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.     

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.     

In vivo stimulation of vitellogenesis in Aedes aegypti with juvenile hormone,juvenile hormone analogue (ZR 515) and 20-hydroxyecdysone

Decapitated blood-fed Aedes aegypti mosquitoes do not undergo normal oöcyte maturation. Topical application of 1.25 ng JH analogue (ZR 515) or 250 ng JH-I restored ovarian development in 70–80% of the treated females. The rate of vitellogenin synthesis in these animals was 80% of normal blood-fed controls.When ligated abdomens were treated, 125 pg ZR 515 or 12.5 ng JH-I were sufficient to restore ovarian development in 80% of the animals. The rate of vitellogenin synthesis in these animals was 70% of normal blood-fed controls. On the other hand, injection of 1.25 μg 20-hydroxyecdysone was needed to restore ovarian development and vitellogenin synthesis in decapitated and abdominally ligated females.These experiments indicate that JH concentrations closer to the physiological norm than 20-hydroxyecdysone, can restore ovarian development and vitellogenin synthesis in vivo.     

Response of cultured Aedes aegypti fat bodies to 20-hydroxyecdysone

Fat bodies from non-blood-fed Aedes aegypti, stimulated in vitro by 10⁻⁴ M and 10⁻⁶ M of 20-hydroxyecdysone, were found to synthesize and release vitellogenin into the culture medium. Vitellogenin-specific monoclonal antibodies were utilized in an enzyme-linked immunosorbent assay procedure for quantification of vitellogenin in small aliquots of medium taken periodically from the culture. A minimal exposure of 5 h to 20-hydroxyecdysone was shown to be needed before the fat bodies would respond. Time-course of vitellogenin production in vitro was found to be identical to that observed in vivo. Vitellogenin-titre profiles were also investigated in cultured fat bodies from blood-fed A. aegypti. In all cases, response patterns were not affected by the presence or absence of 20-hydroxyecdysone after the fat bodies had been stimulated by blood meal to produce vitellogenin. We suggest here that initiation and control of vitellogenin synthesis is a programmed response to 20-hydroxyecdysone.     

Role of the fat body in the regulation of host-seeking behaviour in the mosquito,Aedes aegypti

Following a blood meal that initiates oöcyte development, the host-seeking behaviour of Aedes aegypti mosquitoes is inhibited by a haemolymph-borne factor that is released in response to a humoral signal from a vitellogenic ovary. This inhibition is accompanied by a decrease in the sensitivity of the peripheral lactic acid receptors. Implantation of corpora allata, medial neurosecretory cells, or terminal abdominal ganglia from blood-fed donors could not induce the inhibition in sugar-fed recipients. However, fat body transplanted from blood-fed into sugar-fed females suppressed host-seeking behaviour as well as the sensitivity of lactic acid receptors, suggesting that the source of the behavioural inhibitor is the fat body. Resting-stage ovaries from other mosquito species inhibited host-seeking after the A. aegypti host was fed on blood only if the fat body was activated by the donor ovary.     

Expression and regulation of vitellogenin messenger RNA in the mosquito,Aedes aegypti

Vitellogenin (yolk protein) gene expression in the mosquito was investigated at the level of mRNA using a subcloned fragment (403-1c) of the vitellogenin DNA derived from an Aedes aegypti genomic library. Message appeared 1–3 hr after a blood meal, peaked at 36 hr and was rapidly degraded thereafter. Fluctuations in levels of 20-hydroxyecdysone after a blood meal coincided with accumulation of vitellogenin message. Blood-fed, decapitated females injected with 5 μg of 20-hydroxyecdysone accumulated up to 75% of the message found in blood-fed controls. Fat bodies from non-blood-fed females incubated with physiological levels of 20-hydroxyecdysone and the juvenile hormone analog methoprene contained twice as much vitellogenin message as those incubated with 20-hydroxyecdysone alone. Methoprene alone had no effect.     

