Properties,synthesis, and accumulation of storage proteins in Pieris rapae L.

Two kinds of storage proteins (SP-1, SP-2) were confirmed in hemolymph and fat body of Pieris rapae during metamorphosis. Both proteins were present in high concentrations in the hemolymph during the last larval instar. Hemolymph concentrations of SP-1 and SP-2 dropped after pupation as the proteins were being deposited in fat bodies. SP-2 is present in a larger amount than SP-1. Detailed studies on storage proteins determined their properties, mode of synthesis, and accumulation in the fat body. SP-1 has a molecular weight of 500,000 and consists of one type of subunit (M_r 77,000), while SP-2 has a molecular weight of 460,000 and is composed of two types of subunits (M_r 80,000 and 69,000). The pl values of SP-1 and SP-2 were determined to be 6.97 and 7.06, respectively. Fat body cells from 1-day-old fifth instar larvae synthesized storage proteins in large amounts, whereas those from late prepupae exhibited high protein sequestration. Proteins taken up in fat body accumulated in dense granules during the pupal stage but sharply decreased at the adult stage. Morphological changes in the fat body tissues were observed during the larval-pupal transformation; the nuclei of fat body cells became irregularly shaped, and the boundaries between cells seemed to be obscure. Synthesis, storage, or degradation of storage proteins in fat body during development is closely associated with morphological changes in the tissues.     

Hormonal modulation of glycogen reserves In the fat body of castor semilooperAchaea janata Linn (Lepidoptera,Noctuidea)

Histochemical details of the fat body in the fifth instar larval stage, pupa and adult moth of the castor semilooperAchaea janata were elucidated in detail using light and electron microscopy in conjunction with glycogen storage patterns using polyacrylamide gel electrophoresis. The periodic-acid Schiff staining for glycogen in fat body was maximum in the spinning stage of the larva, when compared to the feeding stage and prepupal stages, and higher in the pupa than in the larva and the adult moth. In insulin injected and juvenile hormone treated fat body, glycogen deposition was more than in glucagon injected tissues. The periodic-acid Schiff stained bands in PAGE had electrophoretic mobility similar to the corresponding protein band numbers, indicating their glycoprotein nature.     

The synthesis and uptake of haemolymph storage proteins by the fat body of the greater wax moth Galleria mellonella (L.)

The four storage proteins of Galleria mellonella (L.) exhibit stage-dependent alterations in distribution between haemolymph and fat body during the final larval instar. Autoradiograms of newly synthesized fat body proteins separated on sodium dodecyl sulphate polyacrylamide gels demonstrated that this tissue both synthesizes and releases all four storage proteins until pharate pupal development. Although the capacity for protein biosynthesis by fat bodies fluctuated during this period of development, qualitative alterations in the synthesis of the storage proteins were not found. Fat body from wandering through to spinning stage larvae sequestered radiolabelled storage proteins which had been injected into the haemolymph. This provides evidence for the direct resorption of haemolymph storage proteins by fat body; however, it may only in part account for the storage protein pool accumulated by pharate pupal and pupal fat body tissues.     

Evaluation of thiamethoxam and imidacloprid as seed treatments to control European corn borer and Indianmeal moth (Lepidoptera: Pyralidae) larvae

Efficacy of thiamethoxam (Cruiser) and imidacloprid (Gaucho) were evaluated as seed treatments for controlling European corn borer, Ostrinia nubilalis (Hübner) and Indianmeal moth, Plodia interpunctella (Hübner) larvae in stored grain. At approximately 22-26 degrees C, all fifth instar European corn borers died after two or 4 d of exposure to corn treated with 250 and 500 ppm thiamethoxam, respectively, while mortality of larvae exposed for two and 4 d on corn treated with 6.3-937.5 ppm imidacloprid did not exceed 48% at any concentration. At 29 degrees C, all nondiapausing fifth instars were killed after 3, 4, and 6-d exposure to 400, 300 and 200-ppm thiamethoxam, respectively, while survival increased at successively lower concentrations of 100, 50, 25, and 12.5 ppm. At 29 degrees C, the LC50 decreased from 85.9 to 7.2 ppm as the duration of exposure on treated corn increased from 2 to 6 d. All second and third instar Indianmeal moth larvae died after a 5 d exposure period to corn grain treated with thiamethoxam at 50 ppm or higher, but as the larvae aged, higher concentrations and longer exposure periods were required to give 100% mortality of each larval instar. Similar results were obtained when larval Indianmeal moths were exposed on corn treated with imidacloprid, or on sorghum treated with thiamethoxam. Mature wandering phase fifth instars were the most tolerant larval stage of the Indianmeal moth.     

