Effect of plasma components on the feeding response of the mosquito Aedes aegypti L. to adenine nucleotides

ABSTRACT. The gorging response of Aedes aegypti to the ATP dissolved in platelet-poor plasma is greater than that of ATP dissolved in 0.15 m NaCl. The plasma components NaHCO₃ and albumin account for the full effect of the potentiation. Phosphate or tris buffers do not duplicate the bicarbonate effect. In 0.15 m NaCl with bicarbonate but lacking albumin the concentrations inducing 50% feeding are 58 μM ATP, 140 μM ADP, 460 μM AMP and 1500 μM cAMP. Non-adenine nucleotides such as ITP and GTP and phytic acid, and 2,3-diphosphoglyceric acid had no activity.     

Dopamine levels in the mosquito Aedes aegypti during adult development, following blood feeding and in response to heat stress

Dopamine, a catecholamine neurotransmitter, is important for insect development and is known to be involved in insect stress responses. In the current study, dopamine was analysed in Aedes aegypti heads by HPLC. We found that immediately after adult emergence, males have significantly higher concentrations of dopamine than females, and that dopamine concentrations decrease with age in both sexes. Dopamine levels increase in females following a blood meal suggesting that dopamine might be involved in ovarian- and/or egg-development. We also found that female mosquitoes have a higher tolerance to a short term thermal stress in a water bath than males up to 44 degrees C, however, both sexes die if exposed to short term temperatures between 44 and 45 degrees C. Finally, we did not find any indication that dopamine levels were associated with short time thermal stress response in female mosquitoes.     

Effect of ATP analogues on the gorging response of Aedes aegypti

ABSTRACT. The ATP analogues adenylylimidodiphosphate and adenylylmethylenediphosphate are 3–5-fold more effective than ATP as gorging stimulants for Aedes aegypti. This increased potency is not due to the fact that the two analogues are not hydrolysed by the mosquito salivary apyrase, but most likely to their greater affinity to the mosquito gustatory receptor protein. The analogues 2'd ATP and 3'd ATP are about half as potent as ATP, while 2',3'-dideoxyadenosine triphosphate is 10-fold more potent than ATP in evoking the gorging response. It is proposed that removal of both hydroxyl groups eliminates binding of the stimulant at the ribose moiety, thus allowing the molecule greater freedom to rotate and bind more effectively to its two other binding sites at the amino group on the purine and at the terminal phosphate. Our data demonstrate that ATP activates the gorging response of Ae.aegypti merely by binding to its receptor protein and is not required as an exogenous source of energy. Gorging response to ATP is competitively inhibited by novobiocin.     

A study on the fecundity of male Aedes aegypti

After inseminating about 5 females, the males of Aedes aegypti are sexually depleted and most do not then re-inseminate additional females for the rest of their lives. Sexually depleted males generally replenish spermatozoa in their seminal vesicles and renew the secretion in their accessory glands, i.e. they again become fecund. Many replenished males do not chase or attempt to copulate with virgin females. Those males which do copulate do not ejaculate. Thus, celibate behaviour and failure to ejaculate rather than lack of fecundity are responsible for the inability of sexually depleted males to impregnate females.     

Normal versus alpha-amanitin induced cellular dynamics of the midgut epithelium in female Aedes aegypti L. (Insecta, Diptera) in response to blood feeding.

In midgut epithelial cells (stomach) of untreated female A. aegypti an increase in the surface area of the rough endoplasmic reticulum (rer) and in the ratio of membrane-bound to free ribosomes is morphometrically measured during digestion of the first blood meal. This can be correlated with the synthesis and release of digestive proteases. The dynamics of the ribosomes in A. aegypti are similar to those in A. stephensi. 3 ng alpha-amanitin per mosquito prevent normal blood digestion, the proliferation of the rer and the increase in the ratio of bound to free ribosomes. On the other hand, some synthesis of new ribosomes takes place.     

A genetical study of DDT resistance in the mosquito Aedes aegypti.

Crosses have been carried out to determine the relationship between adult DDT resistance and the three linkage groups of the mosquito Aedes aegypti. Two linkage groups were implicated in the control of DDT resistance. In the Bangkok-HR strain resistance derived mainly from linkage group III, probably with the maor effect from the gene R-DDT2. When resistance was transferred into a susceptible background, by outcrossing Bangkok-HR to strain 64 and reselecting, resistance in the resulting Bangkok-MR strain came from both linkage groups II and III.     

