Histochemical and biochemical investigations concerning the function of larval oenocytes of Tenebrio molitor L. (Coleoptera,Insecta)

Summary Larval oenocytes of Tenebrio molitor were investigated histochemically. In contrast to the lipid droplets of the fat body, they did not stain with Sudan black. A positive reaction for lipoproteins appeared only after destructive oxidation with sodium hypochlorite. These lipoproteins are the remnants of degenerated membranes, as revealed by ultrastructural analysis. Polyphenols could be identified in the exocuticle of exuvia, and in the newly formed procuticle. Endocuticle, epidermis and oenocytes showed no staining reaction. In oenocytes a great amount of lipase is also present which could be detected with several Tweens as substrates. The significance of these lipases remains unclear, since only few glycerides are synthesized in the cells, as shown below. They may play a role in the extended membrane turnover observed in this cell type. In vitro incubation of oenocytes of the larval generation demonstrated that ¹⁴C-labeled acetate was only incorporated into the paraffin fraction. A negligible amount of the label was found in glycerides; wax esters were free of label. Larval epidermis is also capable of paraffin formation, but only to a small degree. Oenocytes of the imaginal generation located between the sternal epidermis cells of pupae and adults do not synthesize paraffins, but other more polar compounds not yet identified. Labeled waxes in cuticular lipids were detected only when ¹⁴C-acetate was injected into whole larvae, and the lipids extracted some hours later. Autoradiographs demonstrated that ¹⁴C-acetate was intensively incorporated into larval oenocytes, the rate varying in different cells. Incorporation into the epicuticle, probably into the wax layer, was clearly shown. Cuticulin and dense layer do not show an intensive label. The lamellated cuticle also seems to be impregnated with acetate derivatives.Supported by the Deutsche Forschungsgemeinschaft     

Moulting hormone,juvenile hormone and the ultrastructure of the fat body of adult Sarcophaga bullata (Diptera)

Summary In the ovoviviparous fly, Sarcophaga bullata, vitellogenesis is cyclic; a process reflected in ultrastructural changes in the fat body cells and oenocytes. At eclosion the larval fat body has not yet completely disappeared. During vitellogenesis the fat body cells are specialized for intensive protein synthesis showing a very extensive RER and numerous invaginations of the plasma membrane. These features disappear when the eggs descend into the oviducts to complete embryogenesis. The predominant feature of the oenocytes is their very prominent SER. The fat body cells of the males are never as specialized for protein synthesis as those of the females. Feeding of ecdysterone to males for 3 or more days induces a rather extensive subcellular apparatus for protein synthesis, i.e., invaginations of the plasma membrane and an extensive RER. Juvenile hormone is completely ineffective in this respect. Both ecdysterone and juvenile hormone have pronounced but different effects on the oenocytes of males.     

Origin and formation of the royal fat body of the higher termite queens

The formation of the royal fat body was studied by electron microscopy in three species of higher termites (Macrotermes bellicosus, Macrotermes subhyalinus, and Cubitermes fungifaber). The swarming alate imago has a storage fat body typical of most insects. In non-physogastric young queens, during the fasting period, the adipocytes deplete their reserves and then, along with the increased vitellogenesis, acquire protein-synthesizing structures (R.E.R.). During the development of physogastry they progressively specialize in protein synthesis and secretion and undergo many cell divisions. The cytological change is paralleled by a spatial reorganization of the fat body. Observations on the transformation of imaginal adipocytes into royal adipocytes show that the royal fat body is derived from the imaginal fat body and not from the tracheal cells as previously claimed.     

Yolk polypeptide synthesis in the fat body of Sarcophaga bullata: Localization,hormonal induction and cell-free translation

The fat body of adult Sarcophaga bullata consists of different cell-types. The yolk polypeptides (YPs) are localized in secretory granules in the cytoplasm of female trophocyte fat body cells while the oenocytes and larval fat body cells are immunonegative. An antiserum against the larval serum protein 1 of Drosophila crossreacts on immunoblotting with several polypeptide bands in the haemolymph with mol. wt ∼80 kD. This antiserum specifically reacts with some storage granules of the persisting larval fat body cells and not with the other fat body cell types. The trophocyte fat body cells of male Sarcophaga treated with 20-OH-ecdysone, display a similar granular type of immunoreaction with an anti-YP antiserum as in vitellogenic females. Moreover, 20-OH-ecdysone induced in the fat body of males, in contrast to methoprene, synthesis of mRNA coding for YPs to a level as high as that in vitellogenic females, as shown in the reticulocyte lysate cell-free system.     

