Alexander S. Raikhel

Previtellogenic development and vitellogenin synthesis in the fat body of a mosquito: An ultrastructural and immunocytochemical study

We describe two phases, previtellogenic and vitellogenic, in the activity of the trophocytes in the fat body of the mosquito Aedes aegypti. The previtellogenic phase, leading to trophocyte competence to synthesize vitellogenin (Vg), occurred during the first 3 days after eclosion. This phase was characterized by enlargement and activation of the nucleoli, proliferation of ribosomes and rough endoplasmic reticulum (RER), development of Golgi complexes, and extensive invaginations of the plasma membrane. During the vitellogenic phase, initiated by a blood meal, Vg was first detected, by immunofluorescence, 1 hr after feeding. The intensity of the immunoreaction increased for the next 24 hr, was declining at 30 hr, and had disappeared by 48 hr. Vg synthesis was characterized ultrastructurally by the enlargement of the RER and the formation of dense secretion granules in Golgi complexes. These secretion granules were two to three times larger at the peak of Vg synthesis than at the beginning. The granules discharged their contents by exocytosis. Two electron microscopical immunocytochemical methods, immunoferritin and peroxidase-antiperoxidase, confirmed this pathway of Vg processing. For the first 12 hr after feeding. Vg synthetic organelles proliferated and the active nucleoli were multilobed; thereafter, while Vg synthesis continued, the nucleoli began to regress into compact bodies. Termination of Vg synthesis was marked by autophagical degradation of Vg synthetic and processing organelles.

Mosquito trypsin: immunocytochemical localization in the midgut of blood-fed Aedes aegypti (L.)

SummaryA polyclonal antibody was raised against trypsin purified from the midgut of blood-fed Aedes aegypti. Using this antibody and our modification of the peroxidase-antiperoxidase immunocytochemical reaction, strong activity was found in the lumen of the midgut at the light-microscopical level. The activity was localized mainly in the posterior part of the distensible, abdominal midgut, along the periphery of the blood bolus and within the peritrophic membrane. Immunoreactivity appeared 8 h after the blood meal and was most prominent around 24 h, coinciding with our previous spectrophotometric determinations of trypsin.At the electron-microscopical level, secretory granules, immunocytochemically labelled with anti-trypsin antibody and protein A-colloidal gold, were first detected about 12 h after the blood meal. At 18 h, the secretory pathway could be followed immunocytochemically from the formation of granules in the Golgi complex until their release by exocytosis in the midgut lumen. By 24 h, there was a reduction in secretory granules, and large lysosomes appeared.The process of secretion described for this mosquito is comparable to similar events in vertebrate secretory systems and the presence of an intracellular trypsinogen is suggested.

The accumulative pathway of vitellogenin in the mosquito oocyte: a high-resolution immuno- and cytochemical study

The accumulative pathway of yolk protein precursor, vitellogenin (VG), in the mosquito oocyte was studied by qualitative and quantitative VG immunolocalization, fixation with tannic acid, and lysosomal enzyme cytochemistry. VG binds to its receptors located on specific membrane microdomains at the base of and between the oocyte microvilli. The VG-receptor complexes then concentrate in coated pits and are internalized by coated vesicles. Dissociation of VG from its receptor occurs soon after transformation of coated vesicles into uncoated vesicular or tubular endosomes. The endosomes then coalesce into transitional yolk bodies (TYB), where VG accumulates and condenses. Narrow tubules without VG, connected to TYB, presumably represent a specific compartment for the recycling of VG receptors. TYB remain a “sink” for incoming VG until their transformation into mature yolk bodies (MYB), whereupon VG is transformed into a crystalline form, presumably vitellin. All compartments of the VG-accumulative pathway lack lysosomal enzymes and, therefore, the final VG destination, the MYB, is also endosomal and prelysosomal compartment, specialized for long-term storage of yolk protein.