Feeding behaviour of female Aedes aegypti: effects of diet temperature, bicarbonate and feeding technique on the response to ATP

Abstract. The effects of variation in diet temperature, mouthpart deployment and the addition of bicarbonate to a saline-ATP diet were investigated for their effects on amount ingested and diet destination in female Aedes aegypti (L.). Mouthpart deployment was achieved by having the insects feed through a membrane, or from a free-liquid surface with mouthparts intact, or with the fascicle separated from the labial groove.
Under the 'feeding through membrane' protocol, a 6-fold increase in the percentage of Aedes feeding on ATP-diets was recorded at 37C compared with 21C. Bicarbonate (9.5 or 19 mM) induced a 3-fold increase in numbers feeding at both 21C and 37C. Neither diet temperature nor bicarbonate content appeared to affect meal size. The pH of all diets was adjusted to 7.0. Diet was directed primarily to the midgut for all diets tested under this regimen, with the exception of the saline-ATP-19 mM NaHC0₃ at 37C which caused the diet to be directed primarily to the crop.
Under the 'feeding without membrane through feeding tube' protocol, females showed little control over diet destination. Female Aedes aegypti exhibited no strong response to ATP when the mouthparts were immersed in 21C diets in feeding tubes and few of these insects ingested large meals. The addition of bicarbonate to the ATP diets did not enhance feeding under these test conditions.     

A new factor in the Aedes aegypti immune response: CLSP2 modulates melanization

Microbial infections in the mosquito Aedes aegypti activate the newly identified CLSP1 and CLSP2 genes, which encode modular proteins composed of elastase-like serine protease and C-type lectin domains. These genes are predominantly regulated by the immune deficiency pathway, but also by the Toll pathway. Silencing of CLSP2, but not CLSP1, results in the activation of prophenoloxidase (PPO), the terminal enzyme in the melanization cascade, suggesting that CLSP2 is a negative modulator of this reaction. Haemolymph PPO activation is normally inhibited in the presence of Plasmodium parasites, but in CLSP2-depleted mosquitoes, the Plasmodium-induced block of melanization is reverted, and these mosquitoes are refractory to the parasite. Thus, CLSP2 is a new component of the mosquito immune response.     

Electrophysiological and behavioural response of Aedes albopictus to n‐heinecosane,an ovipositional pheromone of Aedes aegypti

Aedes aegypti (L.) and Aedes albopictus (Skuse) (Diptera: Culicidae) are highly anthropophilic mosquito species and potential vectors of dengue and yellow fever. The location of suitable sites for oviposition requires a set of visual, tactile, and olfactory cues that influence females before they lay their eggs. In this study, the effect of n‐heneicosane, a recognized oviposition pheromone of Ae. aegypti, on the olfactory receptors of the antennae of Ae. aegypti and Ae. albopictus was studied using electroantennographic detection coupled to gas chromatography (GC‐EAD). A significant electroantennographic response to n‐heneicosane in adult females of both mosquito species was observed. In addition, gravid Ae. albopictus females laid more eggs in substrate treated with n‐heneicosane at 0.1, 1, or 10 p.p.m. than in the control, denoting oviposition attractancy. Conversely, at 30, 50, 100, and 200 p.p.m., more eggs were laid in the control substrate, indicating oviposition repellency. Analysis of the larval cuticle by GC and mass spectrometry confirmed the presence of n‐heneicosane in the cuticles of Ae. albopictus larvae. The species‐specific role of n‐heneicosane as an oviposition pheromone in Ae. aegypti and its significance as a behaviour modifier of Ae. albopictus in breeding sites is discussed.     

Effect of gamma irradiation on the hemocyte-mediated immune response of Aedes aegypti against microfilariae

The effect of gamma irradiation on the melanotic encapsulation response of Aedes aegypti black eye Liverpool strain against inoculated Dirofilaria immitis microfilariae (mff) was assessed at 1, 2, 3, and 6 days postinoculation (PI). Mosquitoes received 6000 rad from a 137Cs source (Shepard Mark I irradiator) at 3 days postemergence and were inoculated with 15-20 mff 24 hr later. These mosquitoes were compared to nonirradiated controls that also were inoculated with 15-20 mff at 3 days postemergence. The immune response was significantly reduced in irradiated mosquitoes as compared with controls at all days PI. Although the response was significantly inhibited compared with controls, irradiated mosquitoes were still capable of eliciting a response against 69% of recovered mff at 6 days PI. External gamma irradiation did not significantly affect the proliferation of hemocytes associated with the melanotic encapsulation response of A. aegypti. The number of circulating hemocytes increased in irradiated mosquitoes in response to inoculated mff in a manner similar to nonirradiated, inoculated controls. Hemocyte monophenol oxidase activity, however, was significantly reduced in gamma-irradiated mosquitoes at 12 hr PI as compared with controls. The reduced immunological capacity of irradiated mosquitoes might be related to an interference with gene activity required for the synthesis or activation of enzymes that are directly or indirectly involved in the biochemical processes associated with the production of melanotic substances that sequester mff.     

