Apolipophorin III in locusts: purification and characterization

Three molecular species of apolipophorin III were purified from adult locust hemolymph by gel filtration and ion-exchange chromatography, and named apo-III-a, apo-III-b, and apo-III-c, respectively. They were indistinguishable by SDS-polyacrylamide gel electrophoresis, immunodiffusion, and in amino acid composition; however, they had different isoelectric points (5.43 for a, 5.11 for b, and 4.98 for c) and, therefore, could be separated by native- or urea-gel electrophoresis. All three apo-IIIs were glycoproteins and contained fucose, mannose, and glucosamine. The total sugar content amounted to about 11% for each of the three apo-IIIs. The molecular weight of apo-III determined by SDS-polyacrylamide gel electrophoresis was approximately 20,000, almost equivalent to the native molecular weight (approximately 19,000) estimated by the sedimentation-equilibrium method. This indicated that the locust apo-III exists in hemolymph as a monomeric form. It was demonstrated that a total 9 moles of apo-III (2 moles apo-III-a, 6 moles apo-III-b, and 1 mole apo-III-c) associate with each mole of lipophorin in response to the action of locust adipokinetic hormone.     

Biosynthesis and secretion of insect lipoprotein.

Biosynthesis of high density lipophorin (HDLp) was studied in larvae and adults of the migratory locust, Locusta migratoria. In an in vitro system, fat bodies were incubated in a medium containing a mixture of tritiated amino acids. Using SDS-PAGE and immunoblotting, it was shown that larval and adult fat bodies secreted both HDLp apoproteins, apolipophorin I (apoLp-I) and apolipophorin II (apoLp-II). Radiolabel was recovered in both apoproteins, indicative of de novo synthesis. The density of the fractions containing the apoproteins synthesized and secreted by larval and adult fat bodies was determined by density gradient ultracentrifugation. A radiolabeled protein fraction was found at density 1.12 g/ml. Using an enzyme-linked immunosorbent assay for detecting apoLp-I and apoLp-II, it was demonstrated that both apoproteins were present in this fraction, which had a density identical to that of circulating HDLp in hemolymph. Lipid analysis revealed that it contained phospholipid, diacylglycerol, sterol, and hydrocarbons. From these results it is concluded that the fat body of the locust synthesizes both apoLp-I and apoLp-II, which are combined with lipids to a lipoprotein particle that is released into the medium as HDLp.     

Site of hemolymph lectin production and its activation in vitro by 20-hydroxyecdysone

To identify the tissues which produce hemolymph lectin in larvae of Bombyx mori, ovary, testis, fat body, and hemocytes from 5th-instar larvae were cultured in vitro and the culture medium was partially purified and assayed for hemagglutinating activity. Among the tissues tested, hemocytes appeared to be a major source of the hemolymph lectins. Ovary produced lectins to about one-tenth of the amount observed for the hemocytes, whereas testis and fat body were not productive. To study the hormonal control of hemolymph lectin production by hemocytes, hemocytes from 4th-instar larvae were cultured in vitro. Hemagglutinating activity in the hemolymph of 4th-instar larvae was immunostainable with the monoclonal antibody raised against 350,000 dalton lectin found in the 5th-instar hemolymph, but their molecular sizes were larger than the 5th-instar hemolymph lectins. When 20-hydroxyecdysone was added into the medium, production of the lectin by the hemocytes was remarkably enhanced, depending upon the hormone concentration.     

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.     

Effects of female wasp accessory secretions, host fat body, and host hemolymph on protein synthesis and egg viability in Microplitis croceipes (Braconidae)

Effects of female wasp reproductive gland secretions, host fat body and hemolymph, and mechanical constriction of the parasitoid egg on protein synthesis were studied in eggs of Microplitis croceipes (Braconidae) dissected from the wasp ovary. Protein synthesis was measured by 35S-methionine incorporation in eggs held in tissue culture medium for 16 h after treatment. Synthesis was stimulated in oocytes obtained from three regions of the ovary (egg tube, reservoir, and calyx) by fat body and venom gland but not by calyx fluid. A combination of fat body, venom gland, and calyx fluid did not enhance the level of synthesis relative to that of fat body or venom gland alone. Host hemolymph inhibited protein synthesis when incubated directly with the dissected eggs but not when the eggs were collected from an artificial oviposition substrate (AOS) containing hemolymph. The inhibitory effect of the hemolymph is thought to be due to the occurrence of melanization. Mechanical constriction did not alter the rate of synthesis, confirming an earlier report that synthesis in newly deposited eggs in ongoing and is not dependent on mechanical activation during the act of oviposition. Mechanisms responsible for sustaining protein synthesis in eggs for 16 h in vitro after their exposure to host hemolymph in the AOSs or fat body and venom gland are not known. Only a small percentage (less than 2%) of dissected ovarial reservoir oocytes that were mechanically constricted and exposed to the venom gland, calyx fluid, and host fat body hatched in vitro. In contrast, an earlier study demonstrated that 38% of eggs oviposited by female wasps into AOSs developed and hatched.     

