A Critical Role of the Nuclear Receptor HR3 in Regulation of Gonadotrophic Cycles of the Mosquito Aedes aegypti

The orphan nuclear receptor HR3 is essential for developmental switches during insect development and metamorphosis regulated by 20-hydroxyecdysone (20E). Reproduction of female mosquitoes of the major vector of Dengue fever, Aedes aegypti, is cyclic because of its dependence on blood feeding. 20E is an important hormone regulating vitellogenic events in this mosquito; however, any role for HR3 in 20E-driven reproductive events has not been known. Using RNA interference (RNAi) approach, we demonstrated that Aedes HR3 plays a critical role in a timely termination of expression of the vitellogenin (Vg) gene encoding the major yolk protein precursor. It is also important for downregulation of the Target-of-Rapamycin pathway and activation of programmed autophagy in the Aedes fat body at the end of vitellogenesis. HR3 is critical in activating betaFTZ-F1, EcRB and USPA, the expressions of which are highly elevated at the end of vitellogenesis. RNAi depletion of HR3 (iHR3) prior to the first gonadotrophic cycle affects a normal progression of the second gonadotrophic cycle. Most of ovaries 24 h post second blood meal from iHR3 females in the second cycle were small with follicles that were only slightly different in length from of those of resting stage. In addition, these iHR3 females laid a significantly reduced number of eggs per mosquito as compared to those of iMal and the wild type. Our results indicate an important role of HR3 in regulation of 20E-regulated developmental switches during reproductive cycles of A. aegypti females.     

Different modes of programmed cell death during oogenesis of the silkmoth Bombyx mori

It is increasingly recognized that programmed cell death includes not only apoptosis and autophagy, but also other types of nonapoptotic cell death, such as paraptosis, which are all characterized by distinct morphological features. Our findings indicate that all three types of programmed cell death occur in the ovarian nurse cell cluster during late vitellogenesis (formation of the egg yolk) of Bombyx mori (Lepidoptera), whereas middle vitellogenesis is exclusively characterized by the presence of a nonapoptotic type of cell death, known as paraptosis. During middle vitellogenesis, nurse cells exhibit clearly cytoplasmic vacuolization, as revealed by ultrastructural examination performed through conventional light and transmission electron microscopy, while no signs of apoptotic or autophagic features are detectable. Moreover, nurse cells of developmental stages 7, 8 and 9 contain autophagic compartments, as well as apoptotic characteristics, such as condensed chromatin, fragmented DNA and activated caspases, as revealed by in vitro assays. We propose that paraptosis precedes both apoptosis and autophagy during vitellogenesis, since its initial activation is detectable during middle vitellogenesis, whereas no apoptotic nor autophagic features are observed. In contrast, at the late stages of Bombyx mori oogenesis, paraptosis, autophagy and apoptosis operate synergistically, resulting in a more efficient elimination of the degenerated nurse cells.     

Target of rapamycin-dependent activation of S6 kinase is a central step in the transduction of nutritional signals during egg development in a mosquito

Female mosquitoes are effective disease vectors, because they take blood from vertebrate hosts to obtain nutrients for egg development. Amino acid signaling via the target of rapamycin (TOR) pathway has been identified as a key requirement for the activation of egg development after a blood meal. We report the characterization of the TOR kinase and one of its major downstream targets, S6 kinase, of the yellow fever mosquito Aedes aegypti during egg development in adult females. Both TOR and S6K mRNA are expressed at high levels in the ovaries and in lower levels in fat body and other tissues. After a blood meal, the subcellular localization of TOR shifts from the cytoplasm to the plasma membrane of fat body cells. By detecting phosphothreonine 388 of mosquito S6 kinase, we show that TOR activity strongly increases in fat body and ovaries after a blood meal in vivo. Furthermore, phosphorylation of S6 kinase increases in in vitro cultured fat bodies after stimulation with amino acids. This increase is sensitive to the TOR inhibitor rapamycin in a concentration-dependent manner but not to the phosphatidylinositol 3-kinase/phosphatidylinositol 3-kinase-related kinase inhibitor LY294002, the MAPK inhibitor PD98059, or the translational inhibitor cycloheximide. RNA interference-mediated reduction of S6 kinase strongly inhibits the amino acid-induced up-regulation of the major yolk protein vitellogenin in vitro and effectively disrupts egg development after a blood meal in vivo. Our data show that TOR-dependent activation of S6 kinase is a central step in the transduction of nutritional information during egg development in mosquitoes.     

