Mosquito Fat Body Research Articles

Lipophorin as a yolk protein precursor in the mosquito, Aedes aegypti

We examined expression of the lipophorin ( Lp) gene, lipophorin (Lp) synthesis and secretion in the mosquito fat body, as well as dynamic changes in levels of this lipoprotein in the hemolymph and ovaries, during the first vitellogenic cycle of females of the yellow fever mosquito, Aedes aegypti. Lipophorin was purified by potassium bromide (KBr) density gradient ultracentrifugation and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Polyclonal antibodies were produced against individual Lp apoproteins, apolipoprotein-I (apoLp-I) and apolipoprotein-II (apoLp-II), with molecular weights of 240 and 75 kDa, respectively. We report here that in the mosquito A. aegypti, Lp was synthesized by the fat body, with a low level of the Lp gene expression and protein synthesis being maintained in pre- and postvitellogenic females. Following a blood meal, the Lp gene expression and protein synthesis were significantly upregulated. Our findings showed that the fat body levels of Lp mRNA and the rate of Lp secretion by this tissue reached their maximum at 18 h post-blood meal (PMB). 20-Hydroxyecdysone was responsible for an increase in the Lp gene expression and Lp protein synthesis in the mosquito fat body. Finally, the immunocytochemical localization of Lp showed that in vitellogenic female mosquitoes, this protein was accumulated by developing oocytes where it was deposited in yolk granules.

Conserved Molecular Mechanism for the Stage Specificity of the Mosquito Vitellogenic Response to Ecdysone

In the mosquito Aedes aegypti, the adult female becomes competent for a vitellogenic response to ecdysone after previtellogenic development. Here, we show that βFTZ-F1, the nuclear receptor implicated as a competence factor for stage-specific responses to ecdysone during Drosophila metamorphosis, serves a similar function during mosquito vitellogenesis. AaFTZ-F1 is expressed highly in the mosquito fat body during pre- and postvitellogenic periods when ecdysteroid titers are low. The mosquito AaFTZ-F1 transcript nearly disappears in mid-vitellogenesis when ecdysteroid titers are high. An expression peak of HR3, a nuclear receptor implicated in the activation of βFTZ-F1 in Drosophila, precedes each rise in mosquito FTZ-F1 expression. In in vitro fat body culture, AaFTZ-F1 expression is inhibited by 20-hydroxyecdysone (20E) and superactivated by its withdrawal. Following in vitro AaFTZ-F1 superactivation, a secondary 20E challenge results in superinduction of the early AaE75 gene and the late target VCP gene. Electrophoretic mobility-shift assays show that the onset of ecdysone-response competence in the mosquito fat body is correlated with the appearance of the functional AaFTZ-F1 protein at the end of the previtellogenic development. These findings suggest that a conserved molecular mechanism for controlling stage specificity is reiteratively used during metamorphic and reproductive responses to ecdysone.

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.

Two distinct subpopulations of ecdysone receptor complex in the female mosquito during vitellogenesis

The native functional ecdysone receptor complex, a heterodimer of the ecdysone receptor (EcR) and ultraspiracle (USP) proteins, was identified in the fat body of adult female mosquitoes, Aedes aegypti, through electrophoretic mobility shift assays (EMSA) using previously characterized Drosophila ecdysone response elements (EcREs). The use of different salt concentrations during preparation of nuclear extracts enabled us to characterize two distinct subpopulations of the receptor complex, one of which was high salt-sensitive and responsive to exogenous 20-hydroxyecdysone (20E), and the other of which was high salt-resistant and refractory to exogenous 20E. Salt-sensitivity correlated with ligand responsiveness. Developmental EMSA analyses demonstrated that previtellogenic fat body nuclei and nuclei from the termination phase of vitellogenesis with low 20E titer contained solely high-salt-sensitive, ligand responsive complexes, which could be recovered in nuclear extracts (NEs) only by low salt tissue homogenization, suggesting these complexes were unliganded. In contrast, the fat body nuclei from stages of active vitellogenesis with high 20E titer contained almost exclusively high salt-resistant, ligand refractory complexes, implying these complexes were liganded; the nuclei from the intermediate stages, early and late phases of vitellogenesis, contained a mixture of the two subpopulations. The developmental profile of fully activated, ligand refractory receptor complexes closely correlated with that of yolk protein expression, suggesting an intimate involvement of the ecdysone receptor complex in both the induction and maintenance of high level expression of yolk protein genes.

