The formation of plasma membrane reticular systems in the oenocytes of an insect

Plasma membrane reticular systems (RSs) are infolds of the plasma membrane found in cells of several insect tissues that are not transporting epithelia. They form a subsurface reticular lymph space that may be involved in the loading and unloading of hemolymph carrier molecules. The development of a new RS during the fifth larval stadium has been studied in the oenocytes of Calpodes ethlius by scanning electron microscopy. The RS forms by the extension and progressive apical fusion of cell processes leaving a reticular lymph space below. Reticular system formation occurs in a front moving over the cell surface. The RS made in the 4th stadium persists through the moult to the 5th stage but diminishes for the next 3 days. A new intermoult RS then forms very quickly. Its time of formation follows the commitment ecdysteroid peak rather than the beginning of secretion by the wax glands. This new 5th stage RS is maintained during the period of intermoult synthesis, after which it declines and is nearly absent by the time of pupation.     

A function for plasma membrane reticular systems

The basal surface in transporting epithelia is infolded in a way that encourages the formation of standing gradients. Many insect cells have a similar infolded reticular system (RS) although they are clearly not transporting epithelia. These cells are like one another metabolically in that they sequester lipid from hemolymph lipophorins (lipid transporting proteins). Dietary lipids enter the hemolymph from the midgut RS which may be an adaptation for lipophorin loading. The plasma membrane reticular system of tissues metabolizing lipids (fat body, wax glands, oenocytes, lenticles) may be an adaptation for lipophorin reception and unloading. Cationic ferritin (pI 8.5) shows all RSs are covered by a lamina functioning as a negatively charged sieve. The basal plasma membrane leading to the RS is also negatively charged. The RS is a container with charged entrances that would be expected to affect the composition of the contents. Midgut cells release lipid particles into their RS. The particles are positively charged since in tracer studies they associate with anionic but not cationic ferritin. Lipophorins are anionic. The electrostatic binding of lipid to lipophorin would make it less anionic and more likely to leave the RS when loaded, thus carrying lipid to the hemolymph. Conversely, at the destination RS, loaded lipophorin would penetrate more easily than unloaded. A change in charge with unloading would be expected to alter the equilibrium between entering and leaving lipophorin, causing protein concentration in the RS of lipid receiving tissues as has been observed in the fat body. Reticular systems may thus be reaction vessels for interactions between carrier proteins and their load.     

Correlation of hemocyte counts with different developmental parameters during the last larval instar of the tobacco hornworm, Manduca sexta

We determined the changes in hemocyte titer and in the abundance of hemocyte types of the tobacco hornworm Manduca sexta during the fourth and fifth larval stadium and the beginning of the pupal stadium. As we analyzed the samples of individual insects at daily intervals, we were able to correlate phenotypical features, body weight, as well as total protein content and lysozyme activity in the hemolymph with the observations on hemocytes. In the course of the fifth larval stadium, the hemocyte titer decreased slightly and declined further after pupation. Using calculated values for total hemocyte numbers, females had about five times and males three times more hemocytes in the circulating population at the beginning of the wandering stage (in the middle of the fifth larval stadium) than immediately after the last larval--larval molt (from the fourth to the fifth larval stadium). This sexual difference was mainly due to an increase in the number of plasmatocytes, which was more prominent in females than in males. Granular cells were dominant in early fifth larval stadium while plasmatocytes were the most abundant cells in pupae. Oenocytoids and spherule cells disappeared during the wandering stage. Lysozyme activity in the hemolymph rose to a maximum during the wandering stage, with females having lysozyme values twice as high as those for males. These changes in lysozyme activity, however, did not correlate with the increase of total hemolymph protein titer which occurred already at the beginning of the wandering stage. We postulate that changes in hemocyte titers are under direct hormonal control, which has to be proven in future experiments.     

