Some effects of allatectomy in the female tsetse,Glossina austeni

The effects of juvenile hormone on the milk gland, ovaries, and fat body of adult female G. austeni were studied by allatectomy and hormone replacement therapy. In the absence of juvenile hormone, milk synthesis is slow, leading to the production, in a few cases, of small larvae over a prolonged inter-larval period. In most cases, no viable larva is produced and the fat body hypertrophies. Replacement of the corpus allatum with C₁₆JH leads to a rapid synthesis of milk, production of normal-sized larvae and a reversal of the effect on the fat body. It is therefore suggested that the milk gland activity is directly influenced by JH. Allatectomy in most cases also results in only one egg being matured. The others do not enter vitellogenesis. Similarly, this effect on the ovaries can be reversed by topical application of C₁₆JH.     

A function for pericardial cells in an insect

The pericardial cells (PCs) of fifth instar Calpodes ethlius larvae are functionally adapted for filtering hemolymph and sequestering and digesting proteins. They also have a structure appropriate for the synthesis of proteins for secretion. PC secretion has been investigated by labelling the cells with [³⁵S]methionine ti vitro with detection of newly synthesized polypeptides appearing in the medium by electrophoresis and fluorography. Sources possibly contributing to the appearance of newly synthesized polypeptides in the medium, such as cell breakdown and fat body contamination have been ruled out. The post-incubation medium of PCs contains at least six newly synthesized polypeptides. Three of these polypeptides, having relative molecular masses of 82, 57 and 43 kDa, react with antibodies to hemolymph. At least one additional polypeptide is similar by two-dimensional analysis to that naturally present in hemolymph. PCs incubated together with the heart to which they are normally attached, secrete additional polypeptides that are presumed to come from the heart. The 82 kDa polypeptide secreted by the PCs is similar to the subunits of arylphorin secreted by fat body and other tissues. We conclude that PCs secrete proteins into the hemolymph although the amount may be small relative to that of the fat body.     

A study of uptake of radiolabeled host proteins and protein synthesis during development of eggs of the endoparasitoid,Microplitis croceipes (Cresson) (Braconidae)

The uptake of radiolabeled haemolymph and fat body proteins from fourth instar larvae of Heliothis zea (Boddie) by eggs of Microplitis croceipes (Cresson) was examined by SDS-polyacrylamide gel electrophoresis and by autoradiography. None of the ¹²⁵I-labeled haemolymph proteins was detected in eggs exposed to the proteins in vivo. Although several of the proteins were observed in eggs incubated with the labeled proteins in vitro, none of these proteins was degraded or resynthesized into new structural proteins during development of the embryo. Similarly, no significant uptake of labeled fat body proteins by the eggs could be detected in vitro. On the other hand, protein synthesis measured by incorporation of [³⁵S]methionine occurred throughout egg development. Proteins were synthesized at least 1 hr after the egg was deposited into the host. The protein patterns of eggs on one-dimensional SDS gels were complex and ranged in size from less than 18,500 to more than 330,000 mol. wt. The protein band patterns of the newly synthesized proteins showed some qualitative differences at 1–8, 16–32 and 40 hr after egg deposition. We conclude that eggs do not absorb or utilize the host apoproteins (or degradation products) but instead synthesize proteins de novo from free amino acids in the host haemolymph.     

Evidences for a stage-specific juvenile hormone binding protein in the hemolymph of the silkworm,Bombyx mori L.: Identification and characterization by photoaffinity labeling and immunological analyses

Two molecular forms of juvenile hormone binding proteins were identified in the larval hemolymph of Bombyx mori by photoaffinity labeling. One form having an Mr of 33 kDa was present constantly in the hemolymph of the third to the fifth instar larvae while the other form having an Mr of 35 kDa was detected in the hemolymph until in the early fifth instar larvae but not in the prewandering larvae and prepupae. A 33 kDa binding protein was purified by hydrophobic interaction chromatography, gel filtration, and native PAGE. Antiserum against 33 kDa binding protein cross-reacted with 35 kDa binding protein on Western blots, suggesting that these binding proteins shared the same epitopes. From the results of saturation binding assays, it was inferred that 33 and 35 kDa binding proteins had a similar binding affinity for JH 1. It was revealed that one of these binding proteins, 35 kDa binding protein, was produced in the fat body in a stage-specific manner: fat body of the early fifth instar larvae synthesized both 33 and 35 kDa binding proteins while that of prewandering larvae synthesized only 33 kDa binding protein. © 1996 Wiley-Liss, Inc.     

