Hemolymph Polypeptides Research Articles

Humoral immune response of Galleria mellonella after repeated infection with Bacillus thuringiensis.

The insect immune system relies on innate mechanisms only. However, there is an increasing number of data reporting that previous immune challenge with microbial elicitors or a low number of microorganisms can modulate susceptibility after subsequent lethal infection with the same or different pathogen. This phenomenon is called immune priming. Its biochemical and molecular mechanisms remain unravelled. Here we present that Galleria mellonella larvae that survived infection induced by intrahemocelic injection of a low dose of Bacillus thuringiensis were more resistant to re-injection of a lethal dose of the same bacteria but not other bacteria and fungi tested. This correlated with enhanced activity detected in full hemolymph as well as in separated hemolymph polypeptides. In addition, we observed differences in the hemolymph protein pattern between primed and non-primed larvae after infection with the lethal dose of B. thuringiensis. Expression of genes encoding inducible defence molecules was not enhanced in the primed larvae after the infection with the lethal dose of B. thuringiensis. It is likely that priming affects the turnover of immune related hemolymph proteins; hence, upon repeated contact, the immune response may be more ergonomic.

Semi-quantitative analysis of changes in the plasma peptidome of Manduca sexta larvae and their correlation with the transcriptome variations upon immune challenge

The tobacco hornworm, Manduca sexta, has been used as a biochemical model for studying insect physiological processes. While the transcriptomes of its fat body, hemocytes, midgut, and antennae have been examined in several studies, limited information is available for proteins in tissues, cells, or body fluids of this insect. In keeping pace with the M. sexta genome project, we launched a pilot study to identify differences in the peptidome of cell-free hemolymph samples from larvae injected with buffer or a mixture of bacteria. At 24 h after injection, plasma was collected and treated with 50% acetonitrile to precipitate large proteins. The supernatants, containing peptides (<25 kDa) and other stable proteins (>25 kDa), were digested with trypsin and analyzed by nano-liquid chromatography and nano-electrospray tandem mass spectrometry (nanoLC-MS/MS) on an LTQ Orbitrap XL mass spectrometer. Known M. sexta cDNA sequences and gene transcripts from the draft genome were translated in silico to generate a database of polypeptides (i.e. peptides and proteins) in this species. By searching the database, we identified 268 hemolymph polypeptides, 50 of which showed 1.67–200 fold abundance increases after the immune challenge, as judged by significant changes in normalized spectral counts between the control and induced plasma. These included a total of 33 antimicrobial peptides (attacins, cecropins, defensins, diapausins, gallerimycin, gloverin, lebocins, lysozymes), pattern recognition receptors, and proteinase inhibitors. Although there was no strong parallel (correlation coefficients: −0.13, 0.11, 0.39 and 0.62) between plasma peptide levels and their transcript levels in control or induced hemocytes or fat body, we observed the mRNA level changes in hemocytes and fat body concurred with their peptide level changes with correlation coefficients of 0.67 and 0.76, respectively. These data suggest that fat body contributed a significant portion of the plasma polypeptides involved in various aspects of innate immunity after the bacterial injection.

Diversification of Ig superfamily genes in an invertebrate.

The freshwater snail Biomphalaria glabrata possesses a diverse family of fibrinogen-related proteins (FREPs), hemolymph polypeptides that consist of one or two amino-terminal immunoglobulin superfamily (IgSF) domains and a carboxyl-terminal fibrinogen domain. Here, we show that the IgSF1 domain of the FREP3 subfamily is diversified at the genomic level at higher rates than those recorded for control genes. All sequence variants are derived from a small set of nine source sequences by point mutation and recombinatorial processes. Diverse FREP3 transcripts are also produced. We hypothesize a mechanism present in snails that is capable of diversifying molecules involved in internal defense.