Sequential structural changes in the fat body of the tobacco hornworm, Manduca sexta, during the fifth larval stadium

Light and electron microscopy revealed a series of structural changes that occur in the fat body of the tobacco hornworm, Manduca sexta, during the fifth, i.e. the final, larval stadium. At each developmental stage studied, the cells of the fat body were homogeneous in structure. We found no evidence suggesting the presence of more than one type of fat body cell. Our structural data are consistent with published observations on biochemical activities of M. sexta fat body at particular developmental stages. Specific points of agreement include: (a) acquisition of Golgi complex (GC) and rough endoplasmic reticulum (RER) concomitant with the time of major protein production; (b) loss of many cellular organelles (such as GC and RER) as protein production drastically decreases; (c) accumulation of protein granules and urate granules after the onset of wandering (i.e. during the pre-pupal period); (d) accumulation of lipid and glycogen throughout the feeding period. In addition we found that (a) the plasma membrane reticular system (PMRS) developed during the period when protein secretion was great; (b) the PMRS was lost abruptly at the onset of wandering; and (c) the nucleus changed in shape from being roughly spherical to elliptoid in the pre-pupal stage. We found that the structure of M. sexta fat body is similar to that published for other Lepidoptera. However, it differs from that of Heliothis zea in that regional differences are not obviously apparent.     

Regulation of juvenile hormone esterase gene expression in the tobacco budworm (Heliothis virescens)

The tissue distribution, developmental control, and induction of juvenile hormone esterase (JHE) mRNA was examined in Heliothis virescens using an 800-base pair fragment of a JHE cDNA clone. Northern hybridization analysis of poly(A)+RNA from fat body and integument of fifth stadium larvae indicated the presence of a single JHE mRNA species having an estimated length of 3 kilobases. On Day 2 of the fifth stadium (L5D2), basal JHE mRNA levels were 3-fold higher in the integument than the fat body, which correlated with the higher specific activity of the enzyme in the integument at this time. However, JHE mRNA levels in the fat body on Day 4 of the fifth stadium were 9-fold higher than on Day 2, while mRNA levels in the integument remained the same. This endogenous increase in JHE mRNA and activity in the fat body occurred at the time of peak hemolymph JHE activity. JHE mRNA was not detected in third stadium larvae which have very low levels of JHE activity. Treatment of L5D2 larvae with the juvenile hormone mimic epofenonane resulted in a 7- and 14-fold increase in the level of JHE mRNA in the integument and fat body, respectively. The mRNA induced in both tissues was of the same estimated length as the constitutively expressed message. The data indicate that the developmental regulation and induction of JHE can occur at the level of mRNA. There is evidence that the fat body secretes more JHE than does the integument and could be the major source of hemolymph JHE.     

Changes in the titer of the female-predominant storage protein (81K) during larval and pupal development of the waxmoth,galleria mellonella

The levels of an 81K storage protein in the waxmoth, Galleria mellonella, were monitored during the course of development using rocket immunoelectrophoresis. During the fifth and sixth larval stadia, 81K protein levels increased during feeding and growth but sharply declined at each larval molt. During the fifth and sixth stadia hemolymph levels of the 81K protein increased to about 1 and 2.5 mg/ml, respectively, with no discernible differences between levels in males and females. Neither the fat body nor the remainder of the carcass contained the 81K protein, indicating that the accumulation of this protein during the intermolt period was exclusively in the hemolymph and redistribution of the 81K protein into other tissues does not occur at the final two larval molts. During the seventh (final) larval stadium the absolute quantities of the 81K protein increased from 23 μg per insect to over 1,600 μg in females and to 300 μg in males. The hemolymph concentration of the 81K protein reached 28 mg/ml in females and 6 mg/ml in males with only low levels found in the remaining tissues. Shortly after pupal apolysis, marked by eyespot retraction, the fat body in both sexes rapidly and quantitatively sequestered the 81K protein from the hemolymph. The 81K protein in the hemolymph of both males and females rapidly dropped to nearly zero concentration by pupation. The 81K storage protein remained localized in the fat body cells after uptake occurred, even though the fat body cells disaggregate and reaggregate during metamorphosis. During pharate adult development the 81K storage protein disappeared from the fat body without entering the hemolymph. At adult eclosion 81K was virtually absent from the tissues of both males and females.     

