Hormonal and nutritional regulation of insect fat body development and function

The insect fat body is an organ analogue to vertebrate adipose tissue and liver and functions as a major organ for nutrient storage and energy metabolism. Similar to other larval organs, fat body undergoes a developmental “remodeling” process during the period of insect metamorphosis, with the massive destruction of obsolete larval tissues by programmed cell death and the simultaneous growth and differentiation of adult tissues from small clusters of progenitor cells. Genetic ablation of Drosophila fat body cells during larval‐pupal transition results in lethality at the late pupal stage and changes sizes of other larval organs indicating that fat body is the center for pupal development and adult formation. Fat body development and function are largely regulated by several hormonal (i.e. insulin and ecdysteroids) and nutritional signals, including oncogenes and tumor suppressors in these pathways. Combining silkworm physiology with fruitfly genetics might provide a valuable system to understand the mystery of hormonal regulation of insect fat body development and function. © 2009 Wiley Periodicals, Inc.     

Endocrine control of oögenesis in the housefly,Musca domestica vicina

The endocrine system involved in the control of oögenesis in the housefly, Musca domestica vicina, was investigated. Allatectomy, decapitation, and starvation of newly emerged females resulted in inhibition of oögenesis, showing a close relationship between enlargement of the corpus allatum and growth of follicles during the first oögenesis. Histological observation of sexually matured females showed active secretion of the corpus allatum and the medial neurosecretory cells of pars intercerebralis. Topical application of juvenile hormone analogues (JHA) to the allatectomized fly induced the growth of ovary, and critical doses of methoprene and methyl-7, 11-diethyl-juvenate for the maturation of the ovary were determined. JHA stimulated initiationof oögenesis in the starved or decapitated flies as well as vitellogenesis in the sugar-fed one; subsequently it was found that juvenile hormone acted not only as a gonadotropin but also as a regulator of vitellogenesis. Furthermore, JHA stimulated cell lysis in pupal fat body of female flies, indicating a possible influence of juvenile hormone upon the process of releasing vitellogenin.     

Modes of cell death in the pupal perivisceral fat body tissue of the silkworm Bombyx mori L.

Cell death is a scheduled event during animal development and tissue turnover. Here, we affirm the presence of two major pathways of programmed cell death (PCD), viz. apoptotic and autophagic cell death, in the disintegrated pupal perivisceral (PV) fat body during pupal-adult metamorphosis. The acridine orange (a vital stain for apoptosis) staining pattern and DNA fragmentation assay have revealed the exact day (6th day of the pupal stage) of disintegration in the PV fat body as represented by chromatin condensation and DNA laddering. Electron microscopy and scanning electron microscopy have demonstrated the presence of cytoplasmic budding and giant autophagic vacuoles and the low numbers of mitochondria, all of which are attributes of autophagic cell death. Immunoblot analysis of proteosomal subunits 20S and 26S has established the involvement of proteolytic activity during PCD of PV tissue. Lysosomal participation during the PCD of PV tissues has been confirmed by the elevated level of the marker enzyme, acid phosphatase, which is distinct on day 6 of the pupal period. The results of the present study have thus ascertained the co-existence of both autophagic and apoptotic cell death in PV fat body tissue.     

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.     

Fine structural alterations with age in the fat body of the adult male housefly,Musca domestica

Summary The fat body of the adult housefly is composed of two types of cells, the lipid-and glycogen-rich fat body cells and the oenocytes. A comparison of the fine structure of the abdominal fat body in 4-day old and 31–35 day old male houseflies indicated an increase in lipid and a decrease in glycogen content in the fat body cells of old flies. Oenocytes of old flies exhibit deteriorative alterations with an accumulation of secondary lysosomes. Both fat body cells and oenocytes in senile flies are ingested by hemocytes.Supported by a grant from the National Science Foundation.     

Cell death and tissue reorganization in Rhynchosciara americana (Sciaridae: Diptera) metamorphosis and their relation to molting hormone titers

Programmed cell death (PCD) is a focal topic for understanding processes underlying metamorphosis in insects, especially so in holometabolous orders. During adult morphogenesis it allows for the elimination of larva-specific tissues and the reorganization of others for their functionalities in adult life. In Rhynchosciara, this PCD process could be classified as autophagic cell death, yet the expression of apoptosis-related genes and certain morphological aspects suggest that processes, autophagy and apoptosis may be involved. Aiming to reveal the morphological changes that salivary gland and fat body cells undergo during metamorphosis we conducted microscopy analyses to detect chromatin condensation and fragmentation, as well as alterations in the cytoplasm of late pupal tissues of Rhynchosciara americana. Transmission electron microscopy and confocal microscopy revealed cells in variable stages of death. By analyzing the morphological structure of the salivary gland we observed the presence of cells with autophagic vacuoles and apoptotic bodies and DNA fragmentation was confirmed with the TUNEL assay in salivary gland. The reorganization of fat body occurs with discrete detection of cell death by TUNEL assay. However, both salivary gland histolysis and fat body reorganization occur under control of the hormone ecdysone.     