Dynamics of vitellogenin uptake in Aedes aegypti as demonstrated by trypan blue

Trypan blue has been shown to be a reliable indicator of the micropinocytotic uptake of vitellogenin by developing oöcytes. Trypan blue was injected into the mosquito Aedes aegypti to determine at what times after the blood meal vitellogenin was taken up. Histological sections examined by light microscopy showed that trypan blue began to be sequestered from 2 to 5 hr after the blood meal. Any association between dye and ovariole ended from 39 to 42 hr after the blood meal, in which period no dye was incorporated into spheres of yolk protein. Of the times investigated in this experiment, the greatest amount of dye was seen in the oöcyte at 24 hr after the blood meal. The onset and conclusion of trypan blue uptake correspond with the related events in the synthesis of vitellogenin by the fat body. The presence of trypan blue in occasional interfollicular spaces suggests that the route of entry of vitellogenin in Aedes aegypti is indeed an interfollicular one.     

The role of the male accessory gland fluid in stimulating vitellogenesis in Aedes taeniorhynchus

Injection of 20-hydroxyecdysone (20-OH-ecdysone) at high concentrations (5.0 μg) into intact or decapitated female Aedes taeniorhynchus induced vitellogenin synthesis, whereas low concentrations (5.0 ng) were ineffective. Injections of male accessory gland fluid (MAGF), however, at a concentration that was equivalent to 0.25 of the content of a pair of accessory glands, into intact or decapitated A. taeniorhynchus induced viteliogenin synthesis only in intact females. Ovariectomized mosquitoes did not synthesize vitellogenin after MAGF injection or blood feeding. Females that were first injected with MAGF and decapitated 12 h later synthesized viteliogenin at a rate that was 80% of intact controls. Egg development neurosecretory hormone (EDNH) activity in the heads of ovariectomized or intact females injected with MAGF was 9.0 pmol/min/head and 2.5 pmol/min/head, respectively, indicating that MAGF does not stimulate the corpus cardiacum (CC) to release EDNH. Incubation of MAGF and EDNH with fat bodies failed to induce vitellogenin synthesis. These results indicate that in A. taeniorhynchus the MAGF induces the ovary to release corpus cardiacum stimulating factor, which then signals CC to release stored EDNH.     

Juvenile hormone and 20-hydroxyecdysone as primary and secondary stimuli of vitellogenesis in Aedes aegypti

Female Aedes aegypti that were fed blood and immediately abdominally ligated did not deposit yolk. Injection of 20-hydroxyecdysone (1.5–5.0 ng) or topical application of juvenile hormone (JH) analogue methoprene (25 pg) did not induce vitellogenesis in these abdomens. When blood-gorged ligated abdomens were treated with both hormones, however, vitellogenesis was stimulated in 60% of treated animals. Rocket immunoelectrophoresis indicated that vitellin concentration per follicle in treated animals was similar to that in intact controls. When ligated abdomens were first treated with methoprene and immediately injected with a crude head extract of egg development neurosecretory hormone, vitellogenin synthesis was induced at a rate similar to that in blood-fed controls. Methoprene at this concentration (25 pg), did not cause an increase in whole-body ecdysteroid titers. Larger amounts of methoprene (1.65 ng) were needed to stimulate egg development and ecdysteroid production. Implantation of ecdysone-secreting ovaries into ligated abdomens did not stimulate vitellogenesis in the recipients. However, in recipients that were first treated with methoprene (25 pg), implantation of ecdysone-secreting ovaries resulted in normal egg development. These experiments indicate that the appearance of JH precedes 20-hydroxyecdysone in stimulating vitellogenesis following blood feeding in Ae. aegypti.     

Isolation of mosquito vitellogenin genes and induction of expression by 20-hydroxyecdysone

Vitellogenic female Aedes aegypti contain abundant, 6500 nucleotide long RNAs that are not present in males or non-vitellogenic females and which were presumed to encode vitellogenin (VG). Three clones that hybridized to cDNA made to poly(A⁺)RNA from vitelogenic females, but not to cDNA made to male RNA, were selected from a genomic library. DNA from each clone hybridized to the 6500 nucleotide RNA species. Restriction enzyme mapping suggests the clones represent three distinct genes. The two that have been characterized share an uninterrupted region of homology about 6.5 kb long. Part of the coding region of one of the cloned genes was inserted into an expression vector, and the resulting polypeptide reacted specifically with antibodies to vitellogenin, thus confirming that the clones contain VG genes. Using one of the cloned genes as a probe on northern hybridizations we found that injection of 20-hydroxyecdysone into non-blood-fed decapitated females stimulated vitellogenin gene expression. The response was much greater in blood-fed decapitated females than in non-blood-fed females.     

