Synthesis of vitellogenic and non-vitellogenic yolk proteins by the fat body and the ovary of Leptinotarsa decemlineata

The neuro-endocrine control of protein metabolism in the migratory grasshopper, Melanoplus sanguinipes

Target of rapamycin (TOR), a serine/threonine protein kinase, is involved in yolk protein synthesis in insects besides regulating a number of growth and developmental processes of an organism. p70 S6 kinase (S6K), a serine/threonine protein kinase, is a downstream target of TOR and an important regulator of protein synthesis responsible for cell growth and reproduction. In the present study, two genes, TOR and S6K were isolated and designated as BdTOR (GenBank accession no. FJ167395) and BdS6K (GQ203802), respectively, from the oriental fruit fly, Bactrocera dorsalis (Hendel). Downregulation of BdTOR activity in vivo resulted in a significant reduction in yolk protein transcripts in both fat body and ovary, with a substantial reduction of ovary size. Downregulation of BdS6K activity in the larval stage resulted in the developmental defects of larvae, pupae, and adults with a curtailed yolk protein expression in the fat body throughout the first reproductive cycle with a substantial reduction in ovary size and also repressed the egg development in female fruit fly. All these observations support the involvement of S6K-mediated TOR signaling in the regulation of the yolk protein synthesis and egg development in B. dorsalis. This study also shows that nutrition-mediated larval S6K signaling is important for adult yolk protein expression and egg production in B. dorsalis. Treatment of the female flies with the reproductive hormone, juvenile hormone (JH), significantly increased BdTOR and BdS6K expression, and also Bdyp1 mRNA only in the presence of BdTOR mRNA, in the fat body, indicating that TOR connects the link between JH and yolk protein production.

Synthesis of yolk proteins in Drosophila melanogaster

Simple SummaryEfficient and proper functioning of processes within living organisms play key roles in times of climate change and strong human pressure. In insects, the most abundant group of organisms, many important changes occur within their tissues, including the fat body, which plays a key role in the development of insects. Fat body cells undergo numerous metabolic changes in basic energy compounds (i.e., lipids, carbohydrates, and proteins), enabling them to move and nourish themselves. In addition to metabolism, the fat body is involved in the development of insects by determining the time an individual becomes an adult, and creates humoral immunity via the synthesis of bactericidal proteins and polypeptides. As an important tissue that integrates all signals from the body, the processes taking place in the fat body have an impact on the functioning of the entire body.The biodiversity of useful organisms, e.g., insects, decreases due to many environmental factors and increasing anthropopressure. Multifunctional tissues, such as the fat body, are key elements in the proper functioning of invertebrate organisms and resistance factors. The fat body is the center of metabolism, integrating signals, controlling molting and metamorphosis, and synthesizing hormones that control the functioning of the whole body and the synthesis of immune system proteins. In fat body cells, lipids, carbohydrates and proteins are the substrates and products of many pathways that can be used for energy production, accumulate as reserves, and mobilize at the appropriate stage of life (diapause, metamorphosis, flight), determining the survival of an individual. The fat body is the main tissue responsible for innate and acquired humoral immunity. The tissue produces bactericidal proteins and polypeptides, i.e., lysozyme. The fat body is also important in the early stages of an insect’s life due to the production of vitellogenin, the yolk protein needed for the development of oocytes. Although a lot of information is available on its structure and biochemistry, the fat body is an interesting research topic on which much is still to be discovered.

Chapter 8 - Oogenesis

Target of rapamycin (TOR), a serine/threonine protein kinase, is involved in regulating a number of growth and developmental processes of an organism, including yolk protein synthesis in insects. In this study, TOR gene was isolated, designated BdTOR (GenBank accession no. FJ167395), from the oriental fruit fly Bactrocera dorsalis (Hendel). Quantitative RT-PCR showed a higher expression of BdTOR in the pupa than in other developmental stages, as well as in ovary than in the fat body. Downregulation of BdTOR activity by rapamycin treatment and RNA interference (RNAi) in vivo resulted in a significant reduction in yolk protein transcripts in both fat body and ovary, with a substantial reduction in ovary size. However, an unexpected increase in the expression of yolk protein gene was observed in adult ovary 9 days after rapamycin treatment. Taken together, the results suggest the involvement of BdTOR in the regulation of yolk protein synthesis in B. dorsalis.