A hexamerin protein, AgSP-1, is associated with diapause in the boll weevil

The objective of this research was to identify a reliable biochemical indicator for diapause (dormancy) in the boll weevil, Anthonomus grandis. Hemolymph polypeptides from reproductive and diapausing weevils were compared using denaturing sodium dodecyl sulpfate (SDS)-polyacrylamide gel electrophoresis (PAGE). A 77-kDa protein, which proved to be a hexamerin (AgSP-1), strongly correlated with morphological diapause characters in both male and female adult weevils. N-terminal sequence analysis identified the first 25 amino acids of the mature protein and was used to develop an antibody to AgSP-1. Anti-AgSP-1 reacted only with hemolymph from diapausing weevils of both sexes but not with hemolymph from reproductive weevils. Also, the yolk protein, vitellogenin (VG), inversely correlated with AgSP-1. When hemolymph VG was high, AgSP-1 was absent or barely perceptible. Juvenile hormone regulates VG synthesis in most insect species. Juvenile hormone is reported to stimulate reproductive maturation in the boll weevil (Physiological Entomology 22 (1997) 261) and to be absent during diapause (Physiological Entomology 22 (1997a) 269). Therefore, the juvenile hormone (JH) mimic, methoprene, was used to assess the role of JH activity in the boll weevil for terminating diapause, stimulating reproductive maturation and possibly influencing AgSP-1 titers. Application of methoprene was not effective in activating reproductive development. Hemolymph from methoprene-treated, females contained VG and AgSP-1 titers that were similar to acetone-treated and untreated control weevils. Using a genomic DNA library and 3′ RACE, two clones were isolated that yielded the complete sequence of AgSP-1 as well as a portion of the 5′ untranslated region. Northern blot analysis confirmed the presence of a 2.5 kB transcript for AgSP-1 in the fat body of diapausing weevils. AgSP-1 was also present in the fat body of reproductive weevils, but to a lesser extent. No sex-related differences in gene expression were observed; diapausing weevils of both sexes showed similar levels of AgSP-1 expression. An inverse correlation was observed between the VG transcript and AgSP-1 mRNA. VG was highly expressed in the fat body of reproductive females and only slightly expressed in tissue from diapausing females. Our data suggests that AgSP-1 is a diapause-specific protein in adult weevils and that JH, alone, is not effective in terminating diapause.

Plasmodium-Induced Changes in Haemolymph Polypeptides During Development and Ageing of the Malaria Vector Anopheles stephensi (Diptera: Culicidae)

Changes in the pattern of haemolymph polypeptides were studied to map the innate immune response of the malaria vector Anopheles stephensi (Diptera: Culicidae) to infection by the parasite Plasmodium yoelii yoelii. In addition, changes in the ontogenetic pattern due to glucose feeding, age, sex and gonotrophic cycle-related proteins were analysed. Six polypeptides were induced and 22 polypeptides disappeared due to glucose feeding during various stages of the mosquito’s adult life. Five polypeptide patterns (91, 100, 108, 133 & 145 KDa) were found exclusively in female haemolymph. The changes in these polypeptide patterns have been correlated with sexual dimorphism in their feeding behaviour. In total, 18 polypeptides were induced in the haemolymph of parasitised mosquitoes during different stages of development. Most of the polypeptides were induced in the early stages of infection, i.e. immediately after a bloodmeal. One polypeptide (25 KDa) was induced in all the stages. The expression of two polypeptides (32.5 and 70 KDa) on day 9 and one polypeptide (42 KDa) on day 15 was also enhanced following parasitism. The different induced proteins may help mosquitoes of different ages cope with parasite infection. The parasite evidently elicits immune responses in multiple tissues of the mosquito, including two epithelia which the parasite must traverse to complete its development. The mechanism of these responses and their significance in malaria transmission are discussed.

Two-dimensional gel analysis of haemolymph proteins from Plasmodium-melanizing and -non-melanizing strains of Anopheles gambiae.