Internal regulation of rate of digestion of blood meals in the mosquito, Aedes aegypti.

Rate of digestion of blood meals proceeded more rapidly in females of Aedes aegypti that were inseminated or injected with matrone (extract of male accessory glands) than in virgin females. Digestion rate was determined by interfacial precipitin tests and immunodiffusion tests for undigested blood-meal proteins. Application of a cervical ligature within 1 hr of blood feeding retarded the digestion rate. Ligated females that received brain transplants from blood-fed donors digested their blood meals at a rate similar to that of unligated controls. When ligatures were not applied until 12 hr after feeding no delay in digestion was observed.     

Biosynthesis and distribution of ecdysone and 20-OH-ecdysone in Aedes aegypti

The distribution and biosynthesis of ecdysone and 20-hydroxyecdysone (20-OH-ecdysone) was followed in sugar- and blood-fed female Aedes aegypti. In both sugar- and early blood-fed animals most of the ecdysteroid determined by radioimmunoassay was found outside the ovary. Twenty-four to 40 h after blood feeding, however, ecdysteroid was distributed between ovary and carcass in the ratio of 1:1.5. Ecdysteroid titer reached a plateau between 18 to 40 h after the blood meal and decreased thereafter. Analysis of the ecdysteroid titer using thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) revealed that both 20-OH-ecdysone and ecdysone were synthesized after the blood meal. The ratio of 20-OH-ecdysone to ecdysone remained essentially constant and fluctuated in parallel throughout egg development. Chromatography of the early ecdysteroid peak (8 h after feeding) using TLC and HPLC indicated that although it cross-reacted with ecdysteroid antibodies, it did not have the same elution times as ecdysone and 20-OH-ecdysone and is, therefore, probably a precursor of these ecdysteroids. Injections of egg development neurosecretory hormone (EDNH) preparation purified to near homogeneity, into ligated abdomens, induced ecdysteroid synthesis only if the abdomens were first treated with methoprene (12.5 pg). Methoprene at this concentration did not stimulate ecdysteroid synthesis in these abdomens. When blood-fed females were treated with [4-¹⁴C] cholesterol and analyzed using TLC and HPLC procedures, both [¹⁴C]labeled ecdysone and [¹⁴C]labeled 20-OH-ecdysone were synthesized in the ratio of 1:1.5. This report is the first to show that both ecdysone and 20-OH-ecdysone are synthesized in vivo in female A. aegypti.     

Urea Synthesis and Excretion in Aedes aegypti Mosquitoes Are Regulated by a Unique Cross-Talk Mechanism

Aedes aegypti mosquitoes do not have a typical functional urea cycle for ammonia disposal such as the one present in most terrestrial vertebrates. However, they can synthesize urea by two different pathways, argininolysis and uricolysis. We investigated how formation of urea by these two pathways is regulated in females of A. aegypti. The expression of arginase (AR) and urate oxidase (UO), either separately or simultaneously (ARUO) was silenced by RNAi. The amounts of several nitrogen compounds were quantified in excreta using mass spectrometry. Injection of mosquitoes with either dsRNA-AR or dsRNA-UO significantly decreased the expressions of AR or UO in the fat body (FB) and Malpighian tubules (MT). Surprisingly, the expression level of AR was increased when UO was silenced and vice versa, suggesting a cross-talk regulation between pathways. In agreement with these data, the amount of urea measured 48 h after blood feeding remained unchanged in those mosquitoes injected with dsRNA-AR or dsRNA-UO. However, allantoin significantly increased in the excreta of dsRNA-AR-injected females. The knockdown of ARUO mainly led to a decrease in urea and allantoin excretion, and an increase in arginine excretion. In addition, dsRNA-AR-injected mosquitoes treated with a specific nitric oxide synthase inhibitor showed an increase of UO expression in FB and MT and a significant increase in the excretion of nitrogen compounds. Interestingly, both a temporary delay in the digestion of a blood meal and a significant reduction in the expression of several genes involved in ammonia metabolism were observed in dsRNA-AR, UO or ARUO-injected females. These results reveal that urea synthesis and excretion in A. aegypti are tightly regulated by a unique cross-talk signaling mechanism. This process allows blood-fed mosquitoes to regulate the synthesis and/or excretion of nitrogen waste products, and avoid toxic effects that could result from a lethal concentration of ammonia in their tissues.     