Mating elicits two reactions in many insect females: egg deposition is increased and receptivity to males is reduced. Central to the control of receptivity and oviposition in Drosophila melanogaster is the sex peptide (SP), a 36-amino-acid peptide sex pheromone synthesized in the male accessory glands and transferred to the female during copulation. To identify regulatory mechanisms involved in the maintenance of the oviposition response, we have compared the effects of mating and SP application with respect to oogenesis. The distribution of the various stages of oogenesis in the ovary, yolk protein (YP) synthesis by the fat body, as well as YP content, uptake and synthesis by the ovary were investigated. Transcripts of the yolk protein genes (yp) were quantified by Northern blotting. Based on our results, we conclude that mating and SP injection into virgin females stimulate yp gene transcription in the fat body only moderately above the background level. However, uptake into the ovary and transcription of the yp genes in the ovary is strongly enhanced after either mating or SP injection. These data are supported by the finding that the abundance of the vitellogenic stage 10 oocytes is also increased. In contrast, early vitellogenic stages 8 and 9 of oogenesis are present in the same numbers in virgin, mated, and SP-injected females, which suggests a control point at about stage 9 determining vitellogenic oocyte progression. The finding that SP can elicit equally all changes observed after copulation suggests that in the sexually mature female it is the major component controlling and stimulating oogenesis after mating.

The role of the ovary and nutritional signals in the regulation of fat body yolk protein gene expression in Drosophila melanogaster

Metamorphic changes in fat body proteins of the southwestern corn borer, Diatraea grandiosella

Adult flies mutant for doublesex dominant (dsxD) are intermediate in phenotype between males and females. The dsxD mutation acts in the heterozygous state to transform only flies with two X chromosomes into intersexes, XY flies are unaffected by the mutation. Yolk-protein synthesis, which normally occurs in the ovaries and fat bodies of females, but not in males unless stimulated with 20-hydroxy-ecdysone, is reduced. The dsxD fat body synthesizes less yolk proteins throughout adult life, and the gonads rarely make yolk proteins. Using cloned yolk-protein genes as probes for measuring transcript levels we have shown that expression of these genes in dsxD is regulated both transcriptionally and post-transcriptionally. We suggest that the dsxD locus regulates the expression of the yolk-protein genes from within the fat body cells and does not operate by modulating ecdysteroid titres in the adults.

Summary1. The appearance of larval fat body as seen under the light or electron microscope depends on the nutritional state of the larva and on the stage of larval development at which the fat body is observed.2. Early in the last larval instar the cells usually possess a well‐developed endo‐plasmic reticulum rich in ribosomes, numerous mitochondria, glycogen granules, a Golgi complex and fat droplets, while later in the instar the endoplasmic reticulum is much reduced and mitochondria are few, but glycogen and fat droplets are present in greater amount together with the appearance of large numbers of proteinaceous spheres.3. Early in the last instar the fat body synthesizes proteins and exports them into the blood, while later in the instar proteins are sequestered from the blood into the fat body.4. The rate of protein synthesis by the fat body is high in the early to mid part of the last instar, but then falls off rapidly to a low level, at which it remains until the larva pupates. In diapausing pupae, protein synthesis remains at this low level.5. The similarity between the electrophoretic patterns of proteins from the fat body and those from the blood provides strong evidence that the fat body is the site of synthesis of many of the blood proteins.6. Some of the blood proteins have been shown to possess enzymic properties, while others are thought to play a role in the transportation of various types of compounds.7. Ecdysone and juvenile hormone both stimulate the rate of protein synthesis by larval fat body. Protein synthesis in fat body from diapausing pupae is stimulated after injury to the pupae.8. The appearance of adult fat body and the amount of protein it contains is often closely linked with the nutritional and reproductive states of the insect.9. An important role of the fat body in the adult female insect is the synthesis of yolk proteins, which are released into the blood and then taken up by the developing oocytes. This synthesis and uptake are under the control of hormones secreted by the corpora allata and by the median neurosecretory cells of the pars intercerebralis.10. The RNA content of fat body in final‐instar larvae is not constant throughout the instar. In some larvae it is at its highest level early in the instar, falling to a low level as the instar progresses, while in other larvae (e.g. Calliphora) the level of RNA in fat body does not decrease as the instar progresses.11. In some dipterous insects the base composition of total RNA is DNA‐like in that the guanine + cytosine content is low, accounting for 40 % of the bases. A similar composition is seen in rapidly labelled RNA isolated from insects of other orders (Coleoptera and Lepidoptera), but the base content of total RNA from these latter insects resembles ribosomal RNA from vertebrate tissues in that it has a high (ca. 60 %) guanine + cytosine content.12. The RNA/DNA ratios in blowfly larval tissues are high compared with those found in any vertebrate tissue.13. In larval fat body, RNA synthesis is low at the time of a moult, increases during the early and mid‐instar period and subsequently falls during the latter part of the instar. During the pupal period, especially during pupal diapause, the rate of RNA synthesis is very low and then increases during the subsequent development of the pharate adult. Injury to diapausing pupae results in an increased rate of RNA synthesis in most of their tissues.14. Ecdysone and juvenile hormone both stimulate RNA and DNA synthesis in larval and adult fat body and in other tissues, although there is evidence that in some tissues these two hormones may act antagonistically to each other. The insecticide DDT also has been shown to stimulate RNA synthesis in tissues of adult insects.