Storage proteins of the fall webworm,Hyphantria cunea drury

Two storage proteins, storage protein-1 (SP1) and storage protein-2 (SP2), were found in hemolymph and fat body during the development of Hyphantria cunea, the fall webworm. Both storage proteins show similiar quantitative changes during development in males and females; however, SP1 is more abundant. The hemolymph of last instar larvae contains high concentrations of the storage proteins. However, following pupation, the storage proteins accumulate in fat bodies. SP1 peaks in the hemolymph of males and females late in last instar larvae (8-day-old 7th instar larvae). SP1 has a native molecular weight of 460,000 and consists of six identical subunits (Mr = 76,700), while SP2 has a molecular weight of 450,000 and is composed of two different subunits (Mr = 74,100 and 72,400). Both SP1 and SP2 are hexamers and are phosphorylated glycolipoproteins. The pl values of SP1 and SP2 were determined to be 5.70 and 5.50, respectively. Antibodies raised against SP1 react positively with vitellogenin and ovary extract, as well as with proteins in the hemolymph from last instar larvae and proteins in pupal fat bodies. Storage protein synthesis starts in fat bodies of a 4-day-old 7th instar larvae and in female peaks at 6–8 days of the 7th instar.     

Developmental changes in the major inclusion bodies of polytene nuclei from larval tissues of the blowfly,Calliphora stygia

The major ribonucleoprotein inclusion bodies of the polytene nuclei from larval ventral nephrocytes, Malpighian tubules, pericardial cells, fat body and salivary glands of Calliphora stygia show characteristic developmental patterns at the light microscope level. Three morphologically and developmentally distinct classes of nuclear inclusions occur in these tissues. In fat body and salivary gland nuclei, morphological changes in the inclusion material can be correlated with the pattern of protein synthesis during the third larval instar, and with conspicuous changes in chromosome morphology. The appearance of cytologically diverse nuclear inclusions, in an ordered tissue-specific sequence, may thus reflect differential activation of the genome.     

Expression and localization of storage protein 1 (SP1) in differentiated fat body tissues of red hairy caterpillar, Amsacta albistriga Walker

The accumulation and utilization of storage proteins are prominent events linked to the metamorphosis of holometabolous insects. The female-specific storage protein 1 (SP1) is the major storage protein found in the hemolymph and fat body of female larvae of the groundnut pest, Amsacta albistriga. Here we show SP1 expression and localization in differentiated fat body tissues using biochemical and immunohistochemistry scrutiny. Comparison of A. albistriga SP1 with that of other species with respect to amino acid composition and N-terminal sequences show that SP1 is a methonine-rich protein and its identity was confirmed by means of immunoblot analysis. Northern blot studies revealed that the SP1 gene demonstrates stage- and tissue-specific expression in the peripheral fat body cells during the mid-larval period of fifth instar of A. albistriga. During the larval pupal transformation, SP1 are sequestered mainly by the perivisceral fat body tissues, until they serve the purpose of supplying amino acids for the production of egg yolk proteins. Further, electron microscopic studies using immunogold tracer techniques confirmed the localization of crystalline SP1 reserves, stored in the perivisceral fat body tissues. Hence, the peripheral fat body is responsible for biosynthesis of storage proteins, whereas the perivisceral fat body is a specialized storage organ.     

