Mosquito Ovaries Research Articles

Identification of plant compounds that disrupt the insect juvenile hormone receptor complex

Insects impact human health through vector-borne diseases and cause major economic losses by damaging crops and stored agricultural products. Insect-specific growth regulators represent attractive control agents because of their safety to the environment and humans. We identified plant compounds that serve as juvenile hormone antagonists (PJHANs). Using the yeast two-hybrid system transformed with the mosquito JH receptor as a reporter system, we demonstrate that PJHANs affect the JH receptor, methoprene-tolerant (Met), by disrupting its complex with CYCLE or FISC, formation of which is required for mediating JH action. We isolated five diterpene secondary metabolites with JH antagonist activity from two plants: Lindera erythrocarpa and Solidago serotina. They are effective in causing mortality of mosquito larvae at relatively low LD50 values. Topical application of two diterpenes caused reduction in the expression of Met target genes and retardation of follicle development in mosquito ovaries. Hence, the newly discovered PJHANs may lead to development of a new class of safe and effective pesticides.

In Memoriam

In Memoriam

Iron deposition and ferritin accretion in the ovaries of the yellow fever mosquito, Aedes aegypti (948.9)

Malaria and yellow fever are transmitted from mosquitoes to humans through a mosquito bite. Mosquitoes require the essential nutrients from a blood meal, including iron, for oogenesis. Very little research has been conducted to elucidate the effect of iron on mosquito fecundity. Understanding the mechanism of iron deposition in the ovary is important to finding potential strategies to interfere with mosquito fecundity, decrease mosquito populations, and reduce transmission rates of vector‐borne diseases. To this end, Aedes aegypti (yellow fever mosquito) ovaries were dissected from non‐blood fed, 24 h post‐blood meal (PBM), and 72 h PBM animals to measure tissue iron accumulation after a blood meal by iron inductively coupled plasma mass spectrometry (ICP‐MS). Dissected ovaries were also stained with Prussian Blue to visualize ferric iron deposition. Our results indicate that iron is present in the ovary prior to a blood meal and accumulates over time PBM, and the deposition of ferric iron is observed in different areas of the tissue before and after a blood meal. Immunohistochemistry imaging of the ovaries shows three different subunits of the iron storage and transport protein, ferritin, are present and differentially localized in the tissue. Coalescence of ferritin and iron in mosquito ovaries may suggest that the role of ferritin in this tissue is for the deposition of iron in oogenesis and ovarian homeostasis.Grant Funding Source: Supported by funds from the Arizona Ag Experiment Station, CALS, UBRP and the Honors College

Relative quantification of the effect of blood meal iron on protein expression in the ovaries of Aedes aegypti (778.8)

Diseases transmitted by mosquitoes are estimated to kill more than 2 million people annually. The vector for dengue, Aedes aegypti (yellow fever mosquito), is found in the desert Southwest of the continental United States. One approach to limiting the spread of disease is to reduce the numbers of mosquito vectors. Our area of research is iron metabolism. Our long term goal is to use molecular techniques to alter the expression of iron related proteins in mosquitoes to reduce mosquito fecundity and survival. Our hypothesis is that female mosquitoes require iron in the blood meal as an essential nutrient to aid in egg development. Although the iron is necessary for fecundity it is also highly oxidative and toxic in biological systems unless the mineral is strictly regulated to mitigate its oxidative potential. We are currently analyzing tissue‐specific, iron regulated proteins of the mosquito ovary 24 h post‐blood meal fed on artificial blood meal diets supplemented with no iron or low iron, or a porcine blood meal (high iron). Measurement of the blood meal iron effect on ovarian protein expression was carried out using Tandem Mass Tags (TMT) which permits simultaneous identification and relative quantification of proteins during LTQ Velos Orbitrap LC‐MS/MS analysis. This research will contribute to our understanding of mosquito development in general, as well as ovarian iron metabolism.Grant Funding Source: Supported by funds from the Arizona Ag Experiment Station and CALS

