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.     

Bacteria-induced protein P4 (hemolin) from Manduca sexta: a member of the immunoglobulin superfamily which can inhibit hemocyte aggregation.

The synthesis of a number of hemolymph proteins is induced in insects in response to bacterial infections. The major induced hemolymph protein in larvae of Manduca sexta is a glycoprotein of Mr = 48,000 known as P4. We have isolated a clone for P4 from a fat body cDNA library constructed from RNA isolated from larvae injected with bacteria. The cDNA has an open reading frame encoding a 411 residue polypeptide with a hydrophobic NH2-terminal sequence, which appears to be a signal peptide. Analysis of the deduced amino acid sequence shows that P4 is a member of the immunoglobulin (Ig) gene superfamily, and is composed largely of four C2 type Ig domains. The M. sexta P4 amino acid sequence is 60% identical with hemolin (P4) from Hyalophora cecropia. The name "hemolin" has also been adopted for the M. sexta P4 protein. Hemolin mRNA levels in fat body begin to increase within 1 h after injection of bacteria into fifth instar larvae and within 4 h after injection of adults. Hemolin associates with the surface of hemocytes and inhibits hemocyte aggregation responses, suggesting a role for the protein in modulating hemocyte adhesion during recognition and response to bacterial infections in insects.     

Changes in the circulating hemocyte population of Manduca sexta larvae following injection of bacteria

A technique for the collection of stable hemolymph from larvae of Manduca sexta has been developed. The method avoids the cell clumping and melanization reactions commonly encountered with insect hemolymph by minimizing contact between hemocytes and surfaces which provoke defensive or repair responses. The circulating hemocyte population of second-day, fifth-instar larvae (2dL5) of M. sexta consisted of 4.5 ± 2.5 × 10⁶ cells/ml (n = 15, range 2–7 × 10⁶ cells/ml) and contained five cell types: prohemocytes, plasmatocytes, granulocytes, spherulocytes, and oenocytoids. Two strains of Pseudomonas aeruginosa which differ in pathogenicity (P11-1 and 9027) and Escherichia coli D31 grew well at 26°C in cell-free hemolymph prepared from naive (nonimmunized) 2dL5 M. sexta. When viable cells of any of the three bacteria were injected into M. sexta larvae, changes in both the total hemocyte count (THC) and differential hemocyte count were observed. Viable bacteria were not required to produce these changes since formalin-killed cells of P. aeruginosa 9027 produced a qualitatively and quantitatively similar response. Following injection of bacteria, the THC increased, reaching a maximal level at 1 hr postinjection, and remained elevated for at least 4 hr after injection. While prohemocytes, plasmatocytes, granulocytes, and spherulocytes all increased in number, 80% of the increased cell population at 1 hr postinjection of bacteria were the latter two cell types. Granulocytes and spherulocytes are cells with recognized defensive capabilities. The increased numbers of these cells in circulation soon after injection of bacteria may confer an advantage on M. sexta larvae in dealing with bacterial infections. This could explain in part the unusual resistance of M. sexta to certain bacterial pathogens.     

Developmental expression of Manduca sexta hemolin.

Hemolin is hemolymph protein that is a member of the immunoglobulin superfamily. Its induced expression after bacterial infection suggests that it functions in the immune response. In this paper, we describe the expression of the Manduca sexta hemolin gene at certain developmental stages in the absence of microbial challenge. Hemolin was present at a very low level in hemolymph of naive larvae until the beginning of the wandering stage prior to pupation, when its concentration in hemolymph increased dramatically. At the same time, hemolin could be found in the fluid contained in the midgut lumen. The appearance of hemolin mRNA in fat body and midgut at the beginning of the wandering stage correlated with the presence of hemolin in the hemolymph and midgut lumen. Hemolin was present in hemolymph through the pupal and adult stages. Hemolin was also present in newly deposited eggs, and persisted in eggs throughout embryonic development. A hemolin cDNA isolated from an adult fat body library had the same sequence as those previously obtained from larval libraries. Hemolin purified from hemolymph of bacteria-injected larvae, from hemolymph of naive wandering stage larvae and adult moths, and from midgut fluid of wandering stage larvae had the same apparent mass, which was consistent with the mass predicted from the hemolin cDNA sequence. Hemolin from hemolymph of wandering stage larvae did not contain any detectable carbohydrate, but hemolin from the hemolymph of bacteria-injected larvae and from naive adult moths was associated with carbohydrate, although of different amounts and composition. These results suggest that a single hemolin gene is developmentally regulated and is also induced when insects are exposed to microbial infection. M. sexta hemolin apparently lacks post-translational covalent glycosylation, but instead is associated under some conditions with non-covalently bound carbohydrates. Arch.     