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.     

Polydnavirus of Microplitis croceipes prolongs the larval period and changes hemolymph protein content of the host,Heliothis virescens

Polydnaviruses from certain parasitoid Hymenoptera have been reported to interfere with both host immunity and host development. Heliothis virescens larvae injected with either calyx fluid or sucrose gradient-purified polydnavirus from Microplitis croceipes (McPDV) gained less weight than saline-injected larvae. The active feeding portion of the fifth stadium larva (time to reach the burrowing-digging stage) was doubled (7.0 vs. 3.4 days) when a 0.25 wasp equivalent (WE) of sucrose gradient-purified McPDV was injected into a newly ecdysed fifth stadium host. Many of the treated larvae were unable to pupate, successfully and died at a point of incomplete larval-pupal ecdysis. Pupae that did result from the treated larvae weighed significantly less than controls, even at 0.025 WE. The rate of weight gain and extent of delay of development were dose-dependent; as little as 0.1 WE extended the time of active feeding by 1.5 days and yielded only 25% adults. A 0.05 WE dose yielded 78% adults compared to 95% for controls. The total protein content of hemolymph from individuals injected with McPDV was significantly less than that of controls at any McPDV dose equal to or greater than 0.1 WE. SDS-PAGE profiles of hemolymph proteins from control and McPDV-injected larvae revealed a marked inhibition of the normal accumulation of storage proteins during the fifth stadium and a lesser reduction of serine protease inhibitor protein. Thus, McPDV-injected larvae exhibited some symptoms (less total hemolymph protein and reduced amounts of storage protein) similar to those shown by both parasitized larvae and by larvae injected with M. croceipes teratocytes. However, McPDV affected development during the active feeding stage of the larva, while teratocytes primarily impacted larvae at the time when larval-pupal transformation processes are initiated. © 1995 Wiley-Liss, Inc.     

The homeodomain protein PBX participates in JH-related suppressive regulation on the expression of major plasma protein genes in the silkworm, Bombyx mori

In the silkworm, Bombyx mori, major plasma proteins referred to as 30K proteins are the most abundant proteins in the hemolymph of final (fifth) instar larvae. Surgical extirpation of corpora allata, the source of a juvenile hormone (JH), causes rapid accumulation of 30K proteins in the hemolymph of fourth instar larvae. The 30K protein 6G1 (30K6G1) gene was repressed in primary cultured fat body cells treated with a JH analog (JHA), methoprene. To identify the JH response element present in the promoter region of the 30K6G1 gene, we performed transfection analyses of the 5'-deletion mutants of the 30K6G1 gene using primary cultured fat body cells, gel retardation assays and in vivo footprinting analysis. The results from those analyses revealed that a JH response element exists in the sequence between positions -147 and -140. When the promoter construct mutated at positions -143, -142, and -141 was transfected to fat body primary cultured cells, the suppression effect on the reporter gene expression caused by JHA was reduced. Gel retardation assay using specific antibody revealed that a PBX protein binds to the JH response element. Northern blot analysis revealed that the gene expression of Bombyx PBX is enhanced in the fat body cells by JHA treatment. These results indicate that PBX proteins are involved in the JH signaling pathway and play an important role in suppressing 30K protein gene expression in the fat body of B. mori.     

家蚕(Bombyx mori)主要血浆蛋白质及其基因表达的研究

本文用SDS-PAGE法观察不同发育阶段蚕血液中主要血浆蛋白质sp、30KP浓度的变化;从不同发育阶段的蚕脂肪体提取RNA和poly(A)~+-RNA,在兔网织红细胞系作体外翻译并检测翻译产物。结果表明,5龄蚕脂肪体mRNA合成蛋白质的速率为初蛹的2倍;5龄及初蛹脂肪体30KP mRNA活性的发育变化与其相应蛋白质在血液中的浓度变化一致;sp-1在5龄幼虫脂肪体内的表达及卵黄原蛋白(Vg)在蚕蛹脂肪体内的表达具有雌特异性,其表达和性特异性大体是在前翻译水平被调节的。     

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.     