BENDEL KESLINGMAS NO 121 TAHUN 2014

Malaria is one of the health problems cause of death. Mosquito longevity is an important factor to estimate the transmission. How to estimate the age of the mosquitoes with the condition of mosquito ovaries. The reseach was conducted in the seven villages malaria endemic of Banyumas Regency that is Karangsalam village, Karanggintung, Ketanda, Bogangin, Selanegara, Banjarpanepen and Watuagung to know about describe the lifetime of the mosquito Anopheles Spp in the seven villages malaria endemic, describing delatasi, parity rate, life chances and the age estimation of mosquito Anopheles Spp. The method used is descriptive by describe te lifetime of the mosquito Anopheles Spp. The data used in this research is a secondary data obtained from health center and the BPS Banyumas regency. The result showed the age estimation of Anopheles Spp mosquitos in the seven village malaria endemic that is Karangsalam, Ketanda, Selanegara, Banjarpanepen and Watuagung unknown. In the Karanggintung village the age estimation An. balabacensis mosquito is 4,24 days, whereas in the Bogangin village the age estimation An. vagus mosquito is 2,16 days. Conclusion from the research is illustration lifetime Anopheles Spp in the seven village malaria endemic is overall obatained the age estimation highest of 4,24 days is An. balabacensis mosquito. So, An. balabacensis mosquito have the ability to live in the wild longer than other Anophles Spp mosquitos were found. From the result expected for the government to increase attention to the case of malaria, especially in malaria endemic areas.

Aedes aegypti juvenile hormone acid methyl transferase, the ultimate enzyme in the biosynthetic pathway of juvenile hormone III, exhibits substrate control

We report on the cloning, sequencing, characterization, 3D modeling and docking of Aedes aegypti juvenile hormone acid methyl transferase (AeaJHAMT), the enzyme that converts juvenile hormone acid (JHA) into juvenile hormone (JH). Purified recombinant AeaJHAMT was extensively characterized for enzymatic activity and the Michaelis Menten kinetic parameters Km, Vmax, kcat (turn over number) and kcat/Km (catalytic efficiency) using JHA and its analogues as substrates. AeaJHAMT methylates JHA III 5-fold faster than farnesoic acid (FA). Significant differences in lower methyl transferase (MT) activities towards the cis/trans/trans, cis/trans/cis and the trans/cis/cis isomers of JHA I (1.32, 4.71 and 156-fold, respectively) indicate that substrate chirality is important for proper alignment at the catalytic cavity and for efficient methyl transfer by S-adenosyl methionine (SAM). Our 3D model shows a potential binding site below the main catalytic cavity for JHA analogues causing conformational change and steric hindrance in the transfer of the methyl group to JHA III. These, in silico, observations were corroborated by, in vitro, studies showing that several JHA analogues are potent inhibitors of AeaJHAMT. In vitro, and in vivo studies using [3H-methyl]SAM show that the enzyme is present and active throughout the adult life stage of A. aegypti. Tissue specific expressions of the JHAMT gene of A. aegypti (jmtA) transcript during the life cycle of A. aegypti show that AeaJHAMT is a constitutive enzyme and jmtA transcript is expressed in the corpora allata (CA), and the ovary before and after the blood meal. These results indicate that JH III can be synthesized from JHA III by the mosquito ovary, suggesting that ovarian JH III may play an important physiological role in ovarian development and reproduction. Incubating AeaJHAMT with highly pure synthetic substrates indicates that JHA III is the enzyme’s preferred substrate, suggesting that AeaJHAMT is the ultimate enzyme in the biosynthetic pathway of JH III.