Physiological and biochemical response of Aedes aegypti tolerance to Bacillus thuringiensis

Bacillus thuringiensis subsp. israelensis (Bti) represents the only eco-friendly bio-degradable insecticide for mosquito-borne disease control. Our research aims to identify if mosquito detoxification enzymes play an important role in Bti tolerance mechanisms in the dengue vector, Aedes aegypti. Several enzymes, such as amylase, cytochromes P450, Na⁺/K⁺-ATPase, acetylcholinesterase, protease and glutathione S-transferase (GST) were analysed and level of activity determined in Ae. aegypti larvae after Bti treatment. Bti exposure significantly increased the level of amylase (183.2%) as well as the activity of cytochromes P450 (177.5%), and Na⁺/K⁺-ATPase (142.9%). On the other hand, there was a decrease of 8.6% and 11.4% in acetylcholinesterase and GST activity, and no significant effect in the total level of protease activity. We suggest that the variation in amylase, cytochromes P450, Na⁺/K⁺-ATPase, acetylcholinesterase, protease and GST activity may be associated with the Bti insecticidal mechanism. This study provides the basis of detoxifying enzymes in Bti tolerance.     

A secure semi-field system for the study of Aedes aegypti

Background

New contained semi-field cages are being developed and used to test novel vector control strategies of dengue and malaria vectors. We herein describe a new Quarantine Insectary Level-2 (QIC-2) laboratory and field cages (James Cook University Mosquito Research Facility Semi-Field System; MRF SFS) that are being used to measure the impact of the endosymbiont Wolbachia pipientis on populations of Aedes aegypti in Cairns Australia.

Methodology/Principal Findings

The MRF consists of a single QIC-2 laboratory/insectary that connects through a central corridor to two identical QIC-2 semi-field cages. The semi-field cages are constructed of two layers of 0.25 mm stainless steel wire mesh to prevent escape of mosquitoes and ingress of other insects. The cages are covered by an aluminum security mesh to prevent penetration of the cages by branches and other missiles in the advent of a tropical cyclone. Parts of the cage are protected from UV light and rainfall by 90% shade cloth and a vinyl cover. A wooden structure simulating the understory of a Queenslander-style house is also situated at one end of each cage. The remainder of the internal aspect of the cage is covered with mulch and potted plants to emulate a typical yard. An air conditioning system comprised of two external ACs that feed cooled, moistened air into the cage units. The air is released from the central ceiling beam from a long cloth tube that disperses the airflow and also prevents mosquitoes from escaping the cage via the AC system. Sensors located inside and outside the cage monitor ambient temperature and relative humidity, with AC controlled to match ambient conditions. Data loggers set in the cages and outside found a <2°C temperature difference. Additional security features include air curtains over exit doors, sticky traps to monitor for escaping mosquitoes between layers of the mesh, a lockable vestibule leading from the connecting corridor to the cage and from inside to outside of the insectary, and screened (0.25 mm mesh) drains within the insectary and the cage. A set of standard operating procedures (SOP) has been developed to ensure that security is maintained and for enhanced surveillance for escaping mosquitoes on the JCU campus where the MRF is located. A cohort of male and female Aedes aegypti mosquitoes were released in the cage and sampled every 3–4 days to determine daily survival within the cage; log linear regression from BG-sentinel trapping collections produced an estimated daily survival of 0.93 and 0.78 for females and males, respectively.

Conclusions/Significance

The MRF SFS allows us to test novel control strategies within a secure, contained environment. The air-conditioning system maintains conditions within the MRF cages comparable to outside ambient conditions. This cage provides a realistic transitional platform between the laboratory and the field in which to test novel control measures on quarantine level insects.     

The use of a chemically defined artificial diet as a tool to study Aedes aegypti physiology

Aedes aegypti mosquitoes obtain from vertebrate blood nutrients that are essential to oogenesis, such as proteins and lipids. As with all insects, mosquitoes do not synthesize cholesterol but take it from the diet. Here, we used a chemically defined artificial diet, hereafter referred to as Substitute Blood Meal (SBM), that was supplemented with cholesterol to test the nutritional role of cholesterol. SBM-fed and blood-fed mosquitoes were compared regarding several aspects of the insect physiology that are influenced by a blood meal, including egg laying, peritrophic matrix formation, gut microbiota proliferation, generation of reactive oxygen species (ROS) and expression of antioxidant genes, such as catalase and ferritin. Our results show that SBM induced a physiological response that was very similar to a regular blood meal. Depending on the nutritional life history of the mosquito since the larval stage, the presence of cholesterol in the diet increased egg development, suggesting that the teneral reserves of cholesterol in the newly hatched female are determinant of reproductive performance. We propose here the use of SBM as a tool to study other aspects of the physiology of mosquitoes, including their interaction with microbiota and pathogens.     