Different modes of programmed cell death during oogenesis of the silkmoth Bombyx mori

It is increasingly recognized that programmed cell death includes not only apoptosis and autophagy, but also other types of nonapoptotic cell death, such as paraptosis, which are all characterized by distinct morphological features. Our findings indicate that all three types of programmed cell death occur in the ovarian nurse cell cluster during late vitellogenesis (formation of the egg yolk) of Bombyx mori (Lepidoptera), whereas middle vitellogenesis is exclusively characterized by the presence of a nonapoptotic type of cell death, known as paraptosis. During middle vitellogenesis, nurse cells exhibit clearly cytoplasmic vacuolization, as revealed by ultrastructural examination performed through conventional light and transmission electron microscopy, while no signs of apoptotic or autophagic features are detectable. Moreover, nurse cells of developmental stages 7, 8 and 9 contain autophagic compartments, as well as apoptotic characteristics, such as condensed chromatin, fragmented DNA and activated caspases, as revealed by in vitro assays. We propose that paraptosis precedes both apoptosis and autophagy during vitellogenesis, since its initial activation is detectable during middle vitellogenesis, whereas no apoptotic nor autophagic features are observed. In contrast, at the late stages of Bombyx mori oogenesis, paraptosis, autophagy and apoptosis operate synergistically, resulting in a more efficient elimination of the degenerated nurse cells.

Addendum to: Mpakou VE, Nezis IP, Stravopodis DJ, Margaritis LH, Papassideri IS. Programmed cell death of the ovarian nurse cells during oogenesis of the silkmoth Bombyx mori. Dev Growth Differ 2006; 48:419–28.     

Juvenile hormone connects larval nutrition with target of rapamycin signaling in the mosquito Aedes aegypti

Anautogenous mosquitoes require blood meals to promote egg development. If adequate nutrients are not obtained during larval development, the resulting "small" sized adult mosquitoes require multiple blood meals for egg development; markedly increasing host-vector contacts and the likelihood of disease transmission. Nutrient-sensitive target of rapamycin (TOR) signaling is a key signaling pathway that links elevated hemolymph amino acid levels derived from the blood meal to the expression of yolk protein precursors in the fat body. Here we report that the blood-meal-induced activation of the TOR-signaling pathway and subsequent egg maturation depends on the accumulation of adequate nutritional reserves during larval development. We have established well-nourished, "standard" mosquitoes and malnourished, "small" mosquitoes as models to address this nutrient sensitive pathway. This regulatory mechanism involves juvenile hormone (JH), which acts as a mediator of fat body competence, permitting the response to amino acids derived from the blood meal. We demonstrate that treatment with JH results in recovery of the TOR molecular machinery, Aedes aegypti cationic amino acid transporter 2 (AaiCAT2), TOR, and S6 kinase (S6K), in fat bodies of small mosquitoes, enabling them to complete their first gonotrophic cycle after a single blood meal. These findings establish a direct link between nutrient reserves and the establishment of TOR signaling in mosquitoes.     

AHR38, a homolog of NGFI-B, inhibits formation of the functional ecdysteroid receptor in the mosquito Aedes aegypti

In anautogenous mosquitoes, vitellogenesis, the key event in egg maturation, requires a blood meal. Consequently, mosquitoes are vectors of numerous devastating human diseases. After ingestion of blood, 20-hydroxyecdysone activates yolk protein precursor (YPP) genes in the metabolic tissue, the fat body. An important adaptation for anautogenicity is the previtellogenic developmental arrest (the state-of-arrest) preventing the activation of YPP genes in previtellogenic females prior to blood feeding. Here, we show that a retinoid X receptor homolog, Ultraspiracle (AaUSP), which is an obligatory partner in the functional ecdysteroid receptor, exists at the state-of-arrest as a heterodimer with the orphan nuclear receptor AHR38, a homolog of Drosophila DHR38 and nerve growth factor-induced protein B. Yeast two-hybrid and glutathione S-transferase pull-down assays demonstrate that AHR38 can interact strongly with AaUSP. AHR38 also disrupts binding of ecdysteroid receptor to ecdysone response elements. Cell co-transfection of AHR38 with AaEcR and AaUSP inhibits ecdysone-dependent activation of a reporter gene by the ecdysteroid receptor. Co-immunoprecipitation experiments indicate that AaUSP protein associates with AHR38 instead of AaEcR in fat body nuclei at the state-of-arrest.     

Nutritional regulation of vitellogenesis in mosquitoes: implications for anautogeny

Anautogeny is a successful reproductive strategy utilized by many mosquito species and other disease-transmitting arthropod vectors. Developing an understanding of the mechanisms underlying anautogeny in mosquitoes is very important because this reproductive strategy is the driving force behind the transmission of disease to millions of people. Information gained from mosquito studies may also be applicable to other blood feeding insect vectors. The conversion of protein from blood into yolk protein precursors for the developing oocytes is an essential part of the reproductive cycle, and understanding how this process is regulated could lead to safe, specific, and effective ways to block reproduction in blood feeding insects. Great gains have been made in elucidating the mechanisms that regulate vitellogenesis in mosquitoes, especially Ae. aegypti. However, a number of questions remain to be answered to make the picture more complete. In this review, we summarize what is currently known about the nutritional regulation of vitellogenesis in mosquitoes and the questions that remain to be answered about this important biological phenomenon.     

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.     

