radioisotope remained completely with the blood meal and was not passed in the clear excreta. When blood meal volumes were estimated using the radioisotope method, significantly greater values were obtained compared to those calculated by the classical weighing method. A direct relationship was demonstrated between blood meal volume and the quantity of clear fluid excreted. Consequently, the error inherent in the weighing method was increased in mosquitoes taking large blood meals. Ce-144 did not label red blood cells but remained in the plasma. The radioisotope was completely expelled with the blood meal excreta.This technique is compared with related methods previously described. Its applications to problems in mosquito physiology and vector potential are discussed.     

Juvenile hormone and juvenile hormone esterase in adult females of the mosquito Aedes aegypti

Juvenile hormone III levels and juvenile hormone esterase activity were measured in whole body extracts and haemolymph, respectively, of female Aedes aegypti. The amount of juvenile hormone, determined by coupled gas chromatography-mass spectrometry, rose over the first 2 days after emergence from 0.7 to 7.5 ng/g, and then slowly fell over the next 5 days in females not given a blood meal. In females fed blood, juvenile hormone levels fell during the first 3 h to 2.3 ng/g. The rate of decline then slowed so that levels had reached their lowest point (0.4 ng/g) by 24 h after the blood meal. By 48 h, levels started to rise again until 96 h when they were equivalent to pre-blood meal levels.Juvenile hormone esterase activity in the haemolymph of females was measured with a partition assay. The esterase activity showed small fluctuations in unfed animals. In females fed blood on the 3rd day after emergence, the juvenile hormone esterase activity rose slowly to a peak at 36 h. At 42 h it began to decline, and by 66 h it had returned to pre-blood meal levels. Thus, juvenile hormone levels and juvenile hormone esterase activity were inversely correlated after a blood meal. Both the ovary and fat body produce juvenile hormone esterase in organ culture.Juvenile hormone III acid was the only metabolite produced after incubation of haemolymph with racemic-labelled juvenile hormone III. Juvenile hormone acid, diol, and acid diol were the main metabolic products seen in whole animal extracts after topical application of labelled hormone. About 25% of topically applied, labelled juvenile hormone appears in the haemolymph as the acid diol, and 50% of this is excreted in the urine immediately after the blood meal. Topical application of BEPAT (S-benzyl-O-ethyl phosphoramidothiolate), a specific inhibitor of juvenile hormone esterase, resulted in the absence of juvenile hormone acid and a reduction in the acid diol. Both BEPAT and methoprene, a juvenile hormone analogue, caused a reduction in egg hatch when applied topically 30 h after a blood meal, demonstrating that the decline in juvenile hormone levels after a blood meal is necessary for normal egg development and suggesting that the decline is mediated, at least in part, by juvenile hormone esterase.     

Synthetic propeptide inhibits mosquito midgut chitinase and blocks sporogonic development of malaria parasite

Incessant transmission of the parasite by mosquitoes makes most attempts to control malaria fail. Blocking of parasite transmission by mosquitoes therefore is a rational strategy to combat the disease. Upon ingestion of blood meal mosquitoes secrete chitinase into the midgut. This mosquito chitinase is a zymogen which is activated by the removal of a propeptide from the N-terminal. Since the midgut peritrophic matrix acts as a physical barrier, the activated chitinase is likely to contribute to the further development of the malaria parasite in the mosquito. Earlier it has been shown that inhibiting chitinase activity in the mosquito midgut blocked sporogonic development of the malaria parasite. Since synthetic propeptides of several zymogens have been found to be potent inhibitors of their respective enzymes, we tested propeptide of mosquito midgut chitinase as an inhibitor and found that the propeptide almost completely inhibited the recombinant or purified native Anopheles gambiae chitinase. We also examined the effect of the inhibitory peptide on malaria parasite development. The result showed that the synthetic propeptide blocked the development of human malaria parasite Plasmodium falciparum in the African malaria vector An. gambiae and avian malaria parasite Plasmodium gallinaceum in Aedes aegypti mosquitoes. This study implies that the expression of inhibitory mosquito midgut chitinase propeptide in response to blood meal may alter the mosquito's vectorial capacity. This may lead to developing novel strategies for controlling the spread of malaria.     

Nutrient limitation results in juvenile hormone-mediated resorption of previtellogenic ovarian follicles in mosquitoes

Juvenile hormone (JH) is a central hormonal regulator of previtellogenic development in female Aedes aegypti mosquitoes. JH levels are low at eclosion and increase during the first day after adult emergence. This initial rise in JH is essential for female reproductive maturation. After previtellogenic maturation is complete, the mosquito enters a ‘state-of-arrest’ during which JH synthesis continues at a slower pace and further ovary development is repressed until a blood meal is taken. By examining the relationships between juvenile hormone, follicular resorption and nutrition in A. aegypti, we were able to define a critical role of JH during the previtellogenic resting stage. The rate of follicular resorption in resting stage mosquitoes is dependent on nutritional quality. Feeding water alone caused the rate of follicular resorption to reach over 20% by day 7 after emergence. Conversely, feeding a 20% sucrose solution caused resorption to remain below 5% during the entire experimental period. Mosquitoes fed 3% sucrose show rates of resorption intermediate between water and 20% sucrose and only reached 10% by day 7 after emergence. Follicular resorption is related to JH levels. Ligated abdomens separated from a source of JH (the corpora allata) showed an increase in resorption comparable to similarly aged starved mosquitoes (16%). Resorption in ligated abdomens was reduced to 6% by application of methoprene. The application of methoprene was also sufficient to prevent resorption in intact mosquitoes starved for 48 h (14% starved vs. 4% starved with methoprene). Additionally, active caspases were localized to resorbing follicles indicating that an apoptotic cell-death mechanism is responsible for follicular resorption during the previtellogenic resting stage. Taken together, these results indicate that JH mediates reproductive trade-offs in resting stage mosquitoes in response to nutrition.     

Plasmodium sex determination and transmission to mosquitoes

In order to be transmitted by their mosquito vector, malaria parasites undergo sexual reproduction, which occurs between specialized male and female parasites (gametes) within the blood meal in the mosquito. Nothing was known about how Plasmodium determines the sex of its gametocytes (gamete precursors), which are produced in the vertebrate host. Recently, erythropoietin, the vertebrate hormone controlling erythropoiesis in response to anaemia, was implicated in Plasmodium sex determination in animal models of malaria. This review examines the available information and addresses the relevance of such a sex determining mechanism for Plasmodium falciparum transmission to mosquitoes, with special reference to low gametocytaemias.