Fat body cells of female reproductive castes of Attini ants (Hymenoptera: Formicidae): An ultrastructural and chemical analysis

The ultrastructural study on the fat body of gynes (virgin queens) of the basal ant species Cyphomyrmex rimosus and Mycetarotes parallelus and the derived Acromyrmex disciger and Atta laevigata queens showed vesicular rough endoplasmic reticulum, Golgi complex, and mitochondria in trophocytes, suggesting the involvement of these cells in protein synthesis, in addition to digestive vacuoles associated with the digestion of endocytosed compounds or rejected cell organelles. Oenocytes, another cell type present in the fat body of these species exhibit mitochondria, digestive vacuoles, and vesicles, indicating a mobilization of compounds by these cells. In A. laevigata, oenocytes also exhibited large storage sites of glycogen, in addition to a well-developed vesicular rough endoplasmic reticulum, suggesting an intensive participation of these cells in protein synthesis. The ultrastructural cytochemistry study also revealed electrodense granules of basic proteins present throughout the cytoplasm of trophocytes. The same was observed in oenocytes, although with smaller amounts of proteins. In the cytoplasm of trophocytes and oenocytes were also found droplets or electrodense granules of lipids. In oenocytes of A. disciger and in trophocytes of A. laevigata, lipids were observed in mitochondria, suggesting that this organelle might be a site of synthesis of these compounds. The chemical analysis of lipids revealed that in gynes, the main compounds present in fat body cells were saturated fatty acids, while in queens, saturated as well as unsaturated fatty acids were found. In conclusion, the present study showed that the fat body cells of gynes and queens, in general, maintained the same compounds and original features through the evolution process of the Attini tribe.     

Studies on the cytophysiology of the fat body of the American silkmoth

Summary A developmental study at the electron microscopic level was conducted of the fat body cells of Hyalophora cecropia (L.). During the last larval instar the fat body increases in volume and the cells exhibit a well developed rough endoplasmic reticulum and protein bodies of diverse sizes. In the pupal fat body, the protein bodies appear to be enclosed by a double membrane and contain glycogen granules, ribosomes and mitochondrion-like structures. In addition, there are large lipid globules, cytolysomes and rough endoplasmic reticulum. The ultrastructure of the protein bodies suggests the development of large bodies by fusion of smaller protein bodies. Changes in fat body cell ultrastructure were followed during adult development and cytological evidence was obtained for the depletion of protein, glycogen and lipid in the female during this period. The female adult fat body cell contains free ribosomes, protein bodies, many mitochondria, a few lipid globules and glycogen granules. The male moth fat body cells have many mitochondria, a few glycogen granules, essentially no protein bodies, but an abundance of large lipid globules.Studies on the influence of egg maturation on the morphology of the fat body of Hyalophora gloveri (L.) revealed that ovariectomy of pupae yielded adults having more fat body than normal females, and that the fat body cells of the ovariectomized animals contained more glycogen, lipid and protein. Male pupae receiving ovarian implants developed into adults containing eggs and possessed more fat body than normal females but less than normal males. Very few glycogen granules were found in the fat body cells of normal males or males with implanted ovaries.Supported by grant AM-02818 from the National Institutes of Health.We thank Dr. James Oschman for his helpful suggestions and constructive criticisms.     

Histological and cytological studies on the fat body of the cockroach Nauphoeta cinerea during the first reproductive cycle

Summary The central fat body of the ovoviviparous cockroach Nauphoeta cinerea was studied during the first reproductive cycle of the female by means of light microscopy, autoradiography and electron microscopy. Comparative studies in larval stages were also undertaken. The fat body of Nauphoeta contains a large amount of lipid droplets and the remaining cytoplasm is very scarce. The cytological cyclicity of the fat body is consistent with the known biochemical rhythms of vitellogenin production. The proteosynthetic apparatus appears about 3 days after imaginal ecdysis, along with vitellogenin. The ribosomal endoplasmic reticulum (RER) shows a tremendous increase by the 7th day of the first cycle. The most active period of vitellogenin production lasts from day 7 to day 12. The proteosynthetic apparatus then returns to an inactive stage and disappears. This inactive condition lasts to the end of the gestation period. The autoradiographic results are consistent with the cytological features.Work carried out under the scientific direction of Prof. M. Lüscher of the University of Bern and supported in part by a grant from the Swiss National Science Foundation (No 3. 633.71).     

