Nitrogen partitioning and blood meal utilization by Aedes aegypti (Diptera Culicidae)

The utilization of the blood meal by mosquitoes was investigated by first feeding females quantities of blood ranging from 1 to 5 mg, and then analyzing the faeces for the various by-products of protein catabolism that were subsequently eliminated. The nitrogeneous waste products in order of importance were uric acid, histidine, ammonia and arginine. Only traces of the other amino acids were excreted.The total amount of each faecal substance varied linearly with the quantity of blood ingested, however their relative proportions did not change. Regardless of blood meal size the quantily of uric acid and ammonia produced indicates that about 80% of the non-histidine and arginine amino acids are deaminated and utilized for metabolic purposes other than egg protein synthesis.Most of the histidine and about one half of the arginine content of the blood were excreted as free amino acids, but the other amino acids were lost in trace amounts.Nineteen per cent of the total ingested amino acids was incorporated into soluble yolk proteins and this proportion was constant even for small blood meals that result in a reduction in the numbers of eggs produced.The comparative aspects of nitrogen partitioning and blood meal utilization by haematophagous insects, as well as the factors that affect blood meal utilization and fecundity in A. aegypti are discussed.     

Prevalence of parasitism and adult survival time of Aedes albifasciatus (Diptera: Culicidae) parasitized by Strelkovimermis spiculatus (Nematoda: Mermithidae)

We described the carryover of Strelkovimermis spiculatus (Poinar and Camino) (Nematoda: Mermithidae) from mosquito larvae, the primary site of maturation, to adults. We analyzed the survival time of male and female Aedes albifasciatus (Macquart) (Diptera: Culicidae) parasitized by S. spiculatus, the time of emergence of nematodes from adult mosquitoes, and the state of parasitism in the same mosquito cohorts during the immature stages. Mosquito larvae with single and multiple parasitism (up to 11 parasites) were observed. The mortality of mosquito larvae and adults was produced in all cases where at least one mermithid emerged. The mortality of S. spiculatus showed an increasing trend in mosquito larvae with larger numbers of nematodes and was higher in larvae parasitized by eight or more nematodes. Maximum survival of parasitized adult females of Ae. albifasciatus was 38 days, while non‐parasitized adult males and females survived 39 and 41 days, respectively. Strelkovimermis spiculatus mortality was observed in Ae. albifasciatus larvae with single or multiple parasitisms. The spread of mermithid parasitism in adult mosquito populations is discussed.     

Parasitism of Kenyan Mosquito Larvae (Diptera: Culicidae) by Romanomermis culicivorax (Nematoda: Mermithidae)

The ability of Romanomermis culicivorax to infect, develop, and emerge from Kenyan mosquito hosts was evaluated in the laboratory. Host species tested were Aedes aegypti, Ae. dentatus, Ae. hirsutus, Anopheles arabiensis, An. coustani, An. funestus, An. gambiae, An. pharoensis, Culex duttoni, Cu. ethiopicus, Cu. poicilipes, Cu. quinquefasciatus, Cu. tigripes, Cu. univittatus, Coquillettidia metallica, Mansonia africana, Ma. uniformis, Mimomyia splendens, Mi. uniformis, Toxorhyncites brevipalpis, and Uranotaenia balfouri. R. culicivorax penetrated all the host species tested and developed and emerged from most of the hosts. Both penetration and some development, but not nematode emergence, were observed from all instars of Ma. uniformis. T. brevipalpis exhibited signs of resistance in the form of melanization of R. culicivorax within 48 hours of infection in all four instar stages. Nematode melanization, especially in older hosts, was observed in Ae. dentatus, Ae. hirsutus, Cu. duttoni, Cu. tigripes, and Mi. splendens. When melanization occurred, the melanized carcass of the nematode was passed on from instar to instar. The implications for field release of R. culicivorax in Kenya are still good, especially in habitats where different mosquito species occupy the same niche at different times, which would allow for nematode recycling.     