The oocyte is the starting point for a new generation. Most of the machinery for DNA and protein synthesis needed for the developing embryo is made autonomously by the fertilized oocyte. However, in fish and in many other oviparous vertebrates, the major constituents of the egg, i.e. yolk and eggshell proteins, are synthesized in the liver and transported to the oocyte for uptake. Vitellogenesis, the process of yolk protein (vitellogenin) synthesis, transport, and uptake into the oocyte, and zonagenesis, the synthesis of eggshell zona radiata proteins, their transport and deposition by the maturing oocyte, are important aspects of oogenesis. The many molecular events involved in these processes require tight, coordinated regulation that is under strict endocrine control, with the female sex steroid hormone estradiol-17β in a central role. The ability of many synthetic chemical compounds to mimic this estrogen can lead to unscheduled hepatic synthesis of vitellogenin and zona radiata proteins, with potentially detrimental effects to the adult, the egg, the developing embryo and, hence, to the recruitment to the fish population. This has led to the development of specific and sensitive assays for these proteins in fish, and the application of vitellogenin and zona radiata proteins as informative biomarkers for endocrine disrupting effects of chemicals and effluents using fish as test organisms. The genes encoding these important reproductive proteins are conserved in the animal kingdom and are products of several hundred million years of evolution.

The aim of this study was the study the effect of supplementation of hens layer ration with 3% of Nigella sativa seeds on egg characteristics and some biochemical blood parameters . Atotal of 40 Isa brown birds of 30 at age were randomly divided into two groups(20 hens for each group) .The first group (control) feeding on standerd diet along the expermintal period .The second group (treatment) feeding on standerd diet with 3% of Nigella sativa along the expermintal period . The chemical egg charactaries included the percentage of chemical analysis for white and egg yolk in the two groups were moisture,ash,lipid,protein, charbohydrates, cholesterol concentration,high density lipo protein,low density lipo protein and free fatty acid in egg yolk. The biochemical blood parameters included cholesterol concentration ,high and low density lipo proteins ,total protein,uric acid concentration and liver enzymes concentration GOT,GPT,ALP. The data showed there was no significant differences(p>0.05) in the percentage of chemical analysis of egg white between two groups and in the percentage of moisture and ash in the egg yolk in the two treatments.highly significant in the charbohydrates and protein in the egg yolk in the group which treated with 3% of Nigella sativa. The data obtained to the significant reduction in the cholesterol concentration and free fatty acid and low density lipo protein in egg yolk in the treatment group compared with control,higly significant (p<0.05) in high density lipo proteins in the egg yolk of treatment group. In the biochemical blood parameters the result showed to the present of significant reduction in the cholesterol concentration ,low density lipoproteins and liver enzymes GOT,GPT and highly significant in high density lipoproteins,total blood protein and ALP enzyme in the treatment group compared with the control. This study showed to that the addition of 3%Nigella sativa seeds to the hens layer ration lead to reduction in the cholesterol concentration and lipid in eggs and blood and increase in protein concentration in egg and blood .The use of Nigella sativa as feed additives lead to improved the productional and physiological performance in the poultry,in addition to that the safe the customer from the artificial and the chemical materials which added to the poultry ration for improve the production from egg and meat.