Abdominal distention terminates subsequent host-seeking behaviour of Aedes aegypti following a blood meal

The failure of Aedes aegypti females to engage in host-seeking behaviour following a replete blood meal was investigated. Abdominal distention appears to be responsible for this immediate inhibition after feeding. Large enemas of saline had the same effect as blood in terminating host-seeking; this was not due merely to the presence of large amounts of fluid, but rather to the distention produced by these liquids. Since transection of the ventral nerve cord anterior to the 2nd abdominal ganglion did not release the inhibition in blood-fed females, we restricted the degree of distention of abdominal segments with wax. Distention of the abdomen anteriorly by a blood meal more effectively inhibited host-seeking than did distention posteriorly, suggesting that stretch receptors in the anterior portion of the abdomen regulate the response towards a host.     

Effect of dopa-decarboxylase inhibition on Aedes aegypti eggs: evidence for sclerotization

Newly deposited fertilized and unfertilized Aedes aegypti eggs are soft and white. Within a short time they darken and harden. Injection of a potent dopa decarboxylase inhibitor (dl)-3-(3,4-dihydroxyphenyl)-2-hydrazino-2-methylpropionic acid (α-MDH) into females, subsequent to a blood meal, results in oviposited eggs which are pale in colour. Moreover, such fertilized eggs do not hatch. The severity of both effects seems to be positively correlated and is dependent upon the time of α-MDH injection.Extracts of mature ovaries are capable of converting dopa to dopamine in the absence of a pretreatment with α-MDH. Mature ovaries obtained from females who had been previously injected with α-MDH could not accomplish this conversion. The inhibitor does not seem to have any effect on dopa oxidase activity and subsequent melanization. We conclude that dopamine is synthesized by blood-fed females via decarboxylation of dopa by dopa decarboxylase and propose that the normal darkening and hardening of A. aegypti eggs is a result of sclerotization.     

Vitellogenin synthesis in blood-fed Aedes aegypti in the absence of the head,thorax and ovaries

A blood meal initiates oöcyte maturation in Aedes aegypti, and we have used rocket immunoelectrophoresis to investigate the function of midgut, ovaries, and head in the onset of vitellogenin synthesis. Non-blood-fed females and those fed blood (by enema) containing soybean trypsin inhibitor never contained vitellogenin. This demonstrates that the pressure of an undigested blood meal on stretch receptors of the midgut plays no role in the induction of vitellogenin synthesis, rather the stimulus is a digestion product of blood.When females were ovariectomized or decapitated and then fed blood, the haemolymph contained newly synthesized vitellogenin 24 h later. This was also demonstrated in isolated ovariectomized abdomens. Apparently, induction of vitellogenin synthesis does not require factors from either the head, thorax, or ovaries. When ovariectomy or decapitation was postponed after a blood meal, the level of vitellogenin in the haemolymph rose. Therefore, interaction of factors from the head and ovaries maintain the synthesis needed for oöcyte maturation.     

Isolation of trypsin isozymes from the mosquito Aedes aegypti (L.)

Trypsin has been isolated from midgut homogenates of blood-fed females of Aedes aegypti by a simple two-step purification procedure: ion-exchange chromatography followed by affinity chromatography. The resulting mosquito trypsin contains a number of isozymes, among which 5 major SDS-PAGE bands are recognized with molecular weights of 26.7, 28.5, 29.7, 31.0 and 32.0 kdaltons, as are some minor bands above and below this range. The isozymic pattern is comparable to that in crude homogenates. Isoelectric focussing of purified trypsin however, revealed over 20 tryptic isozymes, demonstrating that several isozymes segregate into subforms. A high correlation between TAME-active fractions and their DFP equivalent was demonstrated by using ³H-labelled DFP as a marker for trypsin on native acrylamide gels.The purification factor and the specific activities are discussed with respect to the unusual amounts of protein of dietary origin present in the midgut homogenates. Interference of blood-borne coagulation factors of a tryptic nature is unlikely.     