Site of synthesis,tissue distribution,and lipophorin transport of hydrocarbons in Blattella germanica (L.) nymphs

The site of hydrocarbon (HC) synthesis and the amount of HC in various tissues were investigated in relation to developmental stage in the last larval stadium of the German cockroach, Blattella germanica. Abdominal integument linearly incorporated [1-(14)C]propionate into HC for at least 6h in vitro, whereas other body parts synthesized little or no HC. The third through sixth abdominal sternites and tergites were the principal sites of synthesis. High rates of HC synthesis resulted in a fivefold increase in internal HC during the last stadium. We examined the distribution of HC in the hemolymph, fat body, and the developing imaginal cuticle. Hemolymph HC titer was relatively constant at approximately 8&mgr;g/&mgr;l. However, as hemolymph volume increased from 5 to 11&mgr;l in the first 4days of the last stadium, HC content increased and then remained stable the remainder of the stadium. Lipophorin, immunoprecipitated with adult lipophorin polyclonal antibodies, was the only HC carrier protein in nymphal hemolymph and its HC profile was identical to that of hemolymph and similar to that of the epicuticle. The concentration and total amount of hemolymph lipophorin increased until 3days before adult eclosion and declined immediately after ecdysis. The HC content of non-biosynthetic integument (legs, pronotum) doubled during formation of the imaginal cuticle, as did the HC content of sternites, which synthesize HC. HC content of fat body, however, increased threefold during the same period, suggesting that the fat body serves as a storage site for HC during cuticle formation. We conclude that in the last stadium HC is synthesized by abdominal oenocytes, loaded onto hemolymph lipophorin, and transported to fat body and both nymphal and imaginal cuticle. Hydrocarbons associate with the imaginal integument several days before eclosion.     

Development,Ultrastructure, and Mode of Transmission of Amblyospora sp. (Microspora) in the Mosquito*

SYNOPSIS. Amblyospora sp. in Culex salinarius (Coquillett) is transovarially transmitted and has 2 developmental sequences, one in each host sex. In females, the entire life cycle is restricted to oenocytes which become greatly hypertrophied due to the multiplication of diplokaryotic cells during merogony and come to lie next to ovaries. Sporulation occurs only after a blood meal is taken and is shortly followed by infection of the oocytes and subsequent transmission to the next host generation. In the male host, infections spread from oenocytes to adipose tissue where diplokaryotic cells undergo a 2nd merogony. During this merognic cylce, the number of diplokaryotic cells greatly increases and the infection is spread throughout the body of the larval host. Sporulation is initiated with the physical separation of the 2 members of the diplokaryon and the simulatneous secretion of a pansporoblastic membrane. Subsequent meiotic division and morphogenesis result in the formation of 8 haploid spores enclosed with a pansporoblastic membrane. Buildup of spores and subsequent destruction of host adipose tissue prove fatal to the male host during the 4th larval stage.     

The integument of the Queensland fruit fly,Ddacus tryoni (Frogg.)

Summary During the pupal stage of Dacus tryoni, the hypodermis of the larva is replaced by an imaginal generation of smaller cells. The hypodermal cells of the tergal glands on the fifth abdominal segment of the adult were examined with the electron microscope; they contain slender, membrane-limited bundles of hollow wax filaments that traverse the cuticle in branched pore canals. Outside the glandular areas, the pore canals are narrower. The cuticle of the adult undergoes its greatest increase in thickness soon after emergence; it becomes sclerotized gradually. No epicuticle was detected with either the light or electron microscopes.Early in adult development, bristles are formed over the general surface of the terga. Most of these are innervated by single, bipolar nerve cells, and have more or less enlarged trichogen cells that appear to secrete wax through pore-plates in the cuticle. The bristles in different regions of the abdomen range in function from pure sensory receptors to pure secretors. The sensory bristles on the tergal glands were examined with the electron microscope.For assistance with the electron microscopy, I thank Mr. Tony Webber and Miss Ann Miller of the Electron Microscopy Unit at Sydney University. — Supported by a C.S.I.R.O. Junior Post-Graduate Studentship.     