Analysis of molecular markers for metamorphic competency and their response to starvation or feeding in the mosquito, Aedes aegypti (Diptera: Culicidae)

The nutritional condition of fourth instar larvae of the yellow fever mosquito, Aedes aegypti, governs female longevity and egg production, both are key determinants of pathogen transmission. As well, nutrition provisions larval growth and development and attains its greatest pace in the last larval instar in preparation for metamorphosis to an adult. These developmental processes are regulated by a complex endocrine interplay of juvenile hormone, neuropeptides, and ecdysteroids that is nutrition sensitive. We previously determined that feeding for only 24 h post-ecdysis was sufficient for fourth instar Ae. aegypti larvae to reach critical weight and accumulate sufficient nutritional stores to commit to metamorphosis. To understand the genetic basis of metamorphic commitment in Ae. aegypti, we profiled the expression of 16 genes known to be involved in the endocrine and nutritional regulation of insect metamorphosis in two ways. The first set is a developmental profile from the beginning of the fourth instar to early pupae, and the second set is for fourth instars starved or fed for up to 36 h. By comparing the two sets, we found that seven of the genes (AaegCYP302, AaegJHE43357, AaegBrCZ4, AaegCPF1-2, AaegCPR-7, AaegPpl, and AaegSlif) were expressed during metamorphic commitment in fourth instars and in fed but not starved larvae. Based on these results, the seven genes alone or in combination may serve as molecular indicators of nutritional and metamorphic status of fourth instar Ae. aegypti larvae and possibly other mosquito species in field and laboratory studies to gauge sub-lethal effects of novel and traditional cultural or chemical controls.     

Effect of Aedes aegypti (Diptera:Culicidae) Age on Sex Ratios in Romanomermis culicivorax (Nematoda:Mermithidae)

First, second, third, early and late fourth-instar larvae, and pupae of Aedes aegypti were infected with Romanomerrais culicivorax and reared at 20, 25, and 30 C. An increase in the ratio of male to female nematodes was observed with increase in host age at the time of infection at each temperature. The number of pupal and late fourth-instar infections was low, but R. culicivorax continued to develop in adult A. aegypti. Since male nematodes were recovered from both male and female hosts infected as late fourth instars or pupae, the sex of the host did not influence the sex of the nematode.     

Blood intake by Aedes aegypti not regulated by insemination

A chemical method of determining the amount of blood ingested by mosquitoes was used to test the claim of Adlakha and Pillai (1976) that insemination is involved in the regulation of blood meal size. Inseminated and uninseminated Aedes aegypti do not differ in the amount of blood ingested. The data show that if excretion before, during, and after feeding, is not accounted for, unacceptable errors are introduced in blood meal size determinations.     

A new cytoplasmic polyhedrosis virus from the salt-marsh mosquito, Aedes cantator (Diptera: Culicidae)

The ultrastructure, development, and histopathology of a new cytoplasmic polyhedrosis virus of Aedes cantator are described. Virus particles measure 70 nm in diameter, are icosahedral in shape, and consist of a central electron-dense core surrounded by a capsid with six projections. Occlusion bodies are irregular in size (0.5–3.0 μm) and shape and contain several virus particles. Virus particles are assembled within an interconnecting network of fine filaments and are occluded by the deposition of a proteinaceous crystal around groups of mature virus particles within a virogenic stroma. Infections are confined to cells of the cardia, gastric ceca, and posterior portion of the midgut, which hypertrophy and frequently lyse. Infected larvae die during the fourth larval instar or as pupae. The prevalence of infection in natural field populations is less than 1%.     

Artifactual stimulation of vitellogenesis in Aedes aegypti by 20-hydroxyecdysone

Single or repeated, non-physiological, high doses (0.5–5.0 μg/female) of 20-hydroxyecdysone or ecdysone injected into sugar-fed female Aedes aegypti stimulated follicular growth and deposition of yolk, but suppressed accumulation of protein yolk to approximately one-third, and lipid yolk to one-half that in an equal number of follicles with equivalent yolk length taken from blood-fed controls. Physiological doses (500 pg/female) of ecdysone or 20-hydroxyecdysone or the implantation of ecdysone-secreting ovaries (verified by bioassay), into sugar-fed females failed to induce any yolk deposition. In these experiments, yolk precursors were not the limiting factor, because in decapitated females, digesting a blood meal, the injection of a physiological dose of 20-hydroxyecdysone or the implantation of ecdysone-secreting ovaries still did not stimulate vitellogenesis. Finally, continuous infusion of 500 pg or even 50 ng 20-hydroxyecdysone/hr for 22 hr was as ineffective as single or multiple injections of equivalent doses of hormone. Consequently, rapid excretion or catabolism of 20-hydroxyecdysone by the sugar-fed female does not explain the need for high doses to induce vitellogenesis, or the failure of oöcytes to mature with normal protein and lipid content. Apparently, ovarian ecdysone is not the factor by which normal vitellogenesis is initiated and maintained in this mosquito.     