Manduca sexta serpin-5 regulates prophenoloxidase activation and the Toll signaling pathway by inhibiting hemolymph proteinase HP6

Insect immune responses include prophenoloxidase (proPO) activation and Toll pathway initiation, which are mediated by serine proteinase cascades and regulated by serpins. Manduca sexta hemolymph proteinase-6 (HP6) is a component of both pathways. It cleaves and activates proPO activating proteinase 1 (PAP1) and hemolymph proteinase-8 (HP8), which activates proSpätzle. Inhibitors of HP6 could have the capability of regulating both of these innate immune proteinase cascade pathways. Covalent complexes of HP6 with serpin-4 and serpin-5 were previously isolated from M. sexta plasma using immunoaffinity chromatography with serpin antibodies. We investigated the inhibition of purified, recombinant HP6 by serpin-4 and serpin-5. Both serpin-4 and serpin-5 formed SDS-stable complexes with HP6 in vitro, and they inhibited the activation of proHP8 and proPAP1. Serpin-5 inhibited HP6 more efficiently than did serpin-4. Injection of serpin-5 into larvae resulted in decreased bacteria-induced antimicrobial activity in hemolymph and reduced the bacteria-induced expression of attacin, cecropin and hemolin genes in fat body. Injection of serpin-4 had a weaker effect on antimicrobial peptide expression. These results indicate that serpin-5 may regulate the activity of HP6 to modulate proPO activation and antimicrobial peptide production during immune responses of M. sexta.     

Major hemolymph proteins from larvae of the black swallowtail butterfly,Papilio polyxenes

Three major hemolymph proteins of Papilio polyxenes larvae were isolated and characterized. Density gradient ultracentrifugation of hemolymph resulted in flotation of the major lipoprotein, lipophorin. P. polyxenes larval lipophorin is composed of two apoproteins, apolipophorin-I and apolipophorin-II, plus a mixture of lipids, to give a density of 1.13 g/ml. Immunoblotting experiments using antisera directed against Manduca sexta apolipophorin-I and apolipophorin-II, respectively, revealed cross-reactivity of apoLp-I with Manduca sexta apoLp-I, and apoLp-II with M. sexta apoLp-II. Gel permeation chromatography of the subnatant obtained following density gradient ultracentrifugation revealed the presence of a major protein peak which was shown to contain three major serum proteins, two of which were isolated and characterized. One of these proteins was purified by lectin affinity chromatography. Both proteins have native molecular weights in the range of 450,000 and appear to be hexamers of a single subunit type. Major serum protein-1 is nonglycosylated and has a subunit molecular weight of 75,000. Major serum protein-2 is glycosylated and has a subunit molecular weight of 74,000. Amino acid analysis of this protein revealed a tyrosine plus phenylalanine content of 20 mole percent, characteristic of the arylphorin class of insect storage proteins. Using antibodies against M. sexta larval hemolymph proteins, both the P. polyxenes major serum proteins were shown to be immunologically related to serum proteins of other lepidopteran species.     

Regulation of fat body glycogen phosphorylase activity during refeeding in Manduca sexta larvae

In 12-h-starved larvae of the tobacco hornworm, Manduca sexta, fat body glycogen phosphorylase was quickly inactivated when insects were refed with normal diet and agar which contained 3% sucrose. Only the first 2 min of refeeding were necessary to induce enzyme inactivation. During this short period, larvae did not ingest enough sucrose to increase the hemolymph glucose concentration. This may indicate that the gut released a hormone(s) which directly or indirectly led to the inactivation of fat body glycogen phosphorylase. Inactivation of the enzyme could also be induced by injection of glucose (30 mg) into the hemolymph of starving M. sexta larvae suggesting that there may be separate control from a neuroendocrine site such as the brain or the corpora cardiaca. Trehalose was less effective. Bovine insulin (2 and 4 μg/starved larva) did not induce phosphorylase inactivation over 20 min or decrease hemolymph carbohydrate or lipid concentrations within 60 min. It is, therefore, necessary to screen insect tissues for substances which could bring about inactivation of fat body glycogen phosphorylase. © 1992 Wiley-Liss, Inc.     