Sex before or after blood feeding: Mating activities of Aedes aegypti males under conditions of different densities and female blood feeding opportunities

Blood feeding and mating are critical behaviors that regulate both mosquito population maintenance and disease transmission. However, our understanding of mosquito mating systems remains incomplete. One of the most critical issues is a lack of understanding regarding how and where males and females encounter one another. This study was performed to investigate changes in key mating behaviors of Ae. aegypti relative to female blood feeding opportunities, taking into account male density. We compared courtship latency and copulation activity between single and pooled males in a range of assays performed in the presence or absence of a blood source and after blood feeding. The time taken by grouped males to initiate courtship in the presence of a host was much shorter than that in single males. There was no significant difference in courtship latency between pooled and single males in the absence of a blood source or after blood feeding. At low male density, the presence of the host and blood meal ingestion provided better conditions for copulation. At high male density, however, copulation activity was decreased after blood feeding, but remained high regardless of the presence or absence of the host. In addition to providing insight into the mating ecology of Aedes aegypti, this study indicated that the presence of a blood source influences how males encounter and copulate with females. The observation that copulation activity decreases after blood feeding when males are numerous provides new avenues for improving mass release programs of sterile mosquitoes.     

Anti-mosquito antibodies and their effects on feeding, fecundity and mortality of Aedes aegypti

Anti-mosquito antibodies, produced in mice inoculated with mosquito homogenates or exposed to mosquito bites, reacted with a variety of mosquito antigens including muscle, gut, fat body and nervous tissue; those in anti-mosquito bite sera reacted solely with salivary glands. Mosquitoes fed on restrained immunized mice showed a significant increase in mortality correlated to both the titre and specificity of the anti-mosquito antibodies ingested. No changes in their fecundity or feeding success were noted. Mosquitoes exposed to unrestrained immunized mice or mosquito-bitten mice, however, showed a significant reduction in feeding success, possibly reflecting enhanced host grooming.     

Insulin-like peptides and the target of rapamycin pathway coordinately regulate blood digestion and egg maturation in the mosquito Aedes aegypti

Background

Mosquitoes are insects that vector many serious pathogens to humans and other vertebrates. Most mosquitoes must feed on the blood of a vertebrate host to produce eggs. In turn, multiple cycles of blood feeding promote frequent contacts with hosts and make mosquitoes ideal disease vectors. Both hormonal and nutritional factors are involved in regulating egg development in the mosquito, Aedes aegypti. However, the processes that regulate digestion of the blood meal remain unclear.

Methodology/Principal Findings

Here we report that insulin peptide 3 (ILP3) directly stimulated late phase trypsin-like gene expression in blood fed females. In vivo knockdown of the mosquito insulin receptor (MIR) by RNA interference (RNAi) delayed but did not fully inhibit trypsin-like gene expression in the midgut, ecdysteroid (ECD) production by ovaries, and vitellogenin (Vg) expression by the fat body. In contrast, in vivo treatment with double-stranded MIR RNA and rapamycin completely blocked egg production. In vitro experiments showed that amino acids did not simulate late phase trypsin-like gene expression in the midgut or ECD production by the ovaries. However, amino acids did enhance ILP3-mediated stimulation of trypsin-like gene expression and ECD production.

Conclusions/Significance

Overall, our results indicate that ILPs from the brain synchronize blood meal digestion and amino acid availability with ovarian ECD production to maximize Vg expression by the fat body. The activation of digestion by ILPs may also underlie the growth promoting effects of insulin and TOR signaling in other species.     

Proteome of Aedes aegypti in response to infection and coinfection with microsporidian parasites

Hosts are frequently infected with more than one parasite or pathogen at any one time, but little is known as to how they respond to multiple immune challenges compared to those involving single infections. We investigated the proteome of Aedes aegypti larvae following infection with either Edhazardia aedis or Vavraia culicis, and coinfections involving both. They are both obligate intracellular parasites belonging to the phylum microsporidia and infect natural populations of Ae. aegypti. The results found some proteins only showing modified abundance in response to infections involving E. aedis, while others were only differentially abundant when infections involved V. culicis. Some proteins only responded with modified abundance to the coinfection condition, while others were differentially abundant in response to all three types of infection. As time since infection increased, the response to each of the single parasite infections diverged, while the response to the E. aedis and coinfection treatments converged. Some of the proteins differentially abundant in response to infection were identified. They included two vacuolar ATPases, proteins known to have a role in determining the infection success of intracellular parasites. This result suggests microsporidia could influence the infection success of other intracellular pathogens infecting vector species of mosquito, including viruses, Plasmodium and Wolbachia.