A ribosome-free extract from cultured cells improves recovery of polysomes from the mosquito fat body: analysis of vitellogenin and ribosomal protein rpL34 gene expression

We have examined the association of ribosomal protein rpL34 mRNA with polysomes in Aedes albopictus C7-10 cells in culture using a simple, two-step sucrose gradient. In growing cells, 40-50% of the ribosomes were engaged on polysomes. This proportion could be increased to 80% when metabolism was stimulated by refeeding the cells with fresh medium. Conversely, ribosomes shifted off polysomes when cells were starved with phosphate-buffered saline or cell lysates were treated with puromycin. When similar approaches were used with fat body from blood-fed female Aedes aegypti mosquitoes, we were unable to obtain the polysome fraction that contained vitellogenin mRNA, which is abundantly translated after a blood meal. Addition of post-mitochondrial supernatant from fat body to polysomes from cultured cells shifted the polysome profile towards smaller polysomes and monosomes, in a dose-dependent fashion. Disruption of fat body tissue in a post-ribosomal supernatant from refed cells improved the recovery of polysomes, demonstrating both the engagement of vitellogenin mRNA on polysomes and the mobilization of rpL34 from messenger-ribonuceloprotein particles onto polysomes in blood-fed mosquitoes. These observations suggested that ribonucleases remain active when polysomes are prepared from mosquito fat body, and that cell culture supernatant contains a ribonuclease inhibitor.     

Biosynthesis,purification, and characterization of Aedes aegypti vitellin and vitellogenin

Vitellin, the major egg yolk protein, and vitellogenin, the hemolymph precursor of egg yolk protein, have been purified to apparent homogeneity from the mosquito Aedes aegypti. The purification procedure included chromatography on ion exchange, hydrophobic, and gel filtration columns. Vitellin and vitellogenin have a similar molecular weight (M_r 300,000) on gel filtration columns. However, the molecular weights of vitellin and vitellogenin, as determined from SDS electrophoresis, were 393,000 and 337,000, respectively. Vitellin in sodium dodecyl sulfate released six subunits of molecular weight 116,000, 83,000, 75,000, 54,000, 36,000, and 29,000, whereas vitellogenin released only three subunits (155,000, 120,000, and 62,000). The average molecular weights of vitellin and vitellogenin after gel filtration and SDS electrophoresis were 346,000 and 318,000, respectively. Vitellin has a high content of aspartic acid and glutamic acid, and a low content of histidine, methionine, cysteine, and tryptophan. Vitellin also contains 0.9% mol of glucosamine and no galactosamine. The isoelectric points of vitellin and vitellogenin are at pH 6.4 and 6.3, respectively. Aedes aegypti fat bodies incubated for short intervals in tissue culture medium in the presence of [³H]valine showed incorporation by radio-immunoprecipitation and SDS electrophoresis into three primary vitellogenin polypeptides of molecular weights (± SEM) 156,000 ± 4,000, 114,000 ± 5,000, and 62,000 ± 400 inside the fat body and 162,000 ± 3,000, 118,200 ± 2,000, and 63,000 ± 300 in the medium. These results suggest that the molecular weight of vitellogenin synthesized inside the fat body (M_r 332,000) remains unchanged when secreted into the hemolymph (M_r 343,000). The three vitellogenin subunits are processed by the ovary into six subunits which are then deposited in the yolk granules as vitellin.     

Evidence for hormonal control of diuresis after a blood meal in the mosquito Aedes aegypti

In previous studies we have presented evidence for the role of peptides, isolated from heads of the mosquito Aedes aegypti, in stimulating fluid secretion by isolated Malpighian tubules. In the present study we conducted experiments to investigate whether these peptides are involved in hormone-mediated diuresis after a blood meal. In vivo experiments showed that the head was required to maintain diuresis after the blood meal. Whereas feeding on blood triggered a prompt diuresis in the intact mosquito, subsequent decapitation caused a gradual, not an abrupt, decline in urine excretion rate. Hemolymph collected from mosquitoes fed blood significantly stimulated fluid secretion in vitro by isolated Malpighian tubules, whereas hemolymph from unfed or blood-fed decapitated mosquitoes did not. These results indicate that a diuretic factor was released into the hemolymph after a blood meal. This factor was not present in the hemolymph of decapitated females. We identified the head as a source of diuretic factors. Peptides isolated from a head extract by high-performance liquid chromatography, when injected into the hemocoel of blood-fed decapitated mosquitoes, triggered diuresis in vivo and also stimulated fluid secretion in isolated Malpighian tubules. These studies support the hypothesis that the head is a storage site for diuretic peptides that may be released after a blood meal to control diuresis.