Life cycle of a new species of Amblyospora (Microspora: Amblyosporidae) in the mosquito Aedes taeniorhynchus

A new species of Microspora, Amblyospora polykarya, is described from the mosquito Aedes taeniorhynchus. The parasite is transovarially transmitted for one generation only. Spores in adult females extrude binucleate sporoplasms which infect developing eggs. Merogony occurs in larval oenocytes with diplokaryotic stages in early instars giving rise to plasmodia with many diplokarya. Plasmodia undergo cytokinesis to form diplokaryotic sporonts. In fat body cells, these sporonts secrete pansporoblastic membranes and undergo two nuclear divisions to form octonucleate sporonts. Cytokinesis and differentiation result in uninucleate spores in packets of eight. These spores are not transmissible per os and are of different morphotype from those in adult females. Infected larvae die in the fourth stadium.     

Larval fat body cells die during the early pupal stage in the frame of metamorphosis remodelation in Bombyx mori

In holometabolus insects, morphology of the larval fat body is remodeled during metamorphosis. In higher Diptera, remodeling of the fat body is achieved by cell death of larval fat body cells and differentiation of the adult fat body from primordial cells. However, little is known about remodeling of the fat body at pupal metamorphosis in Lepidoptera. In this study, we found that cell death of the larval fat body in Bombyx mori occurs at shortly after pupation. About 30% of the fat body cells underwent cell death on days 1 and 2 after pupation. The cell death involved genomic DNA fragmentation, a characteristic of apoptosis. Surgical manipulation and in vitro culture of fat body cells revealed that 20-hydroxyecdysone and juvenile hormone had no effect on either initiation or progression of cell death. During cell death, a large increase in activity of caspase-3, a key enzyme of cell death, was observed. Western blot analysis of the active form of caspase-3-like protein revealed that the length of caspase-3 of B. mori was much larger than that of caspase-3 in other species. The results suggest that larval fat body cells of B. mori are removed through cell death, which is mediated by a caspase probably categorized in a novel family.     

Hormonal control of growth and differentiation of insect tissues cultured in vitro

Summary This paper reviews the effects of insect hormones on lepidopteran imaginal discs cultured in vitro.β-ecdysone stimulated both evagination and cuticle deposition of wing discs ofPlodia interpunctella (Hübner). However, evagination required a shorter exposure to ecdysone than did cuticle deposition. Cuticle deposition was obtained under the following conditions: (a) a 24-hr pulse ofβ-ecdysone (0.5–5.0μg/ml); (b) continuous treatment with 0.2μg/mlβ-ecdysone; or (c) continuous treatment with 0.5 to 50.0μg/mlβ-ecdysone in medium conditioned with larval fat body. Investigations of some biochemical effects of ecdysone showed that RNA and protein synthesis was required for evagination and cuticle deposition. In particular, studies with actinomycin D and cycloheximide (at nontoxic levels) showed that RNA and protein synthesis during the ecdysone-dependent period was essential for subsequent development. These findings support the hypothesis that stimulation of macromolecular synthesis is fundamental to the action of ecdysone on imaginal discs. The influence of beta-ecdysone on chitin synthesis was also examined.β-ecdysone stimulated uptake and incorporation of tritiated-glucosamine by culturedP. interpunctella wing discs. Addition of hexosamines to the culture medium had no influence on ecdysone-induced cuticle deposition, but inhibition of glucose-uptake by cytochalasin B prevented the formation of cuticle. The action of ecdysone on particular enzymes in the chitin pathway remains to be elucidated. Presented in the formal symposium on Information Transfer in Eukaryotic Cells, at the 26th Annual Meeting of the Tissue Culture Association, Montreal, Quebec, June 2–5, 1975.     

Autoradiographic study of protein synthesis in abdominal tissues of Glossina austeni

Protein synthesis at various times during the pregnancy cycle of G. austeni was determined by autoradiographic measurement of the incorporation of H³-leucine and H³-tyrosine into the cells of the fat body, oenocytes, milk gland and epidermis. The rate of utilization of these molecules is such that the labelled pool in the haemolymph is depleted before 0.5 hr after injection. The incorporation of both amino acids into fat body and oenocytes is high at eclosion and just after larviposition, with the incorporation of tyrosine by the oenocytes being much higher than that in the fat body. The same pattern of incorporation is observed in the epidermal cells. Label also appears in the endocuticle during the first 10 days of adult life. Except during the first 4 days following emergence, the incorporation of the two amino acids into the milk gland is very high, with periods of less intense protein synthesis at about the time of larviposition. The milk gland represents a highly efficient secretory system, with a t₅₀ of less than 30 min.     