Effects of regional body temperature variation during asphyxial cardiac arrest on mortality and brain damage in a rat model

To date, hypothermia has focused on improving rates of resuscitation to increase survival in patients sustaining cardiac arrest (CA). Towards this end, the role of body temperature in neuronal damage or death during CA needs to be determined. However, few studies have investigated the effect of regional temperature variation on survival rate and neurological outcomes. In this study, adult male rats (12 week-old) were used under the following four conditions: (i) whole-body normothermia (37 ± 0.5 °C) plus (+) no asphyxial CA, (ii) whole-body normothermia + CA, (iii) whole-body hypothermia (33 ± 0.5 °C)+CA, (iv) body hypothermia/brain normothermia + CA, and (v) brain hypothermia/body normothermia + CA. The survival rate after resuscitation was significantly elevated in groups exposed to whole-body hypothermia plus CA and body hypothermia/brain normothermia plus CA, but not in groups exposed to whole-body normothermia combined with CA and brain hypothermia/body normothermia plus CA. However, the group exposed to hypothermia/brain normothermia combined with CA exhibited higher neuroprotective effects against asphyxial CA injury, i.e. improved neurological deficit and neuronal death in the hippocampus compared with those involving whole-body normothermia combined with CA. In addition, neurological deficit and neuronal death in the group of rat exposed to brain hypothermia/body normothermia and CA were similar to those in the rats subjected to whole-body normothermia and CA. In brief, only brain hypothermia during CA was not associated with effective survival rate, neurological function or neuronal protection compared with those under body (but not brain) hypothermia during CA. Our present study suggests that regional temperature in patients during CA significantly affects the outcomes associated with survival rate and neurological recovery.     

Programmed cell death at the periphery of the pupal wing of the butterfly,Pieris rapae

The outline of the adult wing of lepidopteran insects (butterflies and moths) emerges as a result of disappearance of a group of cells at the periphery of the pupal wing. Histological observation of the pupal wing of Pieris rapae showed that, just after apolysis of the wing epithelium from the pupal cuticle, there occurs a rapid and localized decrease of the number of cells at the periphery of the wing. This decrease occurs through cell death, which lasts 1–1.5 days at 20°C. Dying cells lose contact with the neighbouring cells and show condensation of chromatin and cytoplasm. They then appear to be phagocytosed by neighbouring epithelial cells or discharged through the basal surface of the epithelium into the lumen within the wing and taken up by phagocytes. Fragmentation of DNA in the nuclei was detected in the dead cells or their debris. These results indicate that programmed cell death in the lepidopteran wing proceeds through a mechanism closely similar to that of apoptosis in the vertebrate.     

Brain and ring gland cyclic AMP and cyclic GMP levels during initiation and termination of pupal diapause in flesh flies

Brain and ring gland concentrations of cyclic AMP were much higher shortly after pupariation in long day (non-diapause destined) flesh flies than in short day (destined for pupal diapause) flies. This difference was most striking in the ring gland (6 times higher in long day flies). Cholera toxin elevated brain-ring gland cAMP four-fold, thus accounting for its efficacy in averting diapause. No differences in cyclic GMP levels were detected between long and short day flies at pupariation. At diapause termination cAMP and cGMP concentrations in the brain and ring gland and cAMP in whole body homogenates changed only slightly, but whole body concentrations of cGMP rose markedly.     

GENESIS OF MITOCHONDRIA IN INSECT FAT BODY

Electron microscopy and stereological methods have been used to study the time course and mechanism of mitochondrial genesis in the adult fat body of Calpodes ethlius, (Lepidoptera, Hesperiidae). Most of the larval mitochondria are destroyed during a phase of autolysis shortly before pupation, so that pupal and early adult fat body cells have few mitochondria. The number of mitochondria per cell increases rapidly at the end of the 1st day after the adult emerges. Characteristic partitioned mitochondria appear during the period when the number is rapidly increasing. This evidence, coupled with the results of morphometric analyses of mitochondrial diameter, volume, and surface area, confirms the view that the genesis of adult mitochondria involves the growth and division of mitochondria surviving from the larva.     