Nuclear Receptors in Mosquito Vitellogenesis

Vitellogenesis in insects involves the coordinated activity of the fat body, which produces large amounts of yolk protein precursors (YP), and oocytes, which specifically accumulate these proteins. The expression of YP genes is achieved through strict sex-, tissue-, and hormone-specific control in the female fat body. In mosquitoes, expression of YP genes is controlled by 20-hydroxyecdysone (20E). To elucidate the role of 20E in mosquito vitellogenesis, we cloned cDNAs encoding the Aedes aegypti ecdysteroid receptor (AaEcR) and two isoforms of its heterodimeric partner, the Ultraspiracle homologue (AaUSP). The two AaUSP isoforms differ in their A/B domains and have distinct expression patterns. The ecdysone regulation of YP genes likely involves products of early genes. We cloned the gene of the mosquito homologue to the Drosophila early gene E75 (AaE75) belonging to the nuclear receptor superfamily. Kinetics of AaE75 expression correlate with the expression of YP genes, suggesting that AaE75 may have a regulatory role in YP gene expression. A second nuclear receptor superfamily member, the NGFI-B homologue AaHR38 is implicated in repression of the ecdysone-signaling pathway in the fat body of the previtellogenic female mosquito at the state-of-arrest. Finally, three isoforms of the hepatocyte nuclear factor 4 (HNF-4) homologue AaHNF-4 are differentially expressed in the mosquito fat body during vitellogenesis, suggesting their involvement in regulating vitellogenic events in this tissue.

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.

Three Isoforms of a Hepatocyte Nuclear Factor-4 Transcription Factor with Tissue- and Stage-specific Expression in the Adult Mosquito

We cloned three isoforms of hepatocyte nuclear factor-4 (HNF-4) from the mosquito Aedes aegypti, designated AaHNF-4a, AaHNF-4b, and AaHNF-4c. AaHNF-4a and AaHNF-4b are typical members of the HNF-4 subfamily of nuclear receptors with high amino acid conservation. They differ in N-terminal regions and exhibit distinct developmental profiles in the female mosquito fat body, a metabolic tissue functionally analogous to the vertebrate liver. The AaHNF-4b mRNA is predominant during the previtellogenic and vitellogenic phases, while the AaHNF-4a mRNA is predominant during the termination phase of vitellogenesis, coinciding with the onset of lipogenesis. The third isoform, AaHNF-4c, lacks part of the A/B and the entire C (DNA-binding) domains. The AaHNF-4c transcript found in the fat body during the termination of vitellogenesis may serve as a transcriptional inhibitor. Both AaHNF-4a and AaHNF-4b bind to the cognate DNA recognition site in electrophoretic mobility shift assay. Dimerization of AaHNF-4c with other mosquito HNF-4 isoforms or with mammalian HNF-4 prevents binding to the HNF-4 response element. In transfected human 293T cells, AaHNF-4c significantly reduced the transactivating effect of the human HNF-4alpha1 on the apolipoprotein CIII promoter. Electrophoretic mobility shift assay confirmed the presence of HNF-4 binding sites upstream of A. aegypti vg and vcp, two yolk protein genes expressed in the female mosquito fat body during vitellogenesis. Therefore, HNF-4, an important regulator of liver-specific genes, plays a critical role in the insect fat body.

Changes in ribosomal protein rpl8 mRNA during the reproductive cycle of the mosquito, Aedes aegypti

Changes in the abundance of ribosomal protein rpL8 mRNA were compared with 18S rRNA and vitellogenin mRNA in fat body from adult female Aedes aegypti during the reproductive cycle. Levels of rpL8 mRNA began to increase within 2 h after adult eclosion, peaked at about 24 h post-eclosion, and remained high throughout the next 48 h. During this same period, rRNA abundance increased about 2-fold. After the bloodmeal, levels of rpL8 mRNA gradually decreased over the next 48 h, while rRNA levels increased about 4-fold within 16–24 h postbloodfeeding and eventually returned to previtellogenic levels. Vitellogenin mRNA was induced only after the bloodmeal, and disappeared by 48 h after feeding. After oviposition, rpL8 mRNA levels again increased to pre-bloodfeeding levels. These results indicate that rpL8 mRNA transcription in mosquito fat body is independent of rRNA transcription during the previtellogenic acquisition of competence and also during the post-bloodmeal ovarian cycle. Moreover, unlike the vitellogenin gene, the rpL8 gene is under post-transcriptional regulation in blood-fed females.