Differentiation of the membrane system in the cells of imaginai discs

Summary Imaginal discs from developing larvae of the fly Calliphora erythrocephala fixed in permanganate or osmium tetroxide and embedded in Epon 812 were observed by electron microscopy. When the larval growth ceases, the differentiation manifests itself through an enlargement of the endoplasmic reticulum, by which continuous membrane contact is established between all cell organelles. During the same time mitochondria swell up and transform into lipid granules and the intercellular contacts weaken.I am indebted to Mrs Mariann Carleson and Miss Brita Nilsson for technical aid.     

The fate and possible role of arylphorin during the metamorphosis of Mamestra brassicae.

1. Arylphorin, one of the storage proteins has been isolated from the hemolymph of Mamestra brassicae. 2. It has been established that Mamestra arylphorin is the most similar to manducin from among the known storage proteins of other species. 3. A rabbit polyclonal antibody has been developed against arylphorin, and its concentration changes have been determined quantitatively by ELISA in the hemolymph and fat body from the 1st day of the last larval instar to the 3rd day of the imago stage. 4. Histological sections were made on each day during the investigated period and it was shown by immunohistochemical methods that the main quantity of arylphorin was accumulated in the storage protein granules of the fat body and it could be detected even in the imaginal fat body. 5. The uptake of arylphorin by the fat body is induced by 20-hydroxyecdysone. 6. During differentiation of the imaginal cuticle arylphorin is incorporated first in the epidermal cells and it is built in the endocuticular layer of the integument thereafter.     

Life Cycle and Epizootiology of Amblyospora sp. (Microspora: Amblyosporidae) in the Mosquito,Aedes cantator1

In Aedes cantator, Amblyospora sp. is transovarially transmitted and has two developmental sequences. The life cycle is initiated in the adult female with the release of sporoplasms from binucleated spores not bounded by membranes, lying free within host oenocytes. Sporoplasms infect the developing oocytes and are transmitted to the filial generation when the eggs are laid. In some of the female progeny that hatch from infected eggs, diplokaryotic cells infect host oenocytes and divide by binary fission during merogony. Sporulation and spore formation do not occur until a blood meal is taken by the host and they coincide with the development and maturation of the oocytes to complete the cycle. In other female and all male progeny, pathogen development occurs within fat body tissue of the host where diplokaryotic cells divide by multiple fission during merogony to spread the infection. Sporulation in this developmental sequence is characterized by the secretion of an accessory membrane and the meiotic division of diplokaryotic sporonts, which result in the formation of octonucleated plasmodia that undergo cytokinesis to form eight haploid spores which are not perorally infectious to other mosquito larvae. There is no increase in the prevalence of either type of infection in field populations during juvenile development, indicating that there is no direct horizontal transmission of the pathogen within any one generation. Data obtained from laboratory rearings of infected progeny, however, show that infections cannot persist relying solely upon maternal-mediated transmission and that some other mode of transmission must be operative for continued maintenance of this microsporidium in A. cantator.     

Growth, metastasis, and invasiveness of Drosophila tumors caused by mutations in specific tumor suppressor genes

 More than 50 genes have been identified in Drosophila by loss-of-function mutations that lead to overgrowth of specific tissues. Loss-of-function mutations in the lethal giant larvae, discs large, or brain tumor genes cause neoplastic overgrowth of larval brains and imaginal discs. In the present study, the growth and metastatic potential of tumors resulting from mutations in these genes were quantified. Overgrown brains and imaginal discs were transplanted into adults and β-galactosidase accumulation was used as a marker to identify donor cells. Mutations in these three genes generated tumors with similar metastatic patterns. For brain tumors, the metastatic index (a measure we defined as the fraction of hosts that acquired secondary tumors normalized for the amount of primary tumor growth) of each of the three mutants was similar. Analysis of cell proliferation in mutant brains suggests that the tumors arise from a population of several hundred cells which represent only 1–2% of the cells in third instar larval brains. For imaginal disc tumors from lethal giant larvae and brain tumor mutants, it is shown for the first time that they can be metastatic and invasive. Primary imaginal disc tumors from lethal giant larvae and brain tumor mutants formed secondary tumors in 43 and 53% of the hosts, respectively, although the secondary tumors were, in general, smaller than the secondary tumors derived from primary brain tumors. Received: 18 August 1997 / Accepted: 16 October 1997