Haemolymph polypeptides from Plasmodium-refractory and -susceptible mosquitoes were compared by one- and two-dimensional gel electrophoresis. The refractory strain of Anopheles gambiae kills malaria parasites by a humoral melanization mechanism whereas the parasites develop normally in susceptible mosquitoes. The two strains respond in a similar manner to carboxy-methyl-Sephadex beads that have been injected into the thoracic haemocoel, i.e. beads are strongly melanized in refractory but not susceptible mosquitoes. Protein profiles were compared between strains following cold shock (naïve control), saline injection and Sephadex bead injection. Using the susceptible naïve control as the standard, eight constitutively expressed polypeptides were specific to naïve susceptible mosquitoes while twelve other spots were reduced, enhanced or specific to refractory mosquitoes. Several of the strain-specific spots probably comprise related pairs (one in each strain) which vary only in isoelectric focusing point. Nine spots were induced by sham injection or by an injection of beads or saline, but none was reproducibly different between the strains. Amino acid sequence analysis of one of the refractory strain-specific spots identified it as AgSp14D1, an A. gambiae infection-responsive serine protease that is most similar to the Drosophila gene easter and Manduca prophenoloxidase activating enzyme. This gene maps to polytene chromosome division 14, which has been implicated in the melanization phenotype by quantitative trait loci mapping.

Induction of mosquito hemolymph proteins in response to immune challenge and wounding

The rapid induction of proteins in the hemolymph of the mosquito, Anopheles gambiae, was examined after wounding or injection of immune elicitors ( Escherichia coli, lipopolysaccharide, laminarin, zymosan). One-dimensional gel electrophoresis revealed at least six hemolymph polypeptides >25 kDa that consistently appeared after any breech of the cuticle. All of these polypeptides appeared in the hemolymph within 30 min and reached a maximum concentration after approx. 6 h. No proteins were specifically induced by bacteria or bacterial or fungal cell wall products, however two constitutively expressed proteins were repressed by these injections. Patterns of hemolymph proteins were further analyzed by two-dimensional electrophoresis. Seven spots were enhanced or induced 2 h after injection in four replicate experiments. An additional two spots demonstrated some variability between replicates, but were generally responsive to injection. These rapidly induced polypeptides are candidates for regulating and initiating the mosquito’s responses to pathogens and wounding.

Expression of phase-specific haemolymph polypeptides in a laboratory strain and field catches of Schistocerca gregaria

Uvarov's theory of locust phase polymorphism implies a differential phase-specific gene expression. It was the goal of the present investigation to provide data on the molecular level for this concept. We used the technique of 2D gel electrophoresis to generate haemolymph polypeptide maps from mature locust males. Under the given conditions we identified 238 polypeptide spots by their molecular weight and isoelectrical point. Isolated and crowded males from the Mainz strain, all originating from crowded ancesters, differed in 20 spots: three were solitary-specific and 17 crowded-specific. Field catches of solitary and gregarious S. gregaria in Mauritania revealed that those males showed the phase-specific expression of the same 20 polypeptide spots as the Mainz strain. (It should be mentioned that the field catches and the Mainz animals differed in nine spots, but none of them was phase-specific.) As a crosscheck we reared originally solitary field catches for two generations under crowded conditions. This resulted in the repression of one solitary-specific spot and the expression of 14 gregarious/crowded-specific spots. Since JH conceivably plays a decisive role in the manifestation of the solitary phase we applied the JH analogue fenoxycarb to crowded Mainz males. 15 days after the treatment nine of the 17 gregarious-specific polypeptide spots were repressed. The fact that experimental manipulations—limited by time—cannot alter the expression of all phase-specific polypeptides may reflect the observation that, in nature, some phase characteristics can change within hours whereas others need several generations. The results provide evidence that a number of haemolymph polypeptides are expressed and repressed, respectively, in relation to phase.