A proposal for typical eukaryotic meiosis in microsporidians

A review of key publications concerning the biology and life cycles of microsporidians has led us to challenge recent proposals that they exhibit a unique process of meiosis when compared with other eukaryotes. The basic data used to support this challenge are the same as those used by researchers suggesting atypical meiosis, but the data are analyzed from a different viewpoint. Arguments are put forward to support a testable hypothesis that meiosis in microsporidians is identical to that which occurs in other eukaryotes. It is proposed that confusion resulted because two separate cytological developmental sequences for diplokaryotic meronts in mosquito fat body cells were previously interpreted as a single sequence. By rearrangement of the original data into two developmental sequences, one abortive and the other for typical meiosis, a better fit was obtained between cytological stages and microphotometric measurements of nuclear DNA content. This improved data fit and the existence of similar nuclear developmental sequences in the fungi are used to support our hypothesis.Key words: microsporidia, meiosis, eukaryotes.

Cloning of cDNA for mosquito lysosomal aspartic protease. Sequence analysis of an insect lysosomal enzyme similar to cathepsins D and E.

A cDNA coding for the lysosomal aspartic protease from the mosquito (mLAP) was cloned and sequenced. The mLAP cDNA is 1420 base pairs long with an open reading frame of 387 amino acids. The deduced amino acid sequence contains a signal pre-propeptide sequence of 18 amino acids followed by 369 amino acids with a 35-amino acid putative pro-enzyme domain in the NH2-terminal. The amino acid sequence of mLAP is 92 and 81% similar to human cathepsin D and cathepsin E, respectively. Typical cleavage sites for cathepsin D processing into light and heavy chains are lacking in mLAP. A single glycosylation site occurs in the mLAP sequence at a position corresponding to the first glycosylation site of cathepsins D. The mLAP sequence shares putative phosphorylation determinants, which in cathepsins D are linked to the formation of mannose 6-phosphate. In the mosquito fat body, lysosomal enzymes specifically degrade organelles involved in the biosynthesis and secretion of vitellogenin. The mLAP mRNA accumulates to its highest level 24 h after initiation of vitellogenin synthesis and 12 h before the peak of mLAP protein accumulation and its enzymatic activity. Translational regulation of mLAP mRNA may occur. The 5'-untranslated region of mLAP mRNA is similar to elements conferring negative translational control by steroids.

A novel protein produced by the vitellogenic fat body and accumulated in mosquito oocytes.

The fat body of vitellogenic mosquitoes was found to synthesize and secrete another protein in addition to vitellogenin, that accumulated in developing oocytes. In the tissues, this protein has Mr = 53000 on SDS-PAGE under reducing or non-reducing conditions. This protein is glycosylated as shown by [3H]mannose incorporation and experiments with tunicamycin. Polyclonal antibodies were produced using the ovarian 53-kDa peptide. Immunoblot analysis demonstrated the immunological identity of 53 kDa peptides from the fat body and the ovary. Furthermore, the 53-kDa protein (53KP) is synthesized and secreted exclusively by the vitellogenic fat body. Radioimmunoassay showed that 53KP is produced by the female fat body as early as 4 h and reaches its peak near 24 h after the initiation of vitellogenesis. Synthesis then drops to low levels by 36 h and declines to background levels by 48 h. In vitro experiments conducted on fat bodies of previtellogenic females demonstrated that the synthesis and secretion of 53KP can be stimulated by a physiological dose of 20-hydroxyecdysone (10-6 M). Immunocytochemical studies of the ovary demonstrate that 53KP is present in channels between follicle cells, in the perioocytic space and in yolk granules of the developing oocytes. This suggests that 53KP is accumulated in the oocytes by a pathway similar to that of vitellogenin.