Expression profile of cathepsin B in the fat body of Bombyx mori during metamorphosis

Proteolytic enzymes are involved in insect molting and metamorphosis, and play a vital role in the programmed cell death of obsolete organs. Here we show the expression profile of cathepsin B in the fat body of the silkworm Bombyx mori during development. We also compare the expression profiles of B. mori cathepsins B (BmCatB) and D (BmCatD) during normal development and after RNA interference (RNAi)-mediated inhibition. BmCatB is induced by 20-OH-ecdysone, and is expressed in the fat body of B. mori during molting and the larval–pupal and pupal–adult transformations, where its expression leads to programmed cell death. In particular, BmCatB is highly expressed in the fat body of B. mori during the larval–pupal transformation, and BmCatB RNAi treatment resulted in an arrest of the larval–pupal transformation. RNAi-mediated BmCatB knockdown sustained the expression of BmCatD during the larval–pupal transformation. On the other hand, when BmCatD was inhibited via RNAi, the expression of BmCatB was upregulated. Based on these results, we conclude that BmCatB is involved in the programmed cell death of the fat body during B. mori metamorphosis, and that BmCatB and BmCatD contribute to B. mori metamorphosis.     

意大利蜜蜂工蜂脂肪体胚后发育过程中细胞的增殖和凋亡

脂肪体是昆虫体内物质贮备和中间代谢的重要组织。本研究通过显微形态观察、 BrdU免疫组织化学和原位末端转移酶标记(TUNEL)细胞凋亡检测技术, 对意大利蜜蜂Apis mellifera ligustica工蜂脂肪体胚后发育过程中细胞的增殖和凋亡特点进行了比较研究。结果表明： 意大利蜜蜂工蜂脂肪体细胞数量的快速增加集中在幼虫发育前期（1-3龄）, 而细胞的凋亡则集中在蛹发育早期的2-3 d（预蛹-2日龄蛹）时间之内。在变态发育中, 工蜂幼虫脂肪体凋亡降解后重新组建形成成虫的脂肪体。本研究为昆虫脂肪体的功能研究以及昆虫组织细胞自噬和凋亡的机制研究提供一定的证据。     

Diptera--ovary structure and oogenesis in midges and flies

Comparative study of ovary development and oogenesis in the dipterans revealed significant differences between the Nematocera (lower dipterans, midges) and the Brachycera (true flies). The occurrence of these differences emphasizes well the phylogenetic division of the Diptera into these major subgroups. Basic discrepancies were found in the course of ovary development and in the mode of follicular cell differentiation. In contrast to more advanced flies, in midges the initial stages of germ cell differentiation, i.e. divisions of gonial cells, germ cell cluster formation and diversification of cystocytes within clusters take place exclusively in the larval and early pupal stages. Moreover, the formation of cystocyte clusters precedes that of ovarioles. Differences in the behaviour of some follicular cells found between the ovarian follicles of midges and advanced flies suggest that both major dipteran subgroups may differ in the scenario and/or the mechanisms of terminal signalling leading to the determination of the anteriormost part of the body.     

Biochemical evidence of DNA transport from the silk gland to the fat body of the silkworm, Bombyx mori

β-DNA, a component of DNA found in the pupal fat body of the silkworm, Bombyx mori, has the same GC content but a smaller molecular weight than typical silkworm DNA (α-DNA). Its origin and time of synthesis were studied by MAK column chromatography of phenol extracts after labelling with radioactive precursors.The DNA components of the fat body changed greatly during the early pupal stage, the β-DNA showing a striking increase relative to α-DNA. Thymidine-6-³H and phosphoric acid-³²P injected into the animals 1 day before analysis caused labelling of α-DNA, but not of β-DNA of the fat body, indicating that β-DNA was not synthesized during the stage of its appearance in the fat body.On the other hand, injection of thymidine-6-³H into 2-day-old fifth instar larvae, when DNA of the silk gland was being actively synthesized, gave high incorporation of the isotope into β-DNA of the pupal fat body. The sudden appearance of highly labelled β-DNA in the fat body during the early pupal stage as well as the occurrence of β-DNA in both the silk gland and fat body suggested that DNA might move from the silk gland to the fat body.It is possible that the fat body stores DNA as a nutrient from the degenerating silk gland.     