Larvae of an endoparasitoid,Cotesia kariyai (Hymenoptera: Braconidae), feed on the host fat body directly in the second stadium with the help of teratocytes

Larvae of a gregarious endoparasitoid, Cotesia kariyai (Watanabe), grew rapidly during the second stadium in the host. The fat body of a Pseudaletia host parasitized by C. kariyai was completely consumed by 10 d, just before larval emergence. It seemed hard to explain the growth of the second instar parasitoids and the rapid consumption of the fat body only by ingestion of hemolymph converted from the fat body or other organs of the host. Paraffin sections of the parasitized host revealed that many teratocytes were attached to the surface of the fat body in many sites and destroyed the fat body tissue locally. Zymography of proteins released from the teratocytes revealed that the teratocytes 4 to 9 days after parasitization showed collagenase activity (as a gelatinase). Further, 1st instar parasitoids which were transplanted together with teratocytes into unparasitized hosts preconditioned with C. kariyai polydnavirus (CkPDV) plus venom, grew normally to the 2nd stadium. Abnormal growth of parasitoid larvae was observed when parasitoid larvae were transplanted without teratocytes. These results suggest that the teratocytes attach to the outer sheath of the fat body, secrete an enzyme that makes a hole in the matrix of the fat body, thus allowing the second instar parasitoid to ingest the content of the fat body.     

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.     

Effect of metamorphosis on the major hemolymph proteins of the silkworm

During the metamorphosis of the silkworm, Bombyx mori, three major hemolymph proteins (MHPs) (molecular weights 17,000, 25,000, 27,000) were detected and found to be distributed in the hemolymph and in the tissues of several organs, such as the fat body, midgut, ovary, testis, and even eggs. The MHPs in eggs gradually decreased and disappeared during embryogenesis. The formation, distribution, and utilization of MHPs in tissues other than the gonad, however, were not affected by sex. Radioisotope experiments in vivo revealed that the MHPs were synthesized at an early period of the fifth larval instar. The synthesis of at least two of them occurred in the fat body. MHPs in the hemolymph entered the tissues at the onset of the larval-pupal transformation. On the basis of their appearance, distribution, and depletion, the MHPs may be classified as reserve proteins which are synthesized in the larval stage and utilized later in the developmental stages.     

Tyrosine storage vacuoles in insect fat body

The watery vacuoles first described from larval insect fat body (Chironomus, Voinov, 1927; Aedes, Wigglesworth, 1942; Rhodnius, Wigglesworth, 1967) have been studied in 4th and 5th stage Calpodes larvae. The vacuoles arise at the beginning (E+6–24 hr) of the 4th stadium from plasma membrane infolds that separate from the cell surface as provacuoles less than 1 μm in diameter. These provacuoles grow and fuse with one another through the intermolt until about half the volume of each fat body cell is occupied by a single, large vacuole. The vacuoles begin to disappear at molting. Their membrane is either incorporated into the plasma membrane by exocytosis or fragmented into vesicles that fuse to become lamellar bodies where the membranes are presumably digested. All the vacuoles have gone by a few hours after ecdysis.The tyrosine content of the fat body increases and decreases in proportion to the size of the vacuoles. As the vacuoles decrease at molting the titre of tyrosine in the hemolymph is transiently elevated at the time when there is most demand for phenolics for cuticle stabilization. Crystals having the form of tyrosine crystallize out from vacuoles separated from the fat body. In fat body extracts separated by thin layer chromatography, similar crystals occur only in the eluates from spots corresponding to tyrosine. The vacuoles are therefore presumed to be tyrosine stores used in cuticle stabilization at molting. They correspond to a type of aqueous storage compartment that is well known in plants but hitherto little recognized in animal cells.     