The effect of rainstorms on adult Anopheles funestus behavior and survival

We describe the effect that the passage of a cold front, with a subsequent heavy rainstorm ten days later, had on a population of Anopheles funestus mosquitoes collected exiting houses or in light-traps from a village in southern Mozambique. Temperature effects explained 40% (r=0.634; p <0.001) of the variation in numbers of males collected and 19% of the variation in gravid females collected (r=0.437; p=0.033). The age structure of mosquitoes varied according to distance from the breeding site (χ(2) = 64.1, df 6, p <0.001). The proportion of parous insects that were caught in the light-traps with sacs (χ(2) = 6.33, d.f. 2, p=0.042) and young insects that had mated before being collected (χ(2) = 13,3, d.f. 2, p=0.001) were reduced on the night of the rain but this effect was short lived. It is concluded that the effect of rain on mosquito populations depends on the kind of water body used for larval development.

Identification and characterization of a novel ferritin subunit from Aedes aegypti

Mosquitoes consume and absorb significant amount of iron from a blood meal. Ferritin is a protein that is used to store iron. Iron is necessary for egg development in mosquitoes. We wanted to examine the proteome of ovaries from Aedes aegypti (yellow fever mosquito) that were fed an artificial blood meal with or without hemoglobin. The number of proteins identified in ovaries from mosquitoes that were fed an artificial blood meal with or without hemoglobin was 48 and 13 respectively. A previously uncharacterized glycosylated ferritin light chain homologue (G‐LCH) was found in ovaries of mosquitoes that were fed hemoglobin rich diet. Primers were designed specifically for G‐LCH to perform PCR and real‐time PCR. G‐LCH ferritin was cloned and sequenced. Real‐time PCR showed that eggs, larvae and pupae have similar expression levels of G‐LCH. Males and 72h post blood fed (PBF) females have higher expression levels of this message. Real‐time PCR will be performed on tissues of 24h and 72h PBF mosquitoes. The 5’‐UTR region of G‐LCH mRNA will be examined using rapid amplification of cDNA ends to determine if stem loop moieties are present. Characterization of G‐LCH subunit will give further insights into the unique regulation of iron metabolism in mosquitoes. Research support was from NIH(GM056812), the Agricultural Experiment Station and CALS at the University of Arizona.

Regulation of the ribonucleotide reductase small subunit (R2) in the yellow fever mosquito, Aedes aegypti

Ribonucleotide reductase (RNR) catalyzes the formation of deoxyribonucleotides, a rate limiting step in DNA synthesis. Class I RNR is a tetramer that consists of two subunits, R1 and R2; enzymatic activity requires association of R1 with R2. The R2 subunit is of special interest because it dictates the interaction with R1 that is required for enzymatic activity expression, and it is expressed only during the S phase of the cell cycle. We previously sequenced an R2 cDNA clone from the yellow fever mosquito, Aedes aegypti. We found the message was upregulated by blood feeding. We now report the sequence of an R2 genomic clone. The gene consists of 4 introns and 5 exons. Both major and minor transcriptional start sites have been identified, and their use differs in sugar-fed versus blood-fed females. The gene contains putative cis-regulatory sites for E2F, Caudal (Cdx) and Dearolf (Dfd). The mosquito R2 gene contains iron-specific regulatory elements immediately upstream of the minimal promoter region. Binding of a factor to the distal putative Cdx site in the − 400 region is altered by iron treatment of cells. Further, following blood feeding, R2 message is significantly induced in mosquito ovaries (tissues that are involved in oogenesis—a process requiring DNA synthesis).