Manduca sexta lipid transfer particle: Synthesis by fat body and occurrence in hemolymph

Lipid transfer particle (LTP) is present in hemolymph of the tobacco hornworm Manduca sexta. Biosynthesis of LTP, occurrence in hemolymph, and the role of LTP-apoproteins in the lipid transfer reaction were investigated using antibodies specific for LTP or for each of the apoproteins. In vitro protein synthesis followed by immunoprecipitation demonstrated that LTP is synthesized by the fat body and secreted into the medium. In contrast to apolipophorin III, an exchangeable apoprotein of lipophorin (the major lipid transport protein in hemolymph), apoLTP-III could not be detected free in hemolymph. LTP concentrations in the hemolymph were measured by a sandwich ELISA using a mouse monoclonal antibody against apoLTP-III as capturing antibody and rabbit polyclonal antibody against apoLTP-I as detecting antibody. LTP concentration increased during the late fifth instar larval stage, followed by a decrease in the wandering stage. Subsequently, LTP concentrations were strongly increased in hemolymph of adult moths. The role of the three apoproteins of LTP in the lipid transfer reaction was analyzed using apoprotein-specific antibodies. All three, apoLTP-I, -II, and -III, appeared to be important for lipid transfer activity, as shown by inhibition of lipid transfer by antibodies specific for each of the three apoproteins. © 1996 Wiley-Liss, Inc.     

Developmental expression and hormonal regulation of a desiccation stress protein in Tenebrio molitor

In a previous study it has been reported that dsp28 is induced during desiccation in Tenebrio larvae. During that study it was observed that in non-stressed larvae the concentration of dsp28 in hemolymph drops dramatically just prior to pupation. These results suggested that control of dsp28 synthesis is subject to environmental as well as hormonal cues. This study identifies a site of synthesis as fat body, as dsp28 was secreted into the medium during in vitro incubation of larval fat bodies. Using immunoelectrophoresis to determine protein concentration a developmental profile showing changes in levels of dsp28 in hemolymph during larval, pupal and adult stages of Tenebrio molitor was established. The concentration of dsp28 in larval hemolymph dropped from 4 to 5 mg/ml to 1.5 mg/ml just prior to pupation. This lower level was maintained until adults emerged when the concentration of dsp28 rose to prepupal levels again. Hormonal regulation is suggested since application of methoprene to newly-emerged pupae resulted in an increased incorporation of radiolabeled cysteine into dsp28.     

Fat body and hemocyte contribution to the antimicrobial peptide synthesis in <Emphasis Type="Italic">Calliphora vicina</Emphasis> R.-D. (Diptera: Calliphoridae) larvae

Antimicrobial peptides accumulated in the hemolymph in response to infection are a key element of insect innate immunity. The involvement of the fat body and hemocytes in the antimicrobial peptide synthesis is widely acknowledged, although release of the peptides present in the hemolymph from the immune cells was not directly verified so far. Here, we studied the presence of antimicrobial peptides in the culture medium of fat body cells and hemocytes isolated from the blue blowfly Calliphora vicina using complex of liquid chromatography, mass spectrometry, and antimicrobial activity assays. Both fat body and hemocytes are shown to synthesize and release to culture medium defensin, cecropin, diptericins, and proline-rich peptides. The spectra of peptide antibiotics released by the fat body and hemocytes partially overlap. Thus, the results suggest that insect fat body and blood cells are capable of releasing mature antimicrobial peptides to the hemolymph. It is notable that the data obtained demonstrate dramatic difference in the functioning of insect antimicrobial peptides and their mammalian counterparts localized into blood cells’ phagosomes where they exert their antibacterial activity.     