The effect of L-canavanine and L-canaline on the hemolymph amino acid composition of the tobacco hornworm,Manduca sexta (L.) (Sphingidae)

Tobacco hornworm larvae, Manduca sexta (L.) (Sphingidae), were administered L-canaline either by parenteral injection or by dietary consumption. The overt toxicity and the alteration of hemolymph amino acids caused by these nonprotein amino acids were evaluated. The LD₅₀ value for parenterally administered canavanine and canaline is 1.0 and 2.5 mg/g fresh body weight, respectively. A dietary concentration of 5.2 mM for canavanine and over 20 mM for canaline represent the respective LC₅₀ values. A large percentage of the larvae reared on diets supplemented with additional arginine, ornithine, or 2,4-diaminobutyric acid in addition to canavanine or canaline were unable to complete larval-pupal ecdysis. These toxic effects were associated with a decreased glutamic acid hemolymph titer and dramatically elevated ornithine. On the other hand, larvae administered canavanine or canaline alone, either by dietary consumption or parenteral injection, experienced less drastic developmental aberrations. These symptoms were in some cases correlated with increased ornithine and glutamic acid titers. Evidence is presented that even a canavanine- and canaline-sensitive insect such as M. sexta has a marked ability to eliminate these protective allelochemicals.     

Studies on binding and uptake of vitellogenin by follicles of the tobacco hornworm,Manduca sexta

An in vitro system for the uptake of ¹²⁵l-vitellogenin (VG) or vitellin into isolated follicles of the tobacco hornworm, Manduca sexta, is described. After incubation with ¹²⁵l-VG, follicles were disrupted and the internal yolk contents separated from the follicle membranes. The results showed that ¹²⁵l-VG was associated principally with the membranes (92%) after incubation at 4°C. However, at 27°C, ¹²⁵l-VG was mainly in the yolk (92%). Furthermore, trypsin treatment removed approximately 70% of VG bound to the follicles at 4°C. Labeled VG was shown to bind to sonicated follicle membranes with high specificity and affinity (K_D ? 1.3 × 10^?8 M). This binding was sensitive to pH and calcium concentration. The total binding sites were estimated at 4 × 10¹⁴ sites/g of membrane protein. Competition studies showed that binding of ¹²⁵l-VG to follicle membranes was blocked by excess unlabeled vitellin and deglycosylated vitellogenin but not by lipophorin (the major hemolymph lipoprotein), microvitellogenin, a female-specific protein (M_r ～ 31,000) found in both hemolymph and eggs, and the smaller vitellogenin subunit, apovitellogenin-II (M_r ～ 45,000). These results suggest that selective uptake of M. sexta VG from the hemolymph involves binding to specific receptors located on the follicle membranes.     

Multicellular-vesicle-promoting polypeptide from Trichoplusia ni: Tissue distribution and N-terminal sequence

An N-terminal amino acid sequence of a 16.9 kDa hemolymph polypeptide, “Vesicle Promoting Factor” (VPF) from Trichoplusia ni, revealed a high sequence homology (70%) with Manduca sexta apolipophorin-III. A polyclonal antibody developed against VPF, however, was not immunoreactive with either purified M. sexta or T. ni apolipophorin-III. Immunoblots of tissue homogenates of T. ni indicated that VPF was present in imaginal wing discs, central nervous system (CNS), silk glands, midgut and hemocytes from fifth instar larvae, and also in the IAL-TND1 cell line which can grow as either fluid-filled multicellular vesicles or multicellular aggregates. VPF was also detected immunologically in the hemolymph of adults of T. ni, and in hemolymph of adults and larvae of Galleria mellonella and Heliothis virescens. Testes, midgut, hemocytes, and wing discs, but not Malpighian tubules, of T. ni released VPF into tissue culture medium during a 3 h incubation period. © 1995 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Isolation and characterization of lipophorin from Drosophila melanogaster larvae

The hemolymph lipoprotein lipophorin has been isolated from third-instar Drosophila melanogaster larvae by a technique that involves homogenization of whole larvae in a medium containing protease inhibitors and purification of the lipoprotein by density gradient centrifugation. Drosophila lipophorin has a density of 1.16 g/ml and is composed of 62.5% protein, 23.1% phospholipid, 7.4% diacylglycerol, 5.4% triacylglycerol, 0.9% hydrocarbon, and 0.7% sterol. As is the case with other insect lipophorins, Drosophila lipophorin contains two apolipoproteins, apolipophorin-I (M_r ≈ 275,000) and apolipophorin-II (M_r ≈ 76,000). Drosophila apolipophorin-I does not crossreact with antibodies prepared against apolipophorin-I from Manduca sexta.     