QUANTITATIVE ANALYSIS OF LA CROSSE VIRUS TRANSCRIPTION AND REPLICATION IN CELL CULTURES AND MOSQUITOES

La Crosse (LAC) virus (family Bunyaviridae, genus Orthobunyavirus) small (S) segment negative-sense RNA genome (vRNA), positive-sense full-length RNA complement (vcRNA), and subgenomic mRNA were assayed in infected cell cultures and female Aedes (Ochlerotatus) triseriatus mosquito tissues using quantitative PCR (Q-PCR). During persistent infection of C6/36 (Aedes albopictus) and MAT (Aedes triseriatus) cultured cells and cytolytic infection of BHK-21 cultured cells, LAC vRNA was the most abundant RNA species, followed by mRNA and vcRNA. RNA copy numbers per cell were quantified and vRNA correlated to virus titer in cell culture medium. The Q-PCR assay proved more sensitive than reverse transcription (RT)-PCR and immunofluorescence assays (IFA) for detecting LAC virus infection of mosquitoes. After infection of female mosquitoes orally, quantities of LAC RNA increased in ovaries for 6 days, and as ovarian biosynthetic activity quiesced, LAC RNA quantities decreased then remained detectable at a low level. After a second, noninfectious blood meal, quantities of LAC RNA in ovaries increased significantly, quantitatively confirming correlation of LAC virus RNA synthesis with vector metabolic activity. Coregulation of viral replication and mosquito ovary metabolic activity may condition efficient transovarial transmission.

Expression of genes encoding proteins involved in ecdysteroidogenesis in the female mosquito, Aedes aegypti

A blood meal induces the ovaries of female Aedes aegypti mosquitoes to produce ecdysteroid hormones that regulate many processes required for egg maturation. Various proteins involved in the intracellular transport and biosynthesis of ecdysteroid precursors have been identified by analysis of Drosophila melanogaster mutants and by biochemical and molecular techniques in other insects. To begin examining these processes in mosquito ovaries, complete cDNAs were cloned for putative orthologs of diazepam-binding inhibitor ( DBI), StAR-related lipid transfer domain containing protein ( Start1), aldo/keto reductase ( A/KR), adrenodoxin reductase ( AR), and the cytochrome P450 enzymes, CYP302a1 (22-hydroxylase), CYP315a1 (2-hydroxylase) and CYP314a1 (20-hydroxylase). As shown by RT-PCR, transcripts for all seven genes were present in ovaries and other tissues both before and following a blood meal. Expression of these genes likely supports the low level of ecdysteroids produced in vitro (7–10 pg /tissue/ 6 h) by tissues other than ovaries. Ovaries from females not blood fed and up to 6 h post blood meal (PBM) also produced low amounts of ecdysteroids in vitro, but by 18 and 30 h PBM, ecdysteroid production was greatly increased (75–106 pg /ovary pair/ 6 h) and thereafter (48 and 72 h PBM) returned to low levels. As determined by real-time PCR analysis, gene transcript abundance for AedaeCYP302 and AedaeCYP315a1 was significantly greater (9 and 12 fold, respectively) in ovaries during peak ecdysteroid production relative to that in ovaries from females not blood fed or 2 h PBM. AedaeStart1, AedaeA/KR and AedaeAR also had high transcript levels in ovaries during peak ecdysteroid production, and AedaeDBI transcripts had the greatest increase at 48 h PBM. In contrast, gene transcript abundance of AedaeCYP314a1 decreased PBM. This study shows for the first time that transcription of a few key genes for proteins involved in ecdysteroid biosynthesis is positively correlated with the rise in ecdysteroid production by ovaries of a female insect.

Aedes aegyptitransferrin. Gene structure, expression pattern, and regulation

Mosquitoes and all other insects so far examined have an abundant haemolymph transferrin (Tsf). The exact function of these proteins has not been determined, but they may be involved in iron transport, in oogenesis and in innate immune defence against parasites and pathogens. The Tsf gene of Aedes aegypti has been cloned and sequenced. It contains a single small intron, which contrasts it to vertebrate Tsf genes that contain up to sixteen introns. The promoter region of the gene is rich in putative NF-kappaB binding sites, which is consistent with the postulated role of Tsf in insect innate immunity. Tsf message levels are very low in embryos and early larvae, but high in late larvae, pupae and adults. Western blotting experiments revealed high levels of Tsf protein in pupae and adults. Late larvae and ovaries of blood-fed mosquitoes have little intact protein, but two prominent proteolytic degradation products. These may represent biologically active peptides, as has been shown for other organisms. Tsf message is down-regulated by inorganic iron in the diet or environment, but up-regulated by a blood meal in the adult female. The up-regulation following a blood meal may, in part, be due to the decrease in juvenile hormone (JH) that is known to follow blood feeding. Treatment of blood-fed females with methoprene, an analogue of JH, resulted in decrease of the Tsf message.