Internalized proteins directed into accumulative compartments of mosquito oocytes by the specific ligand, vitellogenin

We have investigated the internalization pathways for a specific protein, vitellogenin, and a non-specific protein, horseradish peroxidase, in the mosquito oocyte in vivo. The internalized proteins were localized by electron microscopical immunocytochemistry or autoradiography; the relationship of their destination compartments with lysosomes was monitored by visualization of acid phosphatase. Proteins internalized by the oocyte follow either a specific accumulative route or a lysosomal degradative route. Via coated vesicles, both proteins enter the same compartment, the endosome, where they dissociate from membrane-binding sites. The route to their final destination depends on the presence of the specific ligand. In its absence, the degradative route is followed, and the endosome with non-specific protein fuses with lysosomes. In the presence of the specific ligand, the accumulative route is followed, and both specific and non-specific proteins are delivered into an accumulative compartment, the transitional yolk body. During the transformation of the transitional yolk body into the final storage compartment, a mature yolk body, vitellogenin undergoes crystallization, whereas the non-specific protein is concentrated in small vesicular extensions of the compartmental membrane. These vesicles are separated from the yolk bodies and apparently deliver the non-specific protein into the lysosomal system. We concluded that any protein bound to the membrane would be internalized by the oocyte, but only binding of the specific ligand to its receptor serves as a transmembrane signal stimulating the formation of accumulative compartments.

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.

Culture and analysis of the insect fat body

In insects, the fat body is the centre of intermediate metabolism: it is involved in the metabolism and storage of carbohydrates, lipids, and nitrogenous compounds. Moreover, it is a powerful secretory organ responsible for the production of virtually all haemolymph proteins, including most immune defense factors. The fat body is capable of extremely high levels of protein synthesis and secretion, as exemplified by the massive production of yolk protein precursors during vitellogenesis in female insects. Throughout the life cycle, fat body functions are developmentally or hormonally regulated, accommodating the needs of the developing, growing, metamorphosing, or reproducing insect (Wyatt, 1980; Keeley, 1985; Kanost et al., 1990; Raikhel, 1992).

Monoclonal antibodies as probes for processing of yolk protein in the mosquito; production and characterization

Abstract We have produced a library of monoclonal antibodies against yolk proteins of the mosquito Aedes aegypti . After the initial screening, 45 hybridoma cell lines were selected and cloned. Immunoblot analysis revealed three groups of monoclonal antibodies. One group recognized a 200-kDa polypeptide, the second a 68-kDa, and the third both of these polypeptides. While the affinity of binding by different antibodies varied widely, all monoclonal antibodies recognized these polypeptides only in extracts from vitellogenic fat bodies and ovaries. The antibodies were further characterized by video-enhanced immunofluorescence, which also showed that both yolk polypeptides originated in the fat body and accumulated in the oocytes. The immunolocalization in trophocytes of the fat body suggested that monoclonal antibodies may recognize different stages of the secretory pathway of yolk polypeptides. Similar analysis of oocytes indicated that our panel of antibodies recognizes different steps of processing of both 200-kDa and 68-kDa polypeptides, beginning with internalization by the oocyte and ending with the final crystalline form in mature yolk bodies.

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

Abstract Trophocytes of the mosquito fat body produce large amounts of the yolk-protein precursor, vitellogenin, that is then accumulated by developing oocytes. 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.

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.

Culture and analysis of insect ovaries

In insects, a complex of coordinated processes within the ovary contribute to egg maturation. Among the most important are internalization of large quantities of extra-ovarially produced yolk protein precursors (YPP), massive intra-ovarian synthesis of proteins such as components of chorion, and secretion of ecdysteroid hormones (ecdysteroidogenesis) (Raikhel and Dhadialla, 1992; Dhadialla and Raikhel, 1994; Sappington and Raikhel, 1996). The timing and kinetics of these processes depend on the stage of egg maturation, nutritional status of the female, and environmental conditions. Egg development is mediated through cascades of nervous, endocrine, and intracellular signals. The culturing of insect ovaries is crucial to elucidating the complex web of events comprising egg maturation, as this technique permits the experimental determination and manipulation of individual parameters often difficult or impossible to investigate in vivo.