Storage proteins are present in the hemolymph from larvae and adults of the Colorado potato beetle.

The protein composition of larval and adult hemolymph from the Colorado potato beetle, Leptinotarsa decemlineata, was investigated and some abundant, high molecular weight proteins were identified and characterized. Diapause protein 1, which occurs in the hemolymph of last instar larvae and short-day adults, appeared to be a storage protein. This protein dissociated into two bands due to the high pH used in nondenaturing gels. Its quaternary structure was established by chemical crosslinking. It appeared to be a hexamer. Diapause protein 1 is composed of approximately 82,000 subunits. The amino acid composition and N-terminal sequence of this protein has been determined. Specific antibodies against diapause protein 1 have been developed. Topical application of 1 microgram pyriproxyfen, a juvenile hormone analog, to last instar larvae and short-day adults suppressed the appearance of this protein in the hemolymph. Pyriproxyfen prematurely induced vitellogenin, when applied to last instar larvae. A larval specific protein was also identified in the hemolymph. Its temporary appearance in the hemolymph of last instar larvae, its subunit composition (M(r) approximately 82,000) and its suppression by pyriproxyfen suggests that this protein is a storage protein as well.     

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

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

Basic isoforms of hemolymph storage proteins expressed during larval metamorphosis

The hemolymph of metamorphosing