Juvenile hormone-dependent vitellogenin synthesis in Locusta migratoria fat body: Inducibility related to sex and stage

Juvenile hormone (JH)-dependent vitellogenin (Vg) synthesis in the fat body of Locusta migratoria is normally limited to sexually mature adult females. As a step toward examining the basis of this limitation, we have tested female and male locusts in a series of stages after the third larval molt for inducibility of Vg synthesis by the synthetic JH analog, methoprene. We find that in the fourth and fifth larval instars fat body of both sexes can be induced to produce Vg, but in the adult stage females respond strongly while no more than trace amounts can be induced in males. Quantitative assays show relative responsiveness in the order: adult female > fifth instar female > fifth instar male ? adult male. During the fifth instar of both sexes, maximal vitellogenic response was obtained in midinstar. After the larval-adult ecdysis, female fat body was unresponsive during the first 4 days, then responsiveness increased and by Day 8 after ecdysis fat bodies were fully as competent to produce Vg as at Day 14, the usual maximum of the first vitellogenic cycle due to endogenous JH. Larval and adult female fat bodies implanted into male larvae are competent for Vg synthesis after metamorphosis, so that the differences between adult male and female cannot be imposed by the male milieu intérieur during the larval-adult molt. In male and female precocious adults, produced by treatment of fourth instars with precocene, fat body responded to methoprene as in normal adults. We conclude that factors intrinsic to the fat body cells, determined early in development, are responsible for differential gene programing in males and females, which is partially expressed by the fifth instar but fully manifest only after a molt in the absence of JH.     

In vitro synthesis, release and uptake of storage proteins by the fat body of Manduca sexta: putative hormonal control

1. Two major proteins (P1 and P2) are synthesized by the fifth instar larval fat body of Manduca sexta and then released into the hemolymph. 2. These proteins are later sequestered by the pre-pupal fat body. 20-Hydroxyecdysone does not appear to affect the synthesis of either protein. 3. When day 2 fifth instar larvae are neck-ligated there is an excessive synthesis (supersynthesis) of P2 (arylphorin). 4. Juvenile hormone I (JH I) applications to ligated animals had no effect, but brain homogenate injections resulted in the inhibition of P2 synthesis. 5. Neck ligations of larvae between days 5 and 6 revealed a head critical period between day 5 + 12 hr and day 5 + 18 hr, after which the head is unnecessary for the sequestration of either protein by the fat body. 6. JH I and JH III applications to ligated larvae before the head critical period do not restore the ability of the fat body to sequester the storage proteins. 7. P1 and P2 appear to be synthesized differentially and P2 is sequestered by the fat body to a much lesser extent than P1. 8. P2 is the hemolymph storage protein of both larval and pupal stages, whereas P1 appears to be the storage protein of the pupal fat body. 9. The data indicate that the synthesis of arylphorin and the resorption of both proteins are controlled by a putative head factor(s).     

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.     

Developmental changes of storage proteins and biliverdin-binding proteins in the haemolymph and fat body of the common cutworm,Spodoptera litura

The concentrations of three storage proteins (SL-1,SL-2 and SL-3, hexamers of 70-80kDa subunits) and two biliverdin-binding proteins (BP-A and BP-B, dimers of 165kDa) in the haemolymph and fat body during larval and pupal development of Spodoptera litura were determined by immunodiffusion tests using polyclonal antisera. SL-1 and SL-2 (methionine-rich) first appeared in the haemolymph of one-day-old sixth (final) instar larvae, prominently increased in the haemolymph during the later feeding period and were almost totally sequestered by the fat body after gut purge. SL-3 (arylphorin) was first detected in the haemolymph during the molting period to the final larval ecdysis, increased in concentration throughout the entire feeding period of the final larval instar and was partly sequestered by the fat body several hours later than the other storage proteins. BP-A showed nearly the same pattern in the haemolymph as SL-3: BP-B increased during feeding period and decreased during molting period and attained a maximum level during the penultimate larval instar, however its concentration decreased considerably and remained low in the final larval instar. BP-A was partly and BP-B was almost totally sequestered by the fat body 8 h after sequestration of SL-1 and SL-2, rendering the fat body blue in colour. These facts suggest an additional function of biliverdin-binding proteins as amino acid storage proteins and the results show a differential uptake mechanism for these proteins by the fat body.     