Changes in lipid and carbohydrate metabolism during starvation in adult Manduca sexta

Summary Adult Manduca sexta feed very irregularly in the laboratory, and many adult males never feed. Feeding adults live longer and feeding females lay many more eggs; however, in both feeding (sugar water) and starving adults a decrease of metabolic reserves is observed. Carbohydrates disappear from hemolymph and from fat body. Fat body lipid also decreases, while hemolymph lipid concentration increases strongly in starving adults. The activity of fat body glycogen phosphorylase increases strongly in starving adult M. sexta. The activity of glycogen phosphorylase is correlated inversely with hemolymph sugar concentration. Injected trehalose inactivates glycogen phosphorylase within 2 h, and lowers the hemolymph lipid level within 6 h. In starving adult M. sexta, neither the activation of glycogen phosphorylase nor the increase of hemolymph lipid concentration depends on adipokinetic hormone, since cardiacectomy does not prevent the activation of glycogen phosphorylase nor the increase of hemolymph lipid level.Abbreviations AKH adipokinetic hormone - EDTA ethylenediamine tetraacetate Present address: Department of Biochemistry and Center for Insect Science, The University of Arizona, Tucson, AZ 85721, USA     

Juvenile hormone analog and injection effects on locust hemolymph protein synthesis

In adult female Locusta migratoria, at about day 8 after eclosion, when vitellogenin (Vg) is first produced as a result of induction by juvenile hormone (JH), the intensity of hemolymph protein electrophoretic bands at about 75 kDa and 20 kDa increases sharply, suggesting that JH may induce additional proteins. A major component of the elevated protein is persistent storage protein (PSP; subunit 74 kDa). Administration of the JH analog, methoprene, to precocene-treated adult locusts was followed by a rise in hemolymph levels of PSP but not in apolipophorin III (19 kDa), identified immunochemically and electrophoretically. The synthesis of PSP in adult fat body was confirmed by incorporation of [³H]leucine. At 48 h after treatment with methoprene, Vg synthesis was induced in females (as previously observed) and synthesis of PSP in both sexes was elevated above controls, while synthesis of apolipophorin III was not stimulated. We conclude that in adult locust fat body the synthesis of several proteins responds in different ways to the JH analog: Vg (and a 21 kDa protein described elsewhere) is induced de novo solely in females; PSP (and a 19 kDa protein described elsewhere) is stimulated in both sexes but is not fully JH-dependent; apolipophorin III is not stimulated. In these experiments, methoprene was administered both by injection in mineral oil and topically in acetone. After injection of mineral oil as a vector control, incorporation into secreted proteins was stimulated at 24 h, presumably due to a wound effect; topical application of acetone avoids this effect and is a preferred route for administration of JH analog. © 1992 Wiley-Liss, Inc.     

Identification and characterization of a novel postlarval hemolymph protein from Manduca sexta

The identification, purification and characterization of a new postlarval specific hemolymph protein from Manduca sexta is described. Incorporation of [³⁵S]methionine into Manduca sexta hemolymph proteins in vivo was investigated as a function of development. A major protein band of M_r ≈ 50,000 was highly labeled during the prepupal and adult stage but not in feeding larvae. This postlarval protein (PLP) was isolated from adult male hemolymph and its chemical and immunological properties determined. PLP is a basic protein (pI ～8.6). Electrophoresis under denaturing conditions reveals a subunit M_r ≈ 50,000 while the native protein has an apparent M_r ～ 85,000 by gel permeation chromatography. Anti-PLP serum recognized PLP but not other hemolymph proteins on immunoblots. In vitro translation of fat body mRNA followed by immunoprecipitation revealed that fat body is the site of PLP synthesis. Quantitation of PLP levels in hemolymph throughout development was performed and suggests PLP may play a role in adult development of M. sexta.     

In vitro study of lipid metabolism in the mealworm larval fat body

Lipid metabolism in Tenebrio larval fat body has been studied in vitro. Lipid release required the presence of diluted hemolymph in the incubation medium. This time-dependent release of lipid was strongly stimulated in a dose-dependent manner by Tenebrio corpora cardiaca (CC) extracts or synthetic adipokinetic hormone (AKH I). Furthermore, some glycerol was released when larval fat body was incubated without hemolymph, and this phenomenon was also dose dependent for added CC extracts. Lipid synthesis was estimated in vitro by following the incorporation of radioactivity from [6-¹⁴C] glucose into fatty acids. Lipogenesis occurred in the absence of added carbohydrates in the medium, but it was stimulated by the addition of glucose, and especially trehalose (10 mg ml^?1). Intestinal insulin-like peptide (ILP) also stimulated in vitro lipogenesis in a dose-dependent fashion. We conclude that lipolytic and lipogenetic activities of larval mealworm fat body in vitro are effectively under hormonal control.     