Protein metabolism by the salivary glands and other organs of the larva of the blowfly, Calliphora erythrocephala

Protein metabolism in salivary glands, gut, haemolymph, and fat body during the last larval instar of the blowfly, Calliphora erythrocephala, has been investigated. In salivary glands, protein release, protein synthesis, amylase, and pepsin-like protease activity were maximal in 6 day larvae, this being at a time when the larvae had finished feeding. All these functions declined in glands from the rounded-off white puparial stage (R.O.) while acid phosphatase activity rose throughout the third instar to a maximum at the R.O. stage, Glands from 6 and 7 day larvae released protein which on disk gel electrophoresis separated into four minor bands and two major bands one of the latter possessing protease activity.In the gut, pepsin-like protease activity was maximal in 4 day larvae after which it fell rapidly thus following the feeding pattern of the larva in contrast to that in the salivary glands which did not.In vitro experiments showed that protease was released from 6 day glands through the basal membrane of the cells and not via the duct. A pepsin-like protease was also found in the haemolymph and fat body, the activity in the fat body rising rapidly during the latter part of the third instar, a rise which is attributed to the fat body sequestering protease from the haemolymph. Acid phosphatase activity in the fat body was maximal in 5 day larvae indicating that this enzyme was synthesized early in the third instar. It was shown that fat body sequestered ¹⁴C-labelled protein synthesized by and released from the salivary glands, most of the ¹⁴C activity being associated with a 600 g precipitable, acid-phosphatase rich fraction.It is proposed that in late third instar larvae the salivary glands function as glands of internal secretion, releasing protease into the haemolymph, which is then sequestered by the fat body (and perhaps other tissues) and is subsequently used in the lysis of the tissues at the time of metamorphosis.     

Quantitative morphological changes of the uterine gland cells in relation to physiological events during a pregnancy cycle in Glossina morsitans morsitans

Secretory cells of the uterine gland were investigated morphometrically during the second pregnancy cycle in Glossina morsitans morsitans. In early pregnancy when an egg was present in the uterus, morphometric parameters of the nucleus, the cytoplasm and its organelles were markedly low coincident with low physiological activity in the gland cells. This is the period when a proportion of the assimilated amino acids from the digested blood meals are converted to triglyceride in the fat body and stored. During the second half of pregnancy, when a larva was present in the uterus, there was a striking increase of the cytoplasmic and organelle volumes as well as of the surface areas of rough and smooth endoplasmic reticulum. There was also a marked augmentation of triglyceride-like inclusions, and later of secretory vesicles which released their content into enlarging extracellular reservoirs. This indicated a markedly high activity in the gland cells concomittant with the period of rapid synthesis of large amounts of proteins and lipoproteins destined for the growing intra-uterine larva. Mitochondrial parameters also increased, probably to generate adequate energy for synthesis, transport and secretion of nutriments for the larva. After larviposition, a marked reduction of cellular parameters was measured, and the volume density of lysosome-like structures had increased, indicating that larviposition is followed by degradation of large amounts of cellular organelles involved in biosynthetic processes. Morphometrical investigation of the uterine gland cells demonstrated a temporal correlation of the cellular dynamics with physiological events involved in producing a fully developed third-instar larva at the end of a pregnancy cycle.     

Hormonal control of storage protein synthesis and uptake by the fat body in the silkworm, Bombyx mori

The female silkworm, Bombyx mori, rapidly accumulates two storage proteins, that are synthesized by the fat body, in the haemolymph during the feeding stage of the last-larval instar, and then sequesters them from the haemolymph into fat body during the larval-pupal transformation.The rapid synthesis and uptake of storage proteins by the fat body are shown to be induced by allatectomy in the early-penultimate larval instar. A juvenile hormone analogue, methoprene, is highly effective in inhibiting the allatectomy-induced synthesis, and, in a higher dosage, further blocks the uptake. Allatectomy in the late-penultimate larval instar shortly before moulting does not enhance the storage protein synthesis, but causes the uptake to occur two days earlier in the last-larval instar. Injection of 20-hydroxyecdysone is not stimulatory for synthesis of the proteins, but is effective to induce their uptake. Starvation during the early last-larval instar completely blocks the synthesis.From these results, it is suggested that storage protein synthesis is induced in the absence of juvenile hormone by some supplementary stimulus, possibly the supply of nutrient after feeding, and uptake is induced by ecdysteroids after a decline in the juvenile hormone level.     

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.     

Metabolic shift from lipogenesis to glycogenesis in the last instar larval fat body of the silkworm,Bombyx mori

When [¹⁴C]glucose was injected into the last instar larvae of the silkworm, Bombyx mori, the label was incorporated into various tissues at varying degrees depending on the developmental stages. Fat body exhibited high incorporation rates throughout the feeding periods. Silk glands became active in incorporation but midgut decreased toward larval maturation. The pulse labeling experiment clearly demonstrated that the metabolic shift from lipogenesis to glycogenesis occurred in fat body at the middle of the last instar; a predominant incorporation was found in lipids when [¹⁴C]glucose was injected at the early stage, while at the late stage glycogen synthesis became most active. Incorporation into fat body proteins was not a major factor throughout the instar. Extirpation of silk glands enhanced incorporation into glycogen and proteins at the late stage but did not affect lipid synthesis. Long-term chase showed that fat body lipids and proteins synthesized at the early stage were totally carried over into the pupal fat body, while much glycogen produced at the late stage was used during the larval-pupal transformation with the remainder carried over into the pupa.From these results the metabolic shift from lipogenesis to glycogenesis in fat body is discussed in relation to the storage function of the fat body for pupal metamorphosis.     