Dirofilaria immitis: Effect on hemolymph polypeptide synthesis in Aedes aegypti during melanotic encapsulation reactions against microfilariae

[ 35S]Methionine-labeled hemolymph polypeptides from adult, female Aedes aegypti Liverpool strain mosquitoes inoculated with the microfilariae of the filarial nematode Dirofilaria immitis were compared with those from saline-inoculated and uninoculated controls by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) followed by fluorography. SDS-PAGE analysis of cell-free hemolymph collected via perfusion at 6, 12, 24, 48, 72, and 96 hr postinoculation (PI) detected the enhanced expression of an 84-kDa polypeptide. This polypeptide, expressed constitutively in the hemolymph of all three groups of mosquitoes, increased considerably in inoculated mosquitoes as time progressed as compared with uninoculated controls. Moreover, the 84-kDa polypeptide was expressed at higher levels in D. immitis-inoculated mosquitoes than in saline-inoculated controls. This stimulation of de novo biosynthesis of the 84-kDa polypeptide in inoculated mosquitoes may play a role in the immune response of mosquitoes. Since it is likely that the wound healing response in insects involves many of the same chemical processes as occur in melanotic encapsulation reactions of mosquitoes against filarial worms, the preferential expression of the 84-kDa polypeptide in saline-inoculated mosquitoes seen in this study may reflect a wound healing response. The greater increase in synthesis of this protein in D. immitis-inoculated mosquitoes may reflect production of melanotic material required for parasite destruction as well as for wound healing.

Identification and molecular analysis of storage proteins from Heliothis virescens

Three abundant storage proteins have been detected in larval and pupal hemolymph and pupal fat body of the tobacco budworm, Heliothis virescens. These polypeptides have subunit molecular weights of 74,000, 76,000, and 82,000, as determined by SDS-PAGE and exist as 450,000-Mr hexamers in their native state. A purified 82,000-Mr storage protein fraction has been obtained along with a preparation containing equivalent amounts of the 74,000-Mr and 76,000-Mr subunits, and antisera raised to each of these components have been used to document the developmental profiles of protein accumulation and synthesis by fat body. cDNA clones corresponding to each of three abundant classes of fat body mRNAs have been recovered, and at least one of these has been unambiguously demonstrated to encode the 82,000-Mr storage protein subunit. Northern blot studies with these cDNA clones revealed that the developmental accumulation of transcripts in fat body for each was consistent with the general pattern of storage protein biosynthesis, and more interestingly, that transcripts hybridizing to two of these cDNA sequences are also found in tests. These two cDNAs have also been sequenced revealing that one encodes a polypeptide similar to arylphorins, a class of storage proteins widely distributed in Insecta. The derived amino sequence of the second cDNA, corresponding to the 82,000-Mr protein, had no unusual compositional features and determination of its structural relationship to other hemolymph polypeptides awaits molecular analysis of related genes from other insects.

Hormonal control of vitellogenesis in the gypsy moth, Lymantria dispar (L.): Suppression of haemolymph vitellogenin by the juvenile hormone analogue, methoprene

In most insects, female-specific egg-protein precursors (vitellogenins) are synthesized during pupal or adult stages in response to 20-hydroxyecdysone and/or juvenile hormone. In the gypsy moth, two female-specific haemolymph polypeptides (apo-vitellogenins) having electrophoretic and immunologic identity with Lymantria dispar egg-vitellin apo-proteins begin to accumulate in the haemolymph after day 3 of the fifth larval instar. Head ligation, starvation or treatment with doses of a juvenile hormone analogue, methoprene, >10 μg prior to day 3 block the accumulation of apo-vitellogenin in the haemolymph. Unlike head-ligated or starved larvae which do not gain weight and do no accumulate normal levels of non-vitellogenin haemolymph proteins following treatment, methoprene-treated larvae grow normally and accumulate non-vitellogenin haemolymph proteins. 20-Hydroxyecdysone (2.5 μg) has no effect on the haemolymph accumulation of apo-vitellogenins in normal larvae, and does not restore haemolymph apo-vitellogenins in methoprene-treated, head-ligated or starved larvae. The above results suggest that a low (or declining) juvenile hormone titre during a critical stage early in the last larval instar (prior to day 3) is necessary for the accumulation of vitellogenin in the haemolymph to proceed normally in the gypsy moth. Other factors such as nutrient intake may also be involved. This role of juvenile hormone as an inhibitor of vitellogenin accumulation/synthesis is a novel one for insect species.