Ecdysone 20-hydroxylation and 3-epimerization in larvae of the gypsy moth,Lymantria dispar L.: tissue distribution and developmental changes

The potential for ecdysone metabolism was determined for various larval tissues of the gypsy moth, Lymantria dispar. Homogenates of fat body, midguts, and Malpighian tubules, taken on different days during the second half of the fifth instar, were incubated with [(3)H]ecdysone, and the products were analyzed by reversed-phase and normal-phase HPLC. All tissues showed conversion to 20-hydroxyecdysone, and midguts also produced 3-epiecdysone. Ecdysone 20-monooxygenase (E20MO) activity in the fat body increased from a low level on day 5 to a peak on day 11, coinciding with the peak in the hemolymph ecdysteroid titer on the penultimate day of the instar. Midguts and Malpighian tubules showed E20MO activity only during the last 3 or 4days of the instar, with the highest activity also occurring on the penultimate day. For the midguts, the appearance of the E20MO coincided with the transition from larval to pupal tissue. No activity was detected in larval midguts. 3-Epiecdysone formation, however, was mainly found in larval midguts, with only marginal activity detectable in pupal midguts.     

Stimulated synthesis of the female-specific storage protein in male larvae of the silkworm Bombyx mori treated with juvenile hormone analog

Application of methoprene to fourth (penultimate) instar larvae of the silkworm Bombyx mori induced the appearance of the feeding dauer larvae at the fifth (last) instar and prevented pupal metamorphosis. Methoprene also increased the protein concentrations of hemolymph last instar larvae by preventing sequestration of storage proteins by the fat body. Usually, the female-specific storage protein 1 (SP1)* disappears from the male hemolymph at the time of the last larval instar. However, exposure of male larvae to methoprene at the penultimate instar enhanced the accumulation of SP1 in the hemolymph. The SP1 accumulated in males did not differ in molecular weight and immunoreactivity from the SP1 produced in female larvae. Both sexes of fourth instar larvae allatectomized on day 1 instantly accumulated SP1 in the hemolymph, and methoprene application after allatectomy suppressed the hemolymph accumulation of the SP1. In contrast, if allatectomy was carried out at a later stage of the fourth larval instar, SP1 concentration in hemolymph of fifth instar larvae did not increase, suggesting the different juvenile hormone action for regulation of SP1 synthesis in the penultimate instar larvae of silkworms.     

Regulation of expression of arylphorin and female-specific protein mRNAs in the tobacco hornworm, Manduca sexta

Two non-cross-hybridizing cDNA clones were isolated from a lambda gt11 cDNA library prepared from Day 2 fifth instar female fat body of Manduca sexta and shown by hybrid selection to code respectively for the two storage proteins arylphorin and female-specific protein (FSP). Analysis of the developmental expression of arylphorin showed its presence during the feeding phases of the penultimate (fourth) and final (fifth) larval instars and its absence during the molt. Abdominal ligation of larvae followed by infusion of Grace's medium showed that this amino acid-rich medium was able to maintain arylphorin expression in fourth instar larvae, but not continued high expression in fifth instar larvae. This nutrient medium however was sufficient to allow initiation of expression in newly ecdysed fifth larval abdomens. Infusion of 5 micrograms 20-hydroxyecdysone (20HE) caused a significant reduction of arylphorin RNA in ligated fourth larval abdomens, whereas 50 micrograms was required in Day 2 fifth larval abdomens to suppress this RNA. Thus, both the lack of incoming nutrients and the rising titer of ecdysteroid contribute to the loss of arylphorin mRNA at the molts and at wandering. By contrast, FSP mRNA was first detected in females on Day 2 of the fifth instar, but not in males until wandering, and then was present throughout the prepupal period. In females allatectomy caused the precocious appearance of FSP mRNA which was prevented by application of 10 micrograms methoprene, a juvenile hormone analog. Expression of FSP mRNA in males however appeared to be independent of hormonal milieu.     

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.     

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.     

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.