The dynamics of yolk deposition in the desert locust Schistocerca gregaria

Injection of the protein dye Fast Green or the fluid-phase probe fluorescein dextran into the haemolymph of vitellogenic female desert locusts (Schistocerca gregaria) resulted in their incorporation into oocytes. We used Fast Green to study the physical dynamics of yolk deposition during vitellogenesis. Timed maternal injections of Fast Green reveal that yolk deposition and oocyte growth are inextricably linked during vitellogenesis, and that little or no yolk movement occurs within oocytes prior to embryogenesis. The yolk granules laid down early during vitellogenesis lie at the centre of the egg, with yolk granules deposited later packed around these, such that they lie progressively closer to the eventual egg surface. In contrast, during early embryogenesis yolk granules migrate in a manner that closely resembles the movement of early cleavage nuclei. We find fluorescein dextran to be a clear, robust and developmentally inert marker for the timing of maternal injections relative to vitellogenesis in S. gregaria, and we propose its use in parental RNAi or morpholino knockdown experiments. With such experiments in mind, we show that fluorescein-labelled DNA oligonucleotides are internalized within oocytes during vitellogenesis. However, neither Fast Green, fluorescein dextran nor fluorescein-labelled DNA oligonucleotides are detectably transferred from yolk granules to embryonic cells during embryogenesis, and our initial attempts at parental RNAi using maternal injections of dsRNA targeted to late vitellogenesis have proved unsuccessful.     

A specific radioimmunoassay for vitellogenesis in mosquitoes

Hagedorn's assay for vitellogenesis, in which a crude antigen-antibody complex is collected directly on a Millipore membrane, without prior separation of soluble from insoluble proteins, is unspecific. This was proven as follows.Fat bodies of blood-fed mosquitoes (Aedes aegypti) were incubated in a medium containing ³H valine. The medium was analyzed for synthesis of vitellogenin by incubation with serum of normal rabbits (control) or of rabbits which were immunized against mosquito egg protein (antibody).When these mixtures were filtered directly through Millipore membranes (Hagedorn's method), the radioactivity in the control was about two-thirds of that in the antibody membrane. However, when the antigen-serum and antigen-antibody complex were centrifuged, the antigen-serum precipitate (control) contained only a very small percentage of the radioactivity present in the antigen-antibody precipitate. Apparently, the medium contained a large amount of soluble, nonvitellogenic protein that binds to the membrane.Analysis of the fat body homogenate showed that a large amount of the newly synthesized vitellogenin was stored intracellularly.Since all published data on vitellogenesis in mosquitoes depend on Hagedorn's assay, the validity of current concepts of the control of vitellogenesis in mosquitoes is open to question.     

Isolation and characterization of highly purified mosquito oostatic hormone

Oostatic hormone, the hormone that inhibits vitellogenesis in mosquitoes, was purified 7,000-fold with a recovery of 70% from the ovaries of the mosquito Aedes aegypti. The purification procedure included heat treatment and chromatography on ion exchange and gel filtration columns. The hormone is a small peptidelike molecule of molecular weight 2,200 at pH 4.5, which aggregates into larger molecular species of trimer and octamer at pH 7.0 as determined by gel filtration. The hormone is positively charged at pH 7.8 and has a low Rf at pH 9.4 on disc gel electrophoresis. Injection of purified oostatic hormone (9 ng) into female mosquitoes inhibited yolk deposition and vitellogenin synthesis. Activity of the oostatic hormone in the mosquito ovary increased rapidly following blood feeding and reached a maximum after 48 h. Oostatic hormone of A. aegypti injected into autogenous Aedes taeniorhynchus inhibited egg development. Repeated injections of dilute oostatic hormone at 24 h intervals partially arrested egg development, resulting in 60% reduction in the number of eggs laid. This hormone does not block release of egg development neurosecretory hormone (EDNH) from the mosquito brain but rather appears to act on the ovary.     

SNF4Agamma, the Drosophila AMPK gamma subunit is required for regulation of developmental and stress-induced autophagy

In holometabolous insects including Drosophila melanogaster a wave of autophagy triggered by 20-hydroxyecdysone is observed in the larval tissues during the third larval stage of metamorphosis. We used this model system to study the genetic regulation of autophagy. We performed a genetic screen to select P-element insertions that affect autophagy in the larval fat body. Light and electron microscopy of one of the isolated mutants (l(3)S005042) revealed the absence of autophagic vesicles in their fat body cells during the third larval stage. We show that formation of autophagic vesicles cannot be induced by 20-hydroxyecdysone in the tissues of mutant flies and represent evidence demonstrating that the failure to form autophagic vesicles is due to the insertion of a P-element into the gene coding SNF4Agamma, the Drosophila homologue of the AMPK (AMP-activated protein kinase) gamma subunit. The ability to form autophagic vesicles (wild-type phenotype) can be restored by remobilization of the P-element in the mutant. Silencing of SNF4Agamma by RNAi suppresses autophagic vesicle formation in wild-type flies. We raised an antibody against SNF4Agamma and showed that this gene product is constitutively present in the wild-type larval tissues during postembryonal development. SNF4Agamma is nearly absent from the cells of homozygous mutants. SNF4Agamma translocates into the nuclei of fat body cells at the onset of the wandering stage concurrently with the beginning of the autophagic process. Our results demonstrate that SNF4Agamma has an essential role in the regulation of autophagy in Drosophila larval fat body cells.