Formation of lipid reserves in fat body and eggs of the yellow fever mosquito, Aedes aegypti

We examined the accumulation of lipids in adult females of the mosquito, Aedes aegypti. Females emerged with about 100 μg lipid in the fat body. With access to sugar water lipids increased over seven days to 300 μg. After a blood meal on day five, sugar-fed females accumulated 120-140 μg of lipids in their ovaries within 2 days. At the same time the lipid content of the fat body decreased by 100 μg, indicating transfer of lipids from fat body to oocytes. Experiments in which fat body lipids were prelabelled support this conclusion. Label was transferred to oocytes: in mature oocytes the specific radioactivity of lipids was 80% of the specific radioactivity of prelabeled fat body lipids. Components of blood meals are also used to synthesize oocyte lipids. Fat bodies of females starved for four days had only 27 μg of lipids left. When these females were given a blood meal, they matured oocytes, although the number of ooyctes was reduced and ovaries contained only half the amount of lipids found in ovaries of females which had first fed on sugar water. Fat body lipids of these females had only slightly increased to 36 μg. This demonstrates that female Ae. aegypti use sugar to synthesize lipids, but they can also use components of blood for this purpose.     

Maturation and aging of adult fat body and oenocytes in Drosophila as revealed by light microscopic morphometry

A morphological and cytometric analysis of the adult fat body cells and oenocytes was made on sections of abdomens from immature, mature and senescent Drosophila melanogaster of both sexes. There are about 18,000 fat body cells in abdomens of female and mature male flies. Immature and senescent males have about 12,000 and 15,000 cells, respectively. The size of the cells is almost the same for immature flies of both sexes and increases about six-fold to approximately 2600 micron2, so that mature flies of both sexes have equivalent amounts of fat body tissue. The proportions of lipid, glycogen, and background cytoplasm of fat body cells also remain relatively constant throughout adult life, but dense, proteinaceous granules are observed in cells of senescent flies. The amounts of cellular components change dramatically due to change of cell size with age; the amount of lipid shows the greatest sexual difference with about 2x more in the females at all stages studied. The oenocytes number about 6,000 in the abdomens of all but immature male flies, which have approximately 4,000. Although the cells of both sexes triple in size to about 700 micron 2, the oenocytes of males reach maximum size earlier than those of females. The major features of oenocytes appear to be dense background cytoplasm, putative lipid droplets found only in mature flies, and pigmented granules first seen in the cells of mature flies which accumulate with age to 33% of the cytoplasm. The number of cells and their anticipated capacity for protein synthesis is discussed in relation to the production of yolk protein precursors.     

Effect of diet on the neuroendocrine system and egg development in the sheep blowfly, Lucilia sericata

ABSTRACT. A light microscope study of the endocrine and ovarian systems of Lucilia sericata under two diets revealed that in young females fed on sugar and water, medial neurosecretory cells (MNC) synthesized and stored neurosecretory material (NSM) as the flies matured. The MNC remained filled with NSM as long as the diet was maintained. Following a small increase immediately after emergence, the size of the corpora allata (CA) showed little further change, and the nuclei of nurse cells remained small. However, rapid changes occurred in these tissues soon after a meat meal: NSM was discharged from the MNC, and the CA increased in size. These changes were at a maximum 20 h after a meat meal. 4h later, vitellogenesis was well established and the nurse cell nuclei had increased in size 20-fold. Growth of the nurse cell nuclei continued until approximately 6 h before the completion of vitellogenesis when they are resorbed. Oögenesis took about 48 h at 25°C. When 100 μg of each of three different juvenoids were applied topically to different sugar-fed flies, the nuclei of both MNC and nurse cells became enlarged, whereas the CA were somewhat reduced in size. The relationship between protein ingestion and oogenesis is discussed, and the results obtained with L. sericata are compared with those of other species, especially the blowfly Calliphora erythrocephala.     

Fat-body remodeling in Drosophila melanogaster

The remodeling of the larval fat body is observed in many insects during metamorphosis, but little is known about the physiological importance or the regulation of this process. In Drosophila melanogaster, fat-body remodeling involves the dissociation of the fat body into individual fat cells, which persist throughout pupal development but are later removed by cell death in the young adult. Inhibition of fat-body dissociation is associated with pharate adult lethality and thus is likely to be an essential developmental event. As a start toward understanding the role of fat-body remodeling in the life history of insects, we carried out a detailed study of fat-body disassociation in D. melanogaster using fluorescent microscopy, and tested whether this process is mediated by hemocytes as proposed for fat-body remodeling in Sarcophaga peregrina. We identified and correlated stereotypic events in fat-body dissociation with developmental changes during metamorphosis, and have demonstrated by cell ablation studies that fat-body remodeling in D. melanogaster is a hemocyte independent process.