Juvenile hormone controls previtellogenic proliferation of ribosomal RNA in the mosquito fat body

During the previtellogenic development of mosquito fat body cells, the nucleolus, the organelle responsible for producing ribosomes, enlarges threefold, reaching maximal size between 2 and 3 days after eclosion. The granular component of the nucleolus containing ribosomal precursors increases considerably as well. These signs of nucleolar activation correlate with the synthetic rate and accumulation of poly(A) − RNA (predominantly ribosomal RNA) in the fat body cells. The amount of poly(A) − RNA in fat body cells increases during the first 2 days after eclosion and then declines gradually. The rate of RNA synthesis exhibits similar kinetics, but both the rise and the decline are sharper than for the accumulation of RNA. All the characteristics of nucleolar activation, its enlargement, accumulation of poly(A) − RNA, and the increased rate of RNA synthesis, are blocked by removal of the corpora allata (CA) in newly eclosed adult females but could be restored by either implantation of CA or topical application of juvenile hormone III or its analog, 7- S-methoprene, to allatectomized females. Thus, previtellogenic activation of fat body nucleoli for ribosomal RNA production is controlled by juvenile hormone from the corpora allata.

Lysosomes in the cessation of vitellogenin secretion by the mosquito fat body; selective degradation of Golgi complexes and secretory granules

A massive and selective degradation of Golgi complexes, secretory granules, and RER is the mechanism responsible for the rapid termination of Vg secretion by trophocytes of the mosquito fat body. These cells are involved in an intensive synthesis of a glycoprotein, vitellogenin (Vg), which is accumulated by developing oocytes as yolk protein. Previously, assays for lysosomal enzymes have demonstrated that the cessation of Vg synthesis is characterized by a sharp increase in lysosomal activity; and fluorescent microscopy has shown that, during this intense lysosomal activity, Vg concentrates in lysosomes. In this report, electron microscopy combined with cytochemistry for lysosomal enzymes and localization of Vg with colloidal gold immunocytochemistry has shown that this lysosomal activity is directed towards selective degradation of Vg and organelles associated with its synthesis and secretion. Three organelles undergo lysosomal breakdown: the Golgi complex, Vg-containing secretory granules, and RER. The degradation of Golgi complexes occurs in two steps similar to that for RER: first, the organelle is sequestered by double isolation membranes, and the resulting pre-lysosome then fuses with a primary or secondary lysosome. In contrast, mature Vg-containing secretory granules fuse with lysosomes directly. This combination of crino- and autophagy is a specific, highly intense, and precisely timed event.

Role of lysosomes in regulating of vitellogenin secretion in the mosquito fat body

Trophocytes of the mosquito fat body produce large amounts of the yolk-protein precursor, vitellogenin, that is then accumulated by developing oöcytes. Vitellogenin synthesis is a highly regulated process and is turned on and off according to the stage of the reproductive cycle; it is maximal 27 h after blood feeding and then rapidly declines. Here, I report a new mechanism involved in cellular regulation of vitellogenin secretion, e.g. the lysosomal breakdown of vitellogenin. The activities of four lysosomal enzymes rose sharply about 30 h after blood-feeding and remained high for the next 12 h. Video-enhanced fluorescence, combined with visualization of vitellogenin by immunolabelling and of lysosomes by acridine orange, demonstrated that this intense lysosomal activity was directed toward the specific degradation of vitellogenin; the latter diminished in the cytoplasm and was concentrated in large lysosomes, only later did it disappear entirely from the trophocytes.

The effects of α‐amanitin and cordycepin on vitellogenin synthesis by mosquito fat body

AbstractVitellogenin synthesis by mosquito (Aedes aegypti) fat body is stimulated by incubation with ecdysterone. α‐amanitin and cordycepin prevent the response to ecdysterone. These results support the hypothesis that ecdysterone regulates vitellogenin synthesis at the transcriptional level.

Termination of vitellogenin synthesis by mosquito fat body, a programmed response to ecdysterone

ABSTRACT. In the blood‐fed mosquito, peak vitellogenin synthesis occurs 24–32 h after the meal, dropping to resting levels by 40 h. Challenging fat body with ecdysterone in vitro at various times after a blood meal demonstrated a refractory period at about 50 h, when there was also a drastic decrease in mitochondria, rough endoplasmic reticulum, ribosomes, and glycogen in fat body cells. When fat bodies from sugar‐fed females were incubated with continuous ecdysterone in vitro, vitellogenin synthesis reached a peak at 30 h, but then declined even in the presence of ecdysteroné. This was not due to the in vitro conditions since fat bodies were responsive, even if first exposed to ecdysterone, after 80 h in vitro. If ecdysterone was removed, vitellogenin synthesis ceased. If it was replaced, the fat body responded again only if the initial removal was done during the first 30 h. It is proposed that the falling ecdysterone titre is the major cause of cessation of vitellogenin synthesis, but that synthesis is programmed to decline even if exposure to ecdysterone is abnormally prolonged.