Absence of female-specific protein in the hemolymph of stable fly Stomoxys calcitrans (L.) (Diptera: Muscidae)

Changes, during the reproductive cycle, in fat body, hemolymph, and ovarian proteins of the stable fly Stomoxys calcitrans were characterized quantitatively and qualitatively using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein content of all three tissues increased after blood feeding. Fat body protein increased first, followed by hemolymph and ovarian proteins. SDS-PAGE failed to identify vitellogenin in both female hemolymph and fat body samples. No single protein or group of proteins predominated at any stage of the reproductive cycle. Comparisons between male and female stable fly hemolymph and fat body proteins failed to detect female-specific proteins. Female-specific proteins, however, were detected in the hemolymph of four other species of Diptera.     

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).     

Changes of acid phosphatase content and activity in the fat body and the hemolymph of the flesh fly Neobellieria (sarcophaga) bullata during metamorphosis

Changes in the specific and total activity of the lysosomal marker enzyme acid phosphatase (Acph) and in the amount of enzyme protein were examined in the fat body and the hemolymph from the last larval molt to the larval-pupal apolysis. The specific activity showed minor changes during the last larval period. In contrast, the total activity of the enzyme was low during the feeding period and higher during the wandering stage and strikingly increased at the time of puparium formation. We purified a protein having para-nitrophenyl phosphate phosphatase (Acph) activity and raised antisera against it. The amount of Acph protein in the fat body and hemolymph was examined using an ELISA. The specific Acph content showed little variation, but the total amount of the enzyme protein showed a stepwise increase in both organs during last larval stage and was markedly elevated in the pupal stage in the fat body. In contrast, a considerable decrease in the amount of Acph protein was observed in the hemolymph during this period. These data were in agreement with immunohistochemical observations showing an accumulation of the enzyme protein in fat body cells during the prepupal stage with a concomitant disappearance of the enzyme from the hemolymph. Inhibition of ecdysteroid secretion by water stress prevented the changes both in total enzyme activity and in the amount of Acph protein. However, Acph protein content and enzyme activity could be restored when the water stress was followed by a 20-hydroxyecdysone (20-HE) treatment. Taken together, our data show that Acph is secreted by fat body cells into the hemolymph during the larval stage, where it is stored in an inactive form. Increase in the 20-HE titer at the end of last larval stage reverses this process, and the enzyme is taken up by the fat body cells, where it becomes activated and appears in auto- and heterophagic vacuoles. Arch. Insect Biochem. Physiol. 34:369–390, 1997. © 1997 Wiley-Liss, Inc.     

Dynamics of lipid accumulation by the fat body of Rhodnius prolixus: the involvement of lipophorin binding sites

In insects, lipids are stored in the fat body, mainly as triacylglycerol (TAG). In Rhodnius prolixus, a hematophagous hemipteran, lipids are accumulated after blood meal to be used later on. In adult females, at the second day after feeding, the amount of TAG was 57+/-17 microg/fat body, it increased almost five times and at fourth day it was 244+/-35 microg/fat body. TAG content remained constant until day 13, but it then decreased and, at day 20th it was very low (31+/-4.9 microg/fat body). Radiolabeled free fatty acid was used to follow lipid accumulation by the fat body, as it was previously shown that, in R. prolixus, injected free fatty acids associate with lipophorin, a major hemolymphatic lipoprotein. (3)H-palmitic acid was injected into the hemocoel of R. prolixus females. It disappeared from the hemolymph very rapidly, and radioactivity was incorporated by the fat body. Sixty minutes after injection, radioactivity in the fat body was found mainly in TAGs. The capacity of the fat body to incorporate fatty acids from the hemolymph varied according to the days after blood meal, and it was maximal around the fourth day. Lipophorin binding to specific sites in fat body membrane preparations also showed variation at different days. When membranes obtained from insects at the second, fifth and tenth days were compared, binding was highest at fifth day after feeding.