Tissue- and Stage-specific Expression of Two Lipophorin Receptor Variants with Seven and Eight Ligand-binding Repeats in the Adult Mosquito

We identified two splice variants of lipophorin receptor (LpR) gene products specific to the mosquito fat body (AaLpRfb) and ovary (AaLpRov) with respective molecular masses of 99.3 and 128.9 kDa. Each LpR variant encodes a member of the low density lipoprotein receptor family with five characteristic domains: 1) ligand recognition, 2) epidermal growth factor precursor, 3) putative O-linked sugar, 4) single membrane-spanning domains, and 5) the cytoplasmic tail with a highly conserved internalization signal FDNPVY. Proposed phylogenetic relationships among low density lipoprotein receptor superfamily members suggest that the LpRs of insects are more closely related to vertebrate low density lipoprotein receptors and very low density lipoprotein receptor/vitellogenin receptor than to insect vitellogenin receptor/yolk protein receptors. Two mosquito LpR isoforms differ in their amino termini, the ligand-binding domains, and O-linked sugar domains, which are generated by differential splicing. Polymerase chain reaction and Southern blot hybridization analyses show that these two transcripts originated from a single gene. Significantly, the putative ligand-binding domain consists of seven and eight complement-type, cysteine-rich repeats in AaLpRfb and AaLRov, respectively. Seven cysteine-rich repeats in AaLpRfb are identical to the second through eighth repeats of AaLpRov. Previous analyses (1) have indicated that the AaLpRov transcript is present exclusively in ovarian germ-line cells, nurse cells, and oocytes throughout the previtellogenic and vitellogenic stages, with the peak at 24-30 h after blood meal, coincident with the peak of yolk protein uptake. In contrast, the fat body-specific AaLpRfb transcript expression is restricted to the postvitellogenic period, during which yolk protein production is terminated and the fat body is transformed to a storage depot of lipid, carbohydrate, and protein.

Biosynthesis and control of mosquito gut proteases.

Earlier investigations of the proteases that digest the blood meal in mosquitoes identified the midgut epithelial cells as the source of the main blood digesting protease, trypsin. Surgical manipulations and cytoimmunochemical studies indicated that trypsin in the mosquito is not stored in the epithelial cells as an inactive enzyme, but is synthesized, activated and released into the gut lumen after the blood meal. Using molecular biology techniques the major trypsin genes have been sequenced and some information on the transcription factors that regulate the induction of the genes have been reported. Although all the hormone(s) and the factors that are responsible for the induction of trypsin biosynthesis are not known, the termination of trypsin biosynthesis is controlled by a decapeptide hormone; trypsin modulating oostatic factor (TMOF) that is secreted by the mosquito ovary. Genetic engineering and expression of TMOF in bacteria, yeast and algae is emerging as a potential future larval control of mosquitoes in the field.

Cloning and tissue distribution of two Na+/H+ exchangers from the Malpighian tubules of Aedes aegypti.