Alteration of hemolymph polypeptides in Manduca sexta larvae parasitized by Cotesia congregata: A two‐dimensional electrophoretic analysis and comparison with major bacteria‐induced proteins

AbstractAnalyses using one‐dimensional sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) previously demonstrated that parasitization by the braconid wasp Cotesia congregata significantly alters the normal hemolymph polypeptide profile of host Manduca sexta larvae. In the present study two‐dimensional gel analyses corroborated our earlier findings and provided additional evidence that multiple parasitism‐specific polypeptides were induced, which varied according to the stage of development of the wasps. Parasitization additionally elicited changes in the total protein concentration detected in the blood. Initially an elevation was observed, with newly parasitized larvae exhibiting a twofold elevation in hemolymph protein concentration by 12–24 h postoviposition. In contrast, terminal‐stage hosts with second instar parasites had significantly less protein in the hemolymph, likely due to reduced growth and inhibition of arylphorin synthesis by the fat body during the final stages of parasitism. Comparison of the array of hemolymph polypeptides produced in unparasitized larvae injected with 106cells of the gram‐negative bacterium Enterobacter cloacae with those proteins induced by parasitization indicated the two classes are different. Our findings confirm that the hostresponse to parasitism is a specific one, and not mimicked by bacterial challenge. Duringshort‐term in vitro culture of wasp larvae dissected from the host hemocoel, several proteins were detected in the medium using SDS ‐ PAGE, with their appearance in vitro suggestive of secretion by the wasps in vivo. Moreover, hemolymph from the parasites had significant amounts of putative host proteins, including an arylphorin ‐ like polypeptide and a protein with a mobility similar to that of insecticyanin. Thus, a dynamic interchange of proteins may occur, with the parasites accumulating host proteins while simultaneously secreting a variety of factors into the host hemocoel.

Secretion by insect heart muscle cells

The role of the heart as an endocrine gland has become an expanding field of research in vertebrates, but until recently there has been no suggestion that the hearts of insects might also be secretory. This paper shows that the hearts of Calpodes (Lepidoptera: Hesperiidae) and Rhodnius (Hemiptera: Reduviidae) are like those of vertebrates in having a structure appropriate for secretion through the rough endoplasmic reticulum-Golgi complex secretory vesicle pathway, giving a structural basis for the finding that larval Calpodes hearts synthesize several haemolymph polypeptides and secrete them in vitro into culture media.

Disruptive effects of azadirachtin on development of Cotesia congregata in host tobacco hornworm larvae

AbstractInjection of the insect growth regulator (IGR) azadirachtin into Manduca sexta larvae that were parasitized by Cotesia congregata adversely affected subsequent endoparasite development when the compound was administered prior to the first larval ecdysis of the wasps. Injection of 2.5–10 μg of azadirachtin into newly ecdysed fourth or fifth instar hosts partially inhibited or totally suppressed the first larval ecdysis of the parasites. Instead of initiating rapid growth similar to parasites developing in hosts injected with the solvent carrier (ethanol) only, parasites in azadirachtin‐treated hosts never grew beyond the size normally attained in early terminal stage hosts. The host larvae survived for 2 weeks or longer following injection of azadirachtin, but their parasites never recovered and died encased within one or two exuvial cuticles. The parasitism‐specific hemolymph polypeptides previously shown to be characteristic of terminal stage hosts were undetectable in larvae in which the parasites failed to ecdyse to the second instar. The observed effects of azadirachtin on development of C. congregata differ significantly from those induced by other IGRs in parasitized M. sexta, suggesting azadirachtin has a unique, though as yet undefined, mode of action. Compared to other IGRs, this compound is extremely potent in terms of the minimum doses required to prevent emergence, but affects only the L1‐L2 ecdysis and not the L2‐L3 transition. In contrast, other compounds such as juvenoids and the JH esterase inhibitor BEPAT (S‐benzyl‐0‐ethyl phosphoramidothiolate) permit the parasite to grow and develop normally until the wasps become pharate third instar larvae, then inhibit the second larval ecdysis and emergence from the host.