Oxygen consumption in mosquito larvae parasitized by Romanomermis culicivorax (Nematoda)

The effect of parasitism by Romanomermis culicivorax on the oxygen consumption of three species of mosquito larvae was studied by manometric methods. The Q02 of Culex pipiens, both parasitized and unparasitized, decreased as the host larvae developed from the second to fourth instar. In the early stages there was no significant difference in Q02 in parasitized and unparasitized hosts but the Q02 increased significantly in parasitized fourth instar larvae. The Q02 of parasitized and unparasitized fourth instars of Aedes taeniorhynchus, Anopheles quadrimaculatus and C. pipiens were compared. The Q02 of all parasitized hosts was significantly increased and the Q02 of the two culicine species displayed a dramatic increase in oxygen consumption. The oxygen consumption of the parasitic mermithid larvae was estimated from that obtained earlier for the post-parasitic larvae but did not account for more than 9-36% of the increased Q02 of the parasitized hosts. On this basis it seems probable that the increased metabolic rate as reflected by oxygen consumption of the parasitized mosquito larve was principally a manifestation of the physiological and nutritional stress induced by the mermithid nematodes.     

Release of egg development neurosecretory hormone in Aedes aegypti and Aedes taeniorhynchus induced by an ovarian factor

Ovariectomized Aedes aegypti do not synthesize vitellogenin after a blood meal, unless an ovary from a blood-fed donor is implanted. Decapitation, however, prior to implantation inhibits vitellogenin synthesis. A female ovariectomized and decapitated 6 hr after a blood meal, synthesizes vitellogenin if an ovary from a blood-fed donor is implanted. On the other hand, females that are fed on blood and immediately decapitated can not be stimulated to synthesize vitellogenin with implanted ovaries removed from blood-fed donors. These experiments led to the hypothesis that the blood meal stimulates the ovary to secrete a corpus cardiacum stimulating factor, that in turn promotes release of egg development neurosecretory hormone stored in the corpus cardiacum.Injection of 20-hydroxy-ecdysone or ovarian extract prepared from ovaries removed from unfed females does not release egg development neurosecretory hormone. Thus corpus cardiacum stimulating factor is not 20-hydroxy-ecdysone, and ovaries removed from unfed females do not store it.The rate of inactivation of egg development neurosecretory hormone released from the corpus cardiacum after a blood meal was investigated by implanting an ovary into females that were blood fed for various intervals than decapitated and ovariectomized. Seventy per cent of implants grow when the operation is done 18 hr after feeding, and 30% when the operation is done between 18 and 24 hr after feeding, indicating that egg development neurosecretory hormone is stable for the first 18 hr after a blood meal.Aedes taeniorhynchus females ovariectomized 24 hr after adult emergence do not synthesize vitellogenin. When such a female is implanted with an ovary removed from a sugar-fed or blood-fed Aedes aegypti donor vitellogenin synthesis is initiated, and the implant grows. Decapitation prior to implantation inhibit vitellogenin synthesis and implants do not grow. These results indicate that corpus cardiacum stimulating factor is not species specific.     

Stochastic eco-epidemiological model of dengue disease transmission by Aedes aegypti mosquito

We present a stochastic dynamical model for the transmission of dengue that takes into account seasonal and spatial dynamics of the vector Aedes aegypti. It describes disease dynamics triggered by the arrival of infected people in a city. We show that the probability of an epidemic outbreak depends on seasonal variation in temperature and on the availability of breeding sites. We also show that the arrival date of an infected human in a susceptible population dramatically affects the distribution of the final size of epidemics and that early outbreaks have a low probability. However, early outbreaks are likely to produce large epidemics because they have a longer time to evolve before the winter extinction of vectors. Our model could be used to estimate the risk and final size of epidemic outbreaks in regions with seasonal climatic variations.     