A Na(+)/H(+) exchanger (NHE) on the apical membrane of mosquito Malpighian tubule (MT) cells is believed to participate in the blood-feeding mosquito's vital secretion of Na(+) and fluid. This study presents the molecular cloning, primary structure, and tissue distribution of two cDNAs encoding Aedes aegypti mosquito MT NHEs. The cDNA sequences were obtained from mosquito MT total RNA using reverse transcription-polymerase chain reaction (RT-PCR) and 5' and 3' rapid amplification of cDNA ends (RACE). The two sequences encode proteins of 678 and 1,179 amino acids with calculated molecular weights of 74,473 and 130,276, respectively. When comparing the 678 amino acid protein to the first 678 amino acids of the other protein, the two clones show 98% identity to each other. They also exhibit high identity to Drosophila melanogaster NHEs. Hydropathy analysis reveals that while both clones have 10-13 transmembrane segments, the 1,179 amino acid protein has an extensive carboxy terminus while the 678 amino acid protein has an extremely short carboxy terminus. RT-PCR analysis shows that both clones are expressed in the mosquito Malpighian tubules at the larval and pupal stages, in addition to the adult stage before and after blood-feeding. Expression of both clones was also detected in adult mosquito ovaries, midguts, and hindguts.

Drosophila yolk protein produced in E. coli is accumulated by mosquito ovaries.

Despite similar functions, the yolk proteins of the higher dipteran flies and the vitellogenins found in other insects are unrelated at the sequence level and have evolved from different genes. Both are selectively endocytosed into the ovary via receptors belonging to the LDLR receptor subfamily. We cloned the Drosophila yp1 gene into an E. coli expression vector and showed that the yolk protein produced by E. coli is taken up into ovaries of both Drosophila melanogaster and the malaria mosquito Anopheles gambiae, which normally uses vitellogenin.

Cloning and Characterization of CPVL, a Novel Serine Carboxypeptidase, from Human Macrophages

Carboxypeptidases are proteases that cleave single amino acids from the carboxy termini of proteins or peptides. In addition to degradative functions in the gut, carboxypeptidases activate or inactivate bioactive peptides such as angiotensin, bradykinin, and endothelin I. Using differential display PCR, we cloned a novel carboxypeptidase expressed in human macrophages but not in other leukocytes. The 476-amino-acid gene product has a putative signal sequence but no transmembrane domain and has striking sequence similarity to serine carboxypeptidases, a large family of enzymes in eukaryotes. Only one serine carboxypeptidase, lysosomal protective protein, has previously been reported in mammals. Among known proteins, this gene is most similar (43% amino acid identity) to vitellogenic carboxypeptidase, a serine carboxypeptidase expressed in mosquito ovaries. Therefore, we have named this new gene carboxypeptidase, vitellogenic-like (CPVL). In addition to monocyte/macrophage-rich sources such as spleen, leukocytes, and placenta, CPVL mRNA is abundantly expressed in heart and kidney, suggesting a separate role for CPVL outside the immune system. The CPVL gene contains at least 13 exons spread over more than 150 kb on human chromosome 7p14–p15. An affinity-purified polyclonal antiserum recognized a protein of approximately 57 kDa in macrophage lysates, but not in lysates from lymphocytes, neutrophils, or monocytes. CPVL protein expression was induced during maturation of monocytes into macrophages. Possible functions for CPVL in macrophages include digestion of phagocytosed particles in the lysosome, participation in an inflammatory protease cascade, and trimming of peptides for antigen presentation.

A novel member of the RING-finger gene family associated with reproductive tissues of the mosquito, Aaedes aegypti.

The RING finger is a zinc-binding domain that is found in proteins from viruses, plants and animals. Here we report the characterization and tissue-specific expression of a mosquito gonadal protein gene, mgp, from the mosquito, Aedes aegypti. The putative gene product, MGP, contains two RING fingers, a B-box, and a hydrophobic core. These mosquito MGP structural motifs are highly conserved in proteins found in mouse and nematode. Northern blot analysis and in situ hybridization demonstrated the presence of multiple mgp RNA transcripts in male and female reproductive tissues. Expression of mgp in the ovary is constitutive, but an increase in message was observed in the ovaries of female mosquitoes previously exposed to a blood meal. These results suggest that MGP is a protein that might play a role(s) in mosquito gametogenesis.