The synthesis of hemolymph proteins by the larval fat body of an insect Calpodes ethlius (Lepidoptera: Hesperiidae)

Sheets of the fat body from fifth instar larvae of Calpodes ethlius (Lepidoptera: Hesperiidae) were incubated in artificial hemolymph in the presence of [ 35S]methionine to investigate protein synthesis and secretion. [ 35S]methionine was also injected into whole larvae to determine the nature and number of all the hemolymph proteins being made at this time. The fat body synthesizes and secretes at least 20 polypeptides into the medium. The fat body secretes 14 out of 26 detectable hemolymph polypeptides, amounting to about 90% of the total protein. About 12 polypeptides, amounting to 10% of the total hemolymph protein, are not contributed by the fat body. Antibodies made against whole hemolymph recognized at least 11 polypeptides secreted by the fat body as observed on fluorograms of immunoprecipitates. Arylphorin is secreted by the fat body into the medium. Arylphorin synthesized in vitro has been identified through its precipitation by antibodies to hemolymph arylphorin in the fat body and medium. Only arylphorin is so far unique in being secreted by more than one tissue and the synthesis of arylphorin by other tissues in Calpodes is therefore not related to a decline in arylphorin synthesis by the fat body.

Alterations in Biomphalaria Glabrata plasma Induced by Infection with the Digenetic Trematode Echinostoma paraensei

Alterations in the noncellular hemolymph components of M line Biomphalaria glabrata snails infected with the trematode Echinostoma paraensei for 1, 2, 4, 8, 15, 30, or 60 days were monitored by direct microscopical examination, and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in conjunction with quantitative densitometry. A prominent particulate substance was first noted in the hemolymph of infected snails at 1 day postexposure (PE), persisted through day 15, and subsided by day 30. This substance, which was not observed in control snails, contained 2 major polypeptides of 190-200 and 80-120 kDa. Infection with E. paraensei also induced substantial changes in soluble hemolymph polypeptides. PAGE lanes loaded with plasma samples from M line snails infected for 4, 8, 15, and 30 days exhibited a generalized increase in staining intensity relative to controls. A diffuse band centered at approximately 100 kDa, but of variable width, was selectively enriched relative to control preparations in snails with 4-, 8-, 15-, and 30-day-old infections. Standard protein assays also indicated an increase in total protein content of plasma samples from snails infected for 2-60 days, with significant increases noted at 4, 8, and 30 days. Infected snails of the 10-R2 strain of B. glabrata also contained particulate material in their hemolymph. However, soluble hemolymph components of 10-R2 snails exhibited relatively little change, or declined, as a result of infection. For either strain, no new bands could be detected in plasma samples from infected snails, nor were any bands consistently deleted as a result of infection. Although both snail strains exhibit alterations in hemolymph components as a result of infection, their responses differ qualitatively and quantitatively.

Parasitism-induced hemolymph polypeptides in Manduca sexta (L.) larvae parasitized by the braconid wasp Cotesia congregata (Say)

Parasitization of newly ecdysed third, fourth, or fifth instar Manduca sexta larvae by the gregarious braconid wasp Cotesia congregata induces synthesis of new hemolymph proteins in the host. Analysis of hemolymph from parasitized and unparasitized control larvae using SDS gel electrophoresis showed that a major 33 kd band, plus several minor bands, were synthesized in parasitized but not control larvae; autoradiograms of proteins labeled in vivo for 1 hr with [ 35S]methionine indicated that synthesis of the 33 kd polypeptide began a few hours following oviposition by the wasp. Synthesis of the 33 kd parasitism-specific polypeptide was induced in unparasitized larvae by the injection of ovarian calyx fluid from adult female wasps; this fluid is known to contain two morphologically distinct types of virus particles that are normally injected into the host along with eggs during parasitization. Exposure of calyx fluid to psoralen in the presence of long-wave u.v. light destroyed its capacity to induce synthesis of the 33 kd protein, suggesting that synthesis of this polypeptide may be mediated by viral nucleic acid.