A larval serum protein of the silkworm,Bombyx mori: cDNA sequence and developmental specificity of the transcript

The Bombyx mori larval serum protein (BmLSP) is a major component of larval hemolymph proteins until early in the last instar. The cDNA for BmLSP was cloned from a library constructed from fat body RNA of penultimate instar larvae, and the complete nucleotide sequence of the 909 base pair cDNA insert was determined. The deduced 262 amino acid polypeptide included a 16 amino acid residue signal peptide and a 15 amino acid sequence prosegment. A homology search showed that BmLSP has significant similarity with microvitellogenin of Manduca sexta and the 30K proteins of B. mori. Tissue distribution and developmental profile of BmLSP mRNA were analyzed by northern hybridization. BmLSP mRNA was abundant in fat body but not detected in midgut and silk gland. BmLSP mRNA was present during the feeding periods of the fourth and fifth instar larvae, but absent during the larval molt and after the onset of cocoon spinning.     

Interrelationship between the silk gland and other tissues in protein metabolism in the latest larval stage of the silkworm, Bombyx mori

Gamma irradiation at scheduled embryonic stages of silkworm eggs caused some disturbances in biological activities, i.e. cell proliferation of the silk gland, growth of larva and larval tissues, and cocoon production. The source of precursors for silk formation at the latest age of the fifth instar was found to be mainly in the ‘integument’ in its disintegrating state. Radioactivity in the tissues of the control and irradiated larvae labelled with ¹⁴C-glycine or ¹⁴C-leucine was used for analysis. Disturbance of synthesis of silk protein and of fat body protein with gamma irradiation decreased the degrading ability of the ‘integument’ and gut proteins.     

Differential expression of sex-related fat body proteins during the larval–pupal developmental stages of the silkworm (Bombyx mori)

The silkworm fat body is the site of many intermediary metabolic processes, and a source of sustenance for growth throughout the life cycle. Fat body proteins are responsible for storing nutrients, providing energy, and regulating hormones, and they have been identified using proteomic approaches. However, detailed differential expression of sex-related fat body proteins has not previously been evaluated. In the present study, we characterized the differential expression of sex-related fat body proteins, by using 2-dimensional gel electrophoresis (2-DE) followed by mass spectrometry identification and bioinformatics methods. We extracted the fat body proteins from 5-day-old fifth instar larvae (L5), 10-day-old fifth instar larvae (corresponding to the end of spinning [LE]), and 0-day-old pupae (P0) of the multivoltine silkworm variety “Da Zao”. We confirmed the presence of 11 important sex-specific expression proteins and 14 stage-specific expression proteins. We accurately identified 13 of these specific expression proteins, including actin, calponin-like protein, 75 kDa subunit NADH, receptor for activated protein kinase C from Bombyx mori (BmRACK), IMP (inosine monophosphate) cyclohydrolase, tropomyosin 1, β-tubulin, hypothetical protein, antichymotrypsin precursor, and 30 K protein precursor. We showed that BmRACK was differentially expressed between male and female silkworms. We discuss the biological roles of the specific expression proteins during the larval–pupal developmental stages.     

Nutrient transfer during the reproductive cycle in Glossina austeni Newst.: histology and histochemistry of the milk gland, fat body, and oenocytes

Using thick sections cut from Epon-embedded tissues fixed in Karnovsky's fixative, the cytological changes visible in the light microscope were described for milk gland, fat body and oenocytes during the pregnancy cycle of the female of Glossina austeni. Histochemical procedures on paraffin sections were used to explore changes in nucleic acids, proteins, and tyrosine-containing proteins. The milk gland undergoes a cycle of secretion correlated with the pregnancy cycle and the results are consistent with the view that the protein component of the milk is synthesized in the secretioy cells of the gland. Tyrosine is particularly abundant in the oenocytes which appear to play a crucial role in the metabolism of this amino acid.     

PROTEIN AND NUCLEIC ACID METABOLISM IN INSECT FAT BODY

1. 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.     

Aspects of the nutrition of adult female Glossina morsitans during pregnancy

The adult female Glossina morsitans fed on goat takes, in terms of dry weight, approximately 37·6 mg of blood during a pregnancy period, and at least 84% of this dry blood comprises proteins. In this diet leucine is the most abundant, isoleucine and methionine contents are markedly small, while values for other amino acids differ and are intermediate. Shortly after pupariation the third instar larva produced by goat-fed females contains 67·3% water, 15·3% residual dry weight, 11·0% fat, and 6·4% puparium. The amount of each of the amino acids present in the larva and its puparium comprises a small proportion of that taken by the female fly during a pregnancy period. The fat content of the fully-fed larva is also very small compared with the total nutrient intake by the pregnant female. These results are discussed in terms of the nutrition of intra-uterine larva in relation to feeding by its female parent.