Further study on Ascogregarina culicis in temperate Argentina: Prevalence and intensity in Aedes aegypti larvae and pupae

The bionomics of South American strains of Ascogregarina spp. are poorly known and the first studies were performed a few years ago. Our main objective was to characterize Ascogregarina culicis population in Aedes aegypti immatures in temperate Argentina. A total of 1800 water-filled containers were inspected within a cemetery of Buenos Aires City through a reproductive period of the host (October 2006-June 2007). The parasite was detected in 16.7% (329/1974) of the immatures and 8.5% (15/177) of the breeding sites. The prevalence decreased from 19.9% in larvae to 6.5% in pupae. In those infected breeding sites, about 85% of the immature mosquitoes harbor the parasite with a median intensity of nine trophozoites per larva and six gametocysts per pupa. The prevalence in shaded containers was higher than in sun exposed ones but the intensity of the infection was quite similar between both lighting conditions. Sun-exposed containers recorded water temperatures significantly higher than those under shade throughout the study period. Parasite trophozoites were only found from January to May with a clear seasonal pattern of prevalence. Monthly values of parasite prevalence and mosquito host (percentage of breeding sites and number of immatures) were significantly correlated at p < 0.05 when a temporal delay of two months was considered. Our results suggest that parasite prevalence is spatially and temporally heterogeneous in temperate urban Argentina, and these variations are associated with the host abundance.     

Circadian control of permethrin-resistance in the mosquito Aedes aegypti

Daily fluctuation of permethrin-resistance was found in adult mosquito Aedes aegypti, the major vector of dengue viruses in Taiwan. We hypothesized there is a relationship between resistance and the circadian clock. To test our hypothesis we correlated changes in the knock-down time (KT₅₀) response to permethrin with the expression of the pyrethroid-resistant gene CYP9M9 and the clock gene period (per) during a 12:12 h photoperiodic cycle. Rhythmic expression of per peaked at early scotophase of the light-dark cycle and at early subjective night in constant darkness. The values of KT₅₀ and the expression of CYP9M9 also exhibited circadian rhythms in both susceptible and permethrin-resistant mosquito strains, from which we inferred a link to the circadian clock. The KT₅₀ was significantly longer in the light than in the dark phase, and the level of CYP9M9 mRNA was maximal in early scotophase, dropped to a minimum in the midnight and then slowly increased through the photophase. Existence of a clock control over mosquito sensitivity to permethrin was further indicated by reduced expression of CYP9M9 and reduced mosquito resistance to permethrin after temporal silencing of the per gene. These data provide the first evidence on the circadian control of insect resistance to permethrin.     

Induction of apoptosis in densovirus infected Aedes aegypti mosquitoes

The mechanism of death in densovirus infected mosquitoes remains unexplored. This study investigated the cellular consequences of densovirus infection in Aedes aegypti mosquitoes after a second generation challenge with a densovirus isolated from adult Aedes albopictus mosquitoes in Thailand (AThDNV). Specimens were analyzed by TUNEL assay, fluorescent in situ hybridization (FISH) and a calorimic assay to detect activation of caspase 3-like activity. After challenge, moribund mosquitoes showed considerable evidence of TUNEL positive cells. The caspase 3-like activity assay showed that the presence of TUNEL positive cells was associated with increased levels of activated caspase 3-like activity in AThDNV infected mosquitoes.     

Strelkovimermis spiculatus n. sp. (Mermithidae: Nematoda) Parasitizing Aedes albifasciatus Mac. (Culicidae: Diptera) in Argentina

Strelkovimermis spiculatus n. sp. (Mermithidae: Nematoda), a parasite of the mosquito Aedes albifasciatus Mac. in Argentina, is described. Diagnostic characters of this species include a ventrally shifted mouth opening and spicules fused at the tips. The mermithid has been reared on Culex pipiens L. larvae in the laboratory.     

Fine structure of antennal sensilla of male Aedes aegypti (L.)

The terminal two antennal segments of male Aedes aegypti bear the same variety of sensillar types as the female's antenna, namely, sensilla chaetica, sensilla coeloconica, sensilla ampullacea, grooved pegs, and four types of sensilla trichodea: long and short, pointed-tipped trichodea and blunt-tipped types I and II. Each type of sensillum has a similar fine structure in both sexes. Of the 514 neurones which innervate these sensilla in the male, 91% are olfactory receptors, 7% mechanoreceptors, and 2% thermoreceptors. The total number of neurones in the male is about four times fewer than in the female, but the ratio of those responding to the various stimuli is similar.The sensilla studied herein probably mediate stimuli involved in location of suitable resting sites and nectar meals. In addition they are apparently involved in location of vertebrates as recent studies indicate that certain male mosquitoes are attracted to hosts to bring them into the proximity of the females for mating. This host finding behaviour of males would explain why they have the same sprectrum of sensillar types as do females.     

