Nuclear Receptors in Mosquito Vitellogenesis

Vitellogenesis in insects involves the coordinated activityof the fat body, which produces large amounts of yolk proteinprecursors (YP), and oocytes, which specifically accumulatethese proteins. The expression of YP genes is achieved throughstrict sex-, tissue-, and hormone-specific control in the femalefat body. In mosquitoes, expression of YP genes is controlledby 20-hydroxyecdysone (20E). To elucidate the role of 20E inmosquito vitellogenesis, we cloned cDNAs encoding the Aedesaegypti ecdysteroid receptor (AaEcR) and two isoforms of itsheterodimeric partner, the Ultraspiracle homologue (AaUSP).The two AaUSP isoforms differ in their A/B domains and havedistinct expression patterns. The ecdysone regulation of YPgenes likely involves products of early genes. We cloned thegene of the mosquito homologue to the Drosophila early geneE75 (AaE75) belonging to the nuclear receptor superfamily. Kineticsof AaE75 expression correlate with the expression of YP genes,suggesting that AaE75 may have a regulatory role in YP geneexpression. A second nuclear receptor superfamily member, theNGFI-B homologue AaHR38 is implicated in repression of the ecdysone-signalingpathway in the fat body of the previtellogenic female mosquitoat the state-of-arrest. Finally, three isoforms of the hepatocytenuclear factor 4 (HNF-4) homologue AaHNF-4 are differentiallyexpressed in the mosquito fat body during vitellogenesis, suggestingtheir involvement in regulating vitellogenic events in thistissue.     

Activation in vitro of vitellogenin uptake by the oocytes of the mosquito, Aedes aegypti

A blood meal stimulates the ovaries of the mosquito, Aedes aegypti , to internalize vitellogenin from the haemolymph for storage as yolk. The nature of the stimulation is biphasic, with a pulsed factor from the head released within the first 5 min of feeding stimulating the first phase of uptake, and the continuous release of another head factor initiating the second phase. Using an in vitro assay of [³⁵S]-vitellogenin uptake by ovaries, we sought to determine the intracellular signalling pathways utilized by the ovaries during stimulation of both phases of uptake. The adenylate cyclase activators forskolin and ethanol produced a statistically significant increase in rate of uptake in previtellogenic ovaries to levels characteristic of first-phase activated ovaries. Forskolin also induced the second phase of uptake in ovaries that had been arrested in the first phase of uptake by decapitation of the mosquito within 5 min of ingesting blood. Initiation and up-regulation of the rate of vitellogenin uptake does not depend on new protein synthesis.     

Mitosis of Micronuclei During Division and Regeneration in the Ciliate Stentor coeruleus1

Stages of mitosis of the micronuclei of Stentor coeruleus were described as seen by transmission electron microscopy. Cells in division and those regenerating new oral membranelles were studied. Microtubules were found in early prophase in the karyoplasm and interspersed between the condensing chromatin. A monaxial intranuclear spindle is formed by early metaphase, with kinetochore microtubule attachment sites on the chromosomes. The spindle elongates, separating the daughter nuclei at anaphase. A new nuclear envelope, consisting of two unit membranes, begins to form at late anaphase. Small segments of membrane found in the space between the newly forming and the old micronuclear envelopes appear to fuse to form the new nuclear envelope. No ultrastructural differences were found in the mitotic nuclei of cells in division or regeneration.     

Cell wall architecture: normal development and environmental modification of guard cells of the Cyperaceae and related species*

Abstract. The development and cell wall architecture of guard cells in the Cyperaceae were studied with light and electron microscopy. Development occurs along parallel files and results in a stomatal complex that consists of two guard cells each flanked by a subsidiary cell. The developmental pattern and general morphology are thus similar to that in the Gramineae. Several key differences, however, were observed. Wall synthesis in the Cyperaceae, as observed in the polarization and fluorescence microscope, occurs suddenly, within three to four complexes along a file, but is more gradual in the Gramineae. Mature cell walls in the Cyperaceae predominantly contain microfibrils oriented radially relative to the pore, while those in the Gramineae contain axial microfibrils. This difference was demonstrated in numerous species using freshly-collected as well as preserved material. In Cyperus esculentus, however, the alignment of microfibrils appears to be subject to environmental modification. Plants grown in the greenhouse contain guard cells with axial microfibrils, compared to the radial arrangement found in those grown in the field. In the former, wall is deposited gradually, as in the Gramineae. On return to more stressful conditions, radially micellated guard cells again develop. In each case, the cortical cytoplasm adjacent to areas where the wall is to thicken contains microtubules oriented parallel to the microfibril alignment characteristic of that treatment. These results are discussed in terms of the role of varied wall architecture in stomatal mechanics, the regulation of cell wall biosynthesis, and the evolutionary relationship of the Cyperaceae, Gramineae, and other taxa.     

Abnormal vitelline envelope induced by unphysiological doses of ecdysterone in Aedes aegypti

ABSTRACT. The oocytes of 3-day-old unfed Aedes aegypti mosquitoes are in a state of oogenic arrest, but microgram doses of ecdysterone stimulate their accumulation of a variable amount of yolk. We now find that these doses also induce the deposition of plaques of vitelline envelope by the follicle cells, and with transmission electron microscopy we have compared their formation with that in normal blood-fed females. Plaques in the experimental animals were abnormally large and irregular in shape and distribution. In part, these abnormalities were attributable to the fact that the follicle cells remain in close contact with the oocyte, whereas the space between follicle cells and oocyte increase significantly in the blood-fed female. Deposition of the plaques occurred earliest after the injection of 5 μg ecdysterone, but even at this high dose the amount of plaque material deposited was less than in the blood-fed controls. Induction of the deposition of abnormal vitelline envelope in unfed females was most clearly demonstrated after two injections, 1 μg ecdysterone each, 14h apart; 24h after the second injection, the plaques had prematurely fused into a thin disorganized envelope. When females were injected with ecdysterone immediately after a blood-meal, vitelline envelope plaques formed prematurely, and their structure became increasingly abnormal with time. This early onset of activity was characteristic of follicle cells adjacent both to the oocyte and to nurse cells. Thus, the factors that normally control the formation and organization of the vitelline envelope are absent in the unfed female stimulated with high doses of ecdysterone, while in the blood-fed females, excessive ecdysterone apparently interferes with the timing and orderly sequence of envelope formation.