Yolk protein induction in males of several Drosophila species

The three yolk-protein genes of D. melanogaster (YP1, YP2 and YP3) are normally expressed in the female from the time of eclosion, but not in males. Expression can be induced in males of any age by treatment with a steroid hormone, 20-hydroxyecdysone. Yolk polypeptides can be identified in haemolymph from females of several Drosophila spp by their cross-reactivity to anti-yolk polypeptides antibodies prepared against D. melanogaster yolk proteins. Western blotting was used to show the accumulation of yolk polypeptides in males of several D. melanogaster sibling spp ( D. simulans, D. mauritiana, D. orena, D. erecta, D. yakuba and D. teissieri) and one other spp ( D. virilis), 16 hr after 20-hydroxyecdysone treatment. YP-homologous transcripts were identified in females of the Drosophila melanogaster sibling spp by RNA hybridization to cloned D. melanogaster YP genes (pYP1, pYP2 and pYP3). YP-homologous transcripts accumulated in males of D. melanogaster sibling spp 16 hr after treatment with 20-hydroxyecdysone; but comparisons between hybridizations to a mixed YP1/2/3 gene probe and YP1-specific probes suggested that the yolk polypeptide genes may not always be transcribed in a coordinated fashion in these males. This difference was no longer apparent after translation into yolk polypeptides had occurred. Steroid treatment of males appeared not to stimulate expression of all genes, since the levels of some haemolymph polypeptides did not appear to be affected by 20-hydroxyecdysone, while other such polypeptides were detected at reduced levels after the hormone treatment which led to expression of the yolk polypeptide-genes in this sex.

Translation of the mRNAs coding for the major hemolymph proteins of Ceratitis capitata in cell-free system: Comparison of the translatable mRNA levels to the respective biosynthetic levels of the proteins in the fat body during development

Four major hemolymph polypeptides (ceratitins) with molecular weights between 8.1 × 10 4 and 8.7 × 10 4 daltons have been identified in the fat body of late Ceratitis capitata larvae. Total fat body RNA from late larvae was translated in reticulocyte lysate, and the predominant in vitro translation products were shown to be the ceratitin precursors. The biosynthesis of these proteins during postembryonic development was studied in both tissue culture and cell-free system. Comparison of the biosynthetic patterns obtained in the two systems suggests a linear relationship between messenger concentration and protein synthesis. Three of these polypeptides show a coordinate pattern of synthesis and are immunologically related. After pupation, all four ceratitins are reabsorbed by the fat body where they accumulate.

Developmental changes in the synthesis of haemolymph polypeptides and their sequestration by the prepupal fat body in Calpodes ethlius stöll (Lepidoptera:Hesperiidae)

Developmental changes in haemolymph protein and in its synthesis were examined in Calpodes ethlius from late in the fourth stadium to early pupation. The major haemolymph proteins have mol. wts of 470,000, 580,000 and 720,000. Upon dissociation, the 470 K and 580 K proteins each resolve into single polypeptides with mol. wts of 82,000 and 86,000 respectively. The 720 K protein separates into two distinct polypeptides with mol. wts of 86,000 and 90,000. The 82,000 polypeptide is present in the haemolymph at all times but not synthesized either very early in the fifth stadium or after the second critical period. The 86,000 and 90,000 polypeptides are present and synthesized only between the first and second critical periods. Disappearance of the 86,000 and 90,000 polypeptides from C. ethlius haemolymph is correlated with a decrease in haemolymph protein concentration and an increase in fat body protein granules. Injection of [ 14C]-labelled haemolymph proteins into larvae after the second critical period results in the fat body acquiring radioactive polypeptides having M rs of 86,000 and 90,000. These results suggest that the prepupal fat body of C. ethlius sequesters at least one and probably two (the 720 K and 580 K proteins) of the three major fifth stadium haemolymph proteins.