Vitellogenin synthesis in the mosquito: the role of juvenile hormone in the development of responsiveness to ecdysone

ABSTRACT. Ecdysone stimulates the synthesis of vitellogenin in the fat body of mature female mosquitoes. Preparations from newly emerged animals, however, were found to be unresponsive to ecdysone. Responsiveness developed to a maximal level during a 36‐h post‐emergence period of maturation. This maturation could be accelerated with juvenile hormone application, prevented by allatectomy, and restored by corpora allata implants. It is concluded that the development of fat body responsiveness to ecdysone is dependent upon previous post‐emergence exposure to juvenile hormone.

The Control of Vitellogenesis in the Mosquito,Aedes aegypti

Evidence from several laboratories indicates that oogenesis in mosquitoes is divided into at least two discrete stages. First, emergence triggers development of the oocytes up to the resting stage where they remain until the blood meal. This stage is mediated by the juvenile hormone from the corpora allata. The second stage is triggered by the blood meal and involves the release of a hormone from the brain as well as the vitellogenin stimulating hormone (VSH) from the ovary. The mature oocytes then inhibit development of the penultimate oocytes until the first batch of eggs is laid. The evidence for an ovarian hormone, VSH, controlling vitellogenin synthesis by the mosquito fat body is reviewed. This hormone activates and maintains vitellogenin synthesis by the fat body in vivo and in vitro. The role of VSH in mosquito reproduction and the possibility of similar hormones in other insects is discussed.

Evidence of two inapparent nonoccluded viral infections of Culex tarsalis

Culex tarsalis from a mosquito colony maintained at Bakersfield, California, were infected with two noninclusion cytoplasmic viruses. Both viruses were polyhedral with average diameters of ca. 56 nm and 33 nm for C. tarsalis virus 1 and 2 (CTV1 and CTV2), respectively. CTV1 particles had an average prevalence in the colony of 24% over a 2-year period and CTV2 had a prevalence of 5%. The viruses were first observed in negatively stained preparations of crushed heads using sodium phosphotungstate. Both viruses were seen in thin sectioned material from the salivary glands, fat body, and nervous tissue of infected mosquitoes. CTV1 was retained through six serial passages by needle inoculations of infected mosquito suspensions into virus-free C. tarsalis adults. An estimated total dilution of >;10 18 indicated CTV1 had multiplied in C. tarsalis. The coincident appearance of CTV2 in some of the inoculated mosquitoes through the 5th passage suggested that this virus also multiplied on serial passage. CTV1 had a median incubation period of ca. 6.6 days in adult C. tarsalis kept at 25°C. Preliminary data indicated that the median survival period of male and female C. tarsalis injected with CTV1 was reduced 2 or 3 days over that of inoculated control insects when incubated at 25°C. Infection may cause a degeneration of salivary glands in adult females. CTV1 did not infect Culex pipiens and did not produce plaques in Vero or duck embryo cell cultures.

The migratory pathway of a filarial parasite (Breinlia sergenti) in a mosquito host (Aedes togoi).

A detailed observation on the site of development and the path of migration of the larvae of Breinlia sergenti (a filaria of slow loris) in Aedes togoi is presented. The larvae develop in the fat-body of mosquitoes. They are distributed abundantly in the fat-body of the abdomen and there is a scarcity of the larvae in the fat-body of the thorax and the head region. The majority of the larvae migrate into the head alongside the oesophagus and the thoracic ganglia. After entering the head, the mature larvae congregate in the large subcibarial haemocoelic space situated at the root of the proboscis. They then continue to migrate through the root of the proboscis into the lumen of the labium, where the infective larvae lie parallel to each other among the fascicles of the mouthparts in a unidirectional manner. The head of the larva is always directed towards the labella. In some heavily infected mosquitoes, the infective larvae are found to be coiled in the lumen of the labella.