Mechanistic studies of the degradation of juvenile hormone esterase in Manduca sexta

The mechanisms of degradation of juvenile hormone esterase (JHE) were investigated in larvae of the tobacco hornworm, Manduca sexta. JHE is removed from the hemolymph by the pericardial cells by receptor-mediated endocytosis and is ultimately degraded in the lysosomes. Immunoprecipitation experiments and native PAGE followed by Western blotting showed that JHE associates with a putative heat shock cognate protein (Hsp). Approximately 25% of the active JHE in the pericardial cell complex is associated with the putative Hsp 1 h postinjection of affinity purified JHE. Electron microscope analysis revealed that the putative Hsp is located in the trans-Golgi network of pericardial cells, where it is hypothesized to be involved in sorting of proteins destined for the lysosomes, from those destined for the cell membrane. Data acquired from immunoprecipitation and Western blotting experiments argue against the involvement of ubiquitin in the degradation of JHE. Injection of radiolabeled JHE into larvae of M. sexta followed by SDS-PAGE of pericardial cell homogenates revealed covalent binding of an unidentified protein to JHE in the pericardial cell complex. Arch. Insect Biochem. Physiol. 34:275–286, 1997. © 1997 Wiley-Liss, Inc.     

Biochemical characterization,developmental expression,and induction of the immune protein scolexin from Manduca sexta

The immune protein, scolexin, a bacteria-induced, larva-specific protein from Manduca sexta, was shown to exist in the hemolymph in two isoelectric forms designated herein as scolexin-1 and scolexin-2 (native M_r ～ 72 kd). These two charge isomers appeared to share the same amino acid composition. Scolexin is composed of two subunits (peptide M_r ～ 36 kd) that possess the same N-terminus. Scolexin-2 was subjected to glycosyl composition analysis, revealing the presence of galactose, glucose, mannose, xylose, and sialic acid residues. Hybridization of epidermal RNA with oligonucleotides deduced from the scolexin N-terminal sequence showed a continuous decline in mRNA following day 0 of the 5th larval instar. By employing in vitro protein labelling, it was found that organ cultures of the epidermis from immune larvae showed a greater ability over that of naive epidermal cultures to synthesize scolexin; these data reflected the inducible response seen in the hemolymph, and confirm other data indicating that the epidermis is an important site of scolexin biosynthesis. © 1995 Wiley-Liss, Inc.     

Metabolism of juvenile hormone in cultures of ovaries and fat body in the cockroachperiplaneta Americana

Summary The time course of juvenile hormone (JH) metabolism is examined in cultures ofPeriplaneta americana fat body and ovaries in medium containingManduca sexta carrier protein or cockroach hemolymph. In the absence ofM. sexta carrier protein or cockroach hemolymph, both tissues extensively catabolize exogenous [³H]JH in the medium. Addition of the carrier protein or hemolymph to the culture system prevents the hydrolysis of the hormone in the medium. Within the tissues JH is degraded whether or not carrier protein or hemolymph is present which suggests that the protective role of these molecules is exclusively extracellular. Incubation of [³H]JH with medium preconditioned with tissue results in destruction of the hormone. This suggests that the fat body secretes esterases into the medium. In contrast, the ovarioles hydrolyze the hormone by means of cell-associated enzyme. The relationship of these phenomena to insect development is discussed. This work supported by NSF Grant PCM 76-02229 and University of Kansas Biomedical Sciences Grant RR-07037.     

Mode of action studies on a Manduca sexta diuretic hormone

The mode of action of a diuretic hormone from pharate adult Manduca Sexta heads, which triggers fluid loss in M. sexta larvae and Pieris rapae adults, was studied. In vivo, Mas-DH (M. sexta diuretic hormone) decreased fluid absorption from larval recta, and increased levels of the second messenger cAMP in recta and Malpighian tubules (Mt) from larvae, and in fat body of larvae and adult M. sexta. In vitro, Mas-DH triggered minor changes in fluid loss from adult Mt, but did not affect levels of cAMP in Mt from larvae, pharate adults, or adults, though it elevated cAMP levels in fat body of these stages. © 1992 Wiley-Liss, Inc.     