Egg development in adult insects is characterized by an ecdysone-induced synthesis of vitellogenins (yolk protein precursors) by the fat body and their secretion into the hemolymph and subsequent endocytosis by the ovary (Kunkle and Nordin, 1985). However, in some cyclorraphous dipterans yolk protein synthesis occurs primarily in the ovaries themselves (Brennan et al., 1981; Rina and Mintzas, 1987; Handler and Shirk, 1988). Significant levels of yolk protein synthesis undoubtedly occur in insects with a high biotic potential and fecundity, such as tephritid fruit flies. Given the extensive economic damage caused by fruit flies, our ability to suppress their egg development by capitalizing on the regulatory role of ecdysone in yolk protein synthesis could significantly reduce existing levels of infestation.

This thesis deals with the hormonal control of protein synthesis in the female fat body of Leptinotarsa decemlineata Say. The results are discussed in relation to reproduction (obtained by rearing under long day conditions) and the preparation for diapause (obtained by rearing under short day conditions).The effect of the photoperiod on protein synthesis and storage in the adult fat body is described in Article 1. Protein synthesis is studied with a newlydeveloped in vitro system which measures the rate of lysine incorporation into protein. The synthesis of the released and retained proteins is measured separately. The rates of lysine incorporation into the retained proteins are not affected by the photoperiod. In addition, the behaviour of the RNA/DNA-ratio is the same in long day and short day females. However, the synthesis of the released proteins differs significantly. Fat bodies derived from females reared under long day conditions synthesize many more released proteins. Females reared under short day conditions start storing proteins in the fat body on day 6 after adult ecdysis, but protein storage in haemolymph is also observed.Article 2 deals with the synthesis of five specific released proteins (two vitellogenins and three diapause proteins) which are measured simultaneously. The five proteins are separated by disc-electrophoresis. The relative incorporation of the five investigated proteins produced by the fat body in vitro is the same as in vivo. This demonstrates the important role of the fat body in haemolymph protein synthesis in the entire insect. The large increase in synthesis of released proteins in females reared under long day conditions is mainly attributed to vitellogenin synthesis; vitellogenin synthesis is low in prediapause females. This difference, however, is not expressed in the amount of vitellogenin present in haemolymph. A single increase in the synthesis of one diapause protein is observed in ovipositing females. Massive synthesis of three diapause proteins occurs during the second half of the prediapause period, which results in high levels of these proteins in the haemolymph. The fat body stores two diapause proteins, which are probably taken up from the haemolymph.In Article 3, the histological properties of the female fat body are correlated with the results of Article 1 and 2. The fat body at adult ecdysis is subject to lysis and cell remodeling. All protein granules disappear. The fat body of long day females develops into a tissue with a high capacity for protein synthesis. Rough endoplasmic reticulum is observed around the nucleus and at the periphery of the cell. The rest of the cell is used for fat storage, and the cell surface is invaginated to allow large surface contact between haemolymph and fat body. The development of the fat body of females reared under short day conditions is not essentially different, between day 0 and day 2, from the development of long day females. However, massive storage of fat in short day females occurs during the first half of the prediapause period. At this stage in development, large invaginations at the periphery of the cell are also observed. The storage of protein starts at day 6 after adult ecdysis when the first electron-dense protein granules are seen around the nucleus. On day 10, the first autophagic granules appear. They contain isolated cell organelles, and their appearance correlates with the low rate of protein synthesis during diapause.The hormonal control of vitellogenin synthesis is studied in Article 4. Vitellogenin synthesis increases after the administration of juvenile hormone to allatectomized females in vivo . The synthesis of diapause proteins is not stimulated, however, which shows that the effect of juvenile hormone is specific. Stimulation of the synthesis of vitellogenins and diapause proteins is also achieved in the absence of juvenile hormone. It seems that additional factors are involved in the regulation of vitellogenin synthesis. β-ecdysone has been tested, but injections of 10 μg do not result in an increase of vitellogenin synthesis. The regulation of vitellogenin synthesis by juvenile hormone has also been studied in mated and virgin females. However, vitellogenin synthesis and the juvenile hormone titre do not differ in virgin and mated females. This is rather surprising since a large difference in oviposition rate is observed. Apparently, the female Colorado potato beetle can uncouple the rate of oviposition from the rate of vitellogenin synthesis.