Purification and characterization of a male-specific protein in the hemolymph of the wax moth,Galleria mellonella L.

A male-specific protein (MSP) present only in males was identified from the hemolymph of the wax moth, Galleria mellonella L., by polyacrylamide gel electrophoresis (PAGE) and purified by anion-exchange chromatography. MSP has a native molecular mass of 55 kDa and consists of two 27-kDa subunits. An isoelectric point of MSP was measured to be approximately 5.8. MSP is a glycoprotein that contains 1.7% carbohydrate. The compositional analysis of carbohydrate component indicated a predominance of fructose and glucose. MSP also contains large amounts of asparagine, aspartic acid, glutamine, glutamic acid, and lysine but small amounts of tyrosine, methionine, and tryptophan. Western blot analysis of the hemolymph of each developmental stage indicated that MSP is present in the hemolymph of 8-day-old pupa and adult. Also, results from Western blotting indicated that MSP is not present in the tissues of larvae and of female adults but appears in the fat body of male pupae and adult and testis of adult. The fat body and testis of male pupae and adult were cultured in vitro to trace the place and time of MSP synthesis. The fat body began to synthesize MSP in late pupae and showed active synthesis during the adult stage. The distribution of MSP in the testis was observed by electron microscopic immunogold labeling, using the antibody against MSP. MSP is present between the germinal cysts and is taken up through the basal surface of the seminiferous tubular epithelium. Arch. Insect Biochem. Physiol. 37:257–268, 1998. © 1998 Wiley-Liss, Inc.     

Juvenile hormone action on locust fat body

Production of vitellogenin (Vg) in fat body of adult female Locusta migratoria is abolished by removal or inactivation of the corpora allata and restored by administration of (RS)-methoprene. Juvenile hormone III injection alone has little effect, but when it is injected together with the JH esterase inhibitor OTFP, active Vg synthesis is induced. This supports the assumption that methoprene acts in place of the natural hormone in this system. When fat body from vitellogenic females is maintained in synthetic medium for 48 hr, the proportion of Vg in the secreted protein drops greatly, but when methoprene is present in the medium the proportion of Vg is sustained. When fat body from JH-withdrawn locusts, in which Vg synthesis has declined to zero, is cultured with methoprene, Vg synthesis is re-induced. These results show that the JH analog can act directly on locust fat body to bring about expression of the Vg genes. Experiments to optimize JH action on fat body in vitro are continuing.     

Synthesis and resorption of a humoral chymotrypsin inhibitor, CI-8, by fat body of the silkworm, Bombyx mori

The Bombyx mori hemolymph contains up to 16 chymotrypsin inhibitors (CIs). The present in vitro culture of tissues in Grace's medium indicated that CI-8, which belongs to the largest molecular-size group of CIs with sugar moiety, is synthesized in the fat body and secreted from it during the feeding period. When the fat body from other strain which synthesizes an allelic component (CI-7) instead of CI-8 was incubated in vitro in hemolymph from the strain which has CI-8, the fat body was found to receive CI-8. Thus it was concluded that CI-8, once secreted into the hemolymph, was again sequestered into the fat body after the onset of spinning. Protein granules isolated from the pupal fat body were shown to contain CI-8, indicating that the sequestered CI-8 is present in the protein granules.     

Regulation of hemoglobin synthesis by ecdysterone and juvenile hormone during development of Chironomus thummi (Diptera)