保幼激素类似物及蜕皮甾类对亚洲玉米螟幼虫酚氧化酶活性的影响

分别用1 μg/头、0.1 μg/头和0.01 μg/头浓度的保幼激素类似物methoprene（蒙五一五）体外处理亚洲玉米螟5龄幼虫,测定幼虫体壁组织、血清和血细胞溶离物中酚氧化酶的活性。结果表明： 1 μg/头 methoprene处理组和0.1 μg/头处理组幼虫体壁组织中酚氧化酶活性与对照组相比有显著提高（P<0.01）,血清和血细胞溶离物中酚氧化酶活性也显著上升（P<0.01）。将含有20-羟基蜕皮酮的人工饲料饲喂亚洲玉米螟5龄幼虫,处理组幼虫体壁组织的酚氧化酶活性下降(P<0.05),血清和血细胞溶离物中的酚氧化酶活性均低于对照组 （P<0.01）。这些结果表明methoprene可以诱导亚洲玉米螟5龄幼虫体内酚氧化酶活性的上升,而20-羟基蜕皮酮则抑制了酚氧化酶的活性。     

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     

Phagocytosis and nodule formation by hemocytes of Manduca sexta larvae following injection of Pseudomonas aeruginosa

The time course of clearance of an injected dose of 10⁶ CFU ml^?1 hemolymph of Pseudomonas aeruginosa ATCC 9027 in larvae of the tobacco hornworm, Manduca sexta, has been examined in detail. The clearance process has been subdivided into three stages during which the rates of reduction in concentration of circulating viable bacteria were clearly different. Contributions of hemocyte reactions to bacterial clearance were examined during stages I and II. During stage I (0–2 hr postinoculation (PI), nodule formation produced a dramatic reduction in circulating bacteria by entrapping over 90% of the injected dose in the first 30 min. Phagocytosis of bacteria by circulating hemocytes and subsequent intracellular digestion contributed significantly to reductions in circulating bacteria during stage II (2–8 hr PI). Viable cells of the virulent P. aeruginosa P11-1 were trapped in nodules as efficiently as the less virulent 9027 during the first 30 min after injection into M. sexta. Bacteria of strain P11-1 were also phagocytosed by hemocytes during stage II, however, phagocytosed bacteria were observed less frequently in P11-1-treated insects and intracellular digestion of these bacteria was only rarely observed. The increased virulence of P11-1 in larvae of M. sexta may be due to less efficient phagocytosis by circulating hemocytes and to insensitivity of this strain to killing reactions in nodules and following phagocytosis.     

Dipteran endoparasitoid Exorista bombycis utilizes antihemocyte components against host defense of silkworm Bombyx mori

Dipteran endoparasitoids avoid host immune response; however, antidefense components from the Dipterans are unknown. Infestation of commercial silkworm Bombyx mori Linnaeus (Lepidoptera: Bombycidae) by endoparasitoid Exorista bombycis Louis (Diptera: Tachinidae) induced immune reactions, cytotoxicity, granulation, degranulation, and augmented release of cytotoxic marker enzyme lactate dehydrogenase (LDH), and degranulation-mediator enzyme β-hexosaminidase in hemocytes. In this study, by reverse phase high-performance liquid chromatography, fractions of E. bombycis larval tissue protein with antihemocytic activity are separated. From the fraction, peptides of hemocyte aggregation inhibitor protein (HAIP) and pyridoxamine phosphate oxidase (PNPO) are identified by mass spectrometry. Interacting partners of HAIP and PNPO are retrieved that further enhance the virulence of the parasitoid. PNPO and HAIP genes showed a four- to seven fold increase in expression in the integument of the parasitoid larva. Together, the dipteran endoparasitoid E. bombycis exploit antihemocyte activity to inhibit host defense reactions in addition to defense evasion contemplated.     

Characterization of a diuretic hormone receptor from the tobacco hornworm,Manduca sexta

We have characterized a diuretic hormone receptor from the tobacco hornworm, Manduca sexta. A single high affinity binding site for the 41 amino acid M. sexta diuretic hormone was found in membranes prepared from Malpighian tubules of fifth stadium larvae. The site has a Kd = 79 pM and Bmax = 3.1 pmol/mg protein. The dissociation rate constant was determined to be 0.11 min^?1 with a corresponding half-life of 6.4 min. Receptor binding of the hormone is inhibited by Ca²⁺ and Mg2⁺, while Na⁺ and K⁺ inhibit binding to a lesser extent. Truncated diuretic hormone analogs in which up to 20 amino acids were removed from the N-terminus maintain high affinity for the receptor. A diuretic hormone from Locusta migratoria which has 43% sequence identity with the M. sexta diuretic hormone also possesses a high affinity for the receptor. Conformational analysis of the M. sexta diuretic hormone indicates the core region of the peptide assumes a helical conformation, which may have implications in the binding of the peptide to the receptor. © 1993 Wiley-Liss. Inc.