Plasmodium gallinaceum: Erythrocyte factor essential for zygote infection of Aedes aegypti

Zygotes of Plasmodium gallinaceum, fertilized in vitro and fed to Aedes aegypti mosquitoes through a membrane, formed oocysts only when a substance in the cytoplasm of uninfected erythrocytes was present. The relation between erythrocyte volume and infectivity was linear (1:1.2) up to a 50% hematocrit. The intraerythrocytic substance was both nondialyzable and poorly soluble in plasma. By carboxymethyl cellulose chromatography, cytoplasmic constituents eluted at pH 8.6 supported the same infection as control blood did; but higher and lower pH eluates supported none. Dialyzable factors present in the plasma, but absent from M199, enhanced infection but were not essential. Zygotes developed normally to ookinetes in the gut of plasma-fed mosquitoes, or when cultured in plasma or M199. Ookinetes from culture formed normal oocysts when fed to mosquitoes in blood or when injected with M199 into the hemocoels of unfed females. Mosquitoes fed infected blood containing lima bean or soybean trypsin inhibitor were unable to digest the erythrocytes and, although normal ookinetes developed, no oocysts formed. It appears from this and histological evidence that an erythrocyte substance, released by mosquito digestion, is needed for ookinete invasion of the gut epithelium.     

Evaluation of an experimental product based on Bacillus thuringiensis sorovar. israelensis against Aedes aegypti larvae (Diptera:Culicidae)

The larvicidal activity of an experimental formulation of Bacillus thuringiensis israelensis (Bti) against Aedes aegypti larvae was evaluated under laboratory and simulated field conditions (SFC). Samples of technical powder (TP) were assayed to establish the LC₅₀ and the potency of the product. The larvicidal activity of the TP and the tablet (T) were evaluated under SFC to assess the efficacy and the residual activity, measured against Ae. aegypti larvae. Either a T or 250 mg of TP were added to 50 L of water in plastic containers. Containers were exposed to sunlight or kept in the shade. Results showed a LC₅₀ of 0.26 mg/L and a potency of 750 ITU/mg. In spite of differences in the toxicity amongst TP and T samples, all of them killed 98–100% of the larvae and the mortality remained high for six months, in the shade. The replacement of 20% or 60% of the water volume did not affect the activity of the product. Seasonal differences influenced the persistence of the product in containers exposed to sunlight. Both formulations showed an excellent performance, especially when kept in the shade. The Bti tablet evaluated in this study is potentially very useful in programs to control dengue vectors.     

Diximermis peterseni (Nematoda: Mermithidae): a potential biocontrol agent of Anopheles mosquito larvae

The mermithid nematode Diximermis peterseni was successfully transferred and established in an artificial pond and then in a natural pond. The exposure in the artificial pond initially produced 92% parasitism in introduced larvae of Anopheles quadrimaculatus; then parasite activity gradually decreased and disappeared. However, it reappeared 8 months after the introduction and has been observed in introduced hosts during each of the 40 months since. The release of 2300 Anopheles (85–90% infected) into a natural pond (January–March 1971) produced 12–100% parasitism during the 8-month period between September 1971, and March 1972. Furthermore, during the same months in 1972–1973, an average of 88% of the Anopheles in the pond were parasitized. These data indicate that D. peterseni can be easily established and will produce significant levels of parasitism despite long periods of dry weather and low host densities.     

Ultrastructure of the anterior sense organs of adult Gastromermis boophthorae (Nematoda: Mermithidae)

The amphids of adult Gastromermis boophthorae exhibit an organization unlike any previously reported. Each amphid consists of a distal cuticular channel which opens to the exterior, and a proximal amphidial gland. The cuticular channel is double and 15–18 cilia are held in a tight bundle by the inner channel. The amphidial gland contains an extensive reticulum, and the nerve axons which give rise to the amphidial cilia are arranged around the outside of the gland; anteriorly these axons are characteristically flattened. A granular secretion is produced by the amphidial gland and passes via the reticulum to the double cuticular channel, thus bathing the ciliary bundle. The ultrastructure of the amphids is discussed in relation to functional considerations.