Expression of the early-late gene encoding the nuclear receptor HR3 suggests its involvement in regulating the vitellogenic response to ecdysone in the adult mosquito

The insect steroid hormone, 20-hydroxyecdysone (20E), is a key factor controlling critical developmental events of embryogenesis, larval molting, metamorphosis, and, in some insects, reproduction. We are interested in understanding the molecular basis of the steroid hormone ecdysone action in insect egg development. The yellow fever mosquito, Aedes aegypti, in addition to being an important vector of human diseases, represents an outstanding model for studying molecular mechanisms underlying egg maturation due to stringently controlled, blood meal-activated reproductive events in this insect. To elucidate the genetic regulatory hierarchy controlling the reproductive ecdysone response, we have investigated ecdysone-regulated gene expression in vitellogenic mosquito ovaries and fat bodies. We have previously demonstrated the conservation of a primary ecdysone-triggered regulatory hierarchy, implicated in development of immature stages of Drosophila, represented by the ecdysone receptor/Ultraspiracle complex and an early gene E75 during the reproductive ecdysone response (Wang, S.-F., Miura, K., Miksicek, R.J., Segraves, W.A., Raikhel, A.S., 1998. DNA binding and transactivation characteristics of the mosquito ecdysone receptor — Ultraspiracle complex. J. Biol. Chem. 273, 27531–27540; Pierceall, W.E., Li, C., Biran, A., Miura, K., Raikhel, A.S., Segraves, W.A., 1999. E75 expression in mosquito ovary and fat body suggests reiterative use of ecdysone-regulated hierarchies in development and reproduction. Mol. Cell. Endocrinol. 150, 73–89). The present paper demonstrates that conservation of the factors involved in the ecdysone-responsive genetic hierarchy regulating female reproduction extends beyond the early genes. Here, we identify AHR3, a highly conserved homologue of the Drosophila HR3 early-late ecdysone-inducible gene in the mosquito. We show that AHR3 is expressed in both vitellogenic tissues of the female mosquito, the fat body and the ovary. The expression of AHR3 correlates with the ecdysteroid titer, reaching a peak at 24 h after a blood meal. Moreover, in vitro fat body culture experiments demonstrate that the kinetics and dose response of AHR3 to 20-hydroxyecdysone (20E), an active ecdysteroid in the mosquito, is similar to those of the late vitellogenic genes rather than the early E75 gene. However, as shown for other early and early-late genes, the 20E activation of AHR3 is not inhibited by the presence of cycloheximide, a protein synthesis inhibitor. Taken together, these findings strongly suggest AHR3 involvement in regulating the vitellogenic response to ecdysone in the adult mosquito.

E75 expression in mosquito ovary and fat body suggests reiterative use of ecdysone-regulated hierarchies in development and reproduction

The steroid hormone ecdysone controls genetic regulatory hierarchies underlying insect molting, metamorphosis and, in some insects, reproduction. Cytogenetic and molecular analysis of ecdysone response in Drosophila larval salivary glands has revealed regulatory hierarchies including early genes which encode transcription factors controlling late ecdysone response. In order to determine whether similar hierarchies control reproductive ecdysone response, we have investigated ecdysone-regulated gene expression in vitellogenic mosquito ovaries and fat bodies. Here, we identify the homologue of the Drosophila E75 early ecdysone inducible gene in the yellow fever mosquito Aedes aegypti, and show that, as in Drosophila, the mosquito homologue, AaE75, consists of three overlapping transcription units with three mRNA isoforms, AaE75A, AaE75B, and AaE75C, originating as a result of alternative splicing. All three AaE75 isoforms are induced at the onset of vitellogenesis by a blood meal-activated hormonal cascade, and highly expressed in the mosquito ovary and fat body, suggesting their involvement in the regulation of oogenesis and vitellogenesis, respectively. Furthermore, in vitro fat body culture experiments demonstrate that AaE75 isoforms are induced by 20-hydroxyecdysone, an active ecdysteroid in the mosquito. These findings suggest that related ecdysone-triggered regulatory hierarchies may be used reiteratively during developmental and reproductive ecdysone responses.