Chironomus thummi contains nine soluble hemoglobins (Hbs) in the larval hemolymph which can be resolved by 12.7% acrylamide gel electrophoresis (pH 8.65). Hemoglobins 2 and 3 are stage specific for the 4th instar and are first detected by day 4 of this stage in vivo, being absent in the 3rd instar. Fat-body cultures in the presence of 3H-delta-aminolevulinic acid and 14C-amino acids synthesize and secrete labelled Hbs, as was assayed by acrylamide gel electrophoresis and immunoprecipitation of Hbs recovered from the culture medium. During development from 3rd instar to pupa, Chironomus fat body undergoes functional changes, being actively involved in Hb synthesis in intermolt periods and inactive with respect to Hb production during molting. The repression of Hb synthesis is reversed following the molt from the 3rd instar to the 4th instar. Metamorphosis is related to a gradual and irreversible loss of Hb synthesis and secretion by the fat body. The treatment of fat body in vitro with ecdysterone inhibits Hb synthesis in tissue from intermolt animals, even in the presence of excess methoprene, a potent juvenile hormone analogue. In contrast, immunoprecipitation of the translation products from a wheat-germ cell-free system, using mRNA from ecdysterone-treated 4th-instar fat body as a template, shows significant synthesis of globins, suggesting that ecdysterone does not affect the amount or template activity of globin messages. Methoprene induces the precocious in vitro synthesis of Hbs 2 and 3 in day-2 4th-instar fat body and enhances all Hb synthesis in the absence of ecdysterone. In vitro treatment with methoprene activates newly molted fat body to synthesize Hbs 2 and 3 in vitro. The process of Hb induction by this analogue is completely inhibited by actinomycin D or ecdysterone. Fat body from animals already exposed to high endogeneous ecdysterone titer are insensitive to treatment with this juvenile hormone analogue. Intermolt larvae normally possess stable Hb mRNA molecules, because actinomycin-D administration in vitro does not affect Hb synthesis for as long as 30 h, whereas it effectively inhibits all RNA synthesis in the fat body. Immunoprecipitation of globin translated in vitro from mRNA from 2-day-old 4th-instar larvae treated in vivo with methoprene shows enhanced synthesis of globins 2 and 3, as compared to controls with no treatment. It is suggested that both juvenile hormone and ecdysterone regulate Hb synthesis in Chironomus; juvenile hormone affecting the activity of Hb genes, and ecdysterone modulating the level of Hb gene expression.     

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。     

Synthesis of adipokinetic hormone (AKH-I) in the locust brain

Methanol extracts of vitellogenic female locust brains contain two factors that inhibit protein synthesis in fat body tissue excised from such individuals. One of these factors (BI) elicits lipid mobilization when injected into adult male locusts. The retention times of BI on an RP-18 column and on an RP-4 column are identical to those of synthetic locust adipokinetic hormone (AKH-I) on each of these columns. Half maximal inhibition of protein synthesis in excised adult locust fat bodies is exerted by 0.05 brain extract equivalents of BI, which is equivalent to activity elicited by 1.5 pmol of AKH-I, as previously determined by AKH-radioimmunoassay. Enzymatic hydrolysis of the N-terminal pyroglutamate, followed by amino acid sequence analysis, indicates that the structure of BI is similar to that of the decapeptide AKH-I synthesized in the glandular lobe of the locust corpora cardiaca (CC). Incorporation of [5-³H]tryptophan into BI of locust brains incubated in vitro indicates that the AKH-I present in the brain is synthesized in situ and is not transported from the CC. Similar incorporation of radiolabel into AKH-I is obtained when excised CC are incubated in vitro.     

Quantitative changes and synthesis of cyanoprotein in whole body and tissues during development of the bean bug,Riptortus clavatus

Changes in biliverdin-binding cyanoprotein content in whole body and tissue extracts during development of nymphal and adult (non-diapause) bean bugs, Riptortus clavatus were analyzed by rocket immunoelectrophoresis (RIE). RIE using anti-CPegg serum can be used to determine the content of CP-A (Cp-1, 2 and 3) and CP-B (CP-4) separately. During the nymphal stage CP content of whole body changes cyclically in each instar. In the first nymphal instar, CPegg is the main CP which disappears during the first-second instar ecdysis. In nymphal bugs from the 2nd to 4th instars only CP-B (CP-4) is detected, and at the beginning of each instar the CP content is very low but increases toward the next ecdysis, after which CP decreases and disappears very rapidly. In the 5th nymphal instar, CP-B is the major CP but CP-A (CP-1, 2 and 3) is also detected. These changes in whole body CP content of 5th instar nymphs are observed in both females and males. The content of total CPs in the 5th instar nymph reaches about 1000 μg in the whole insect. During nymphal-adult ecdysis, nymphal CPs decrease and disappear at day 2 after emergence. In female adults CP-A (CP-1 only) increases rapidly after day 4 of adult emergence, while no CP is detected in male adults. In females CPs were detected only in the fat body, hemolymph and ovary. In the mid-5th-instar nymphs, CPs (CP-A and B) are mainly distributed in the hemolymph. CPs in the Hemolymph decrease during nymphal-adult ecdysis, whereas they increase in the fat body. CPs disappear from both the hemolymph and fat body by 2 days after ecdysis. Subsequently in the adult stage only CP-A increases again in the fat body and ovary. By tracer experiments using [³⁵S]-methionine, the fat body was shown to be the site of CP synthesis. CP-A and B synthetic activity was detected in nymphal females whereas, only CP-A synthesis was observed in adult females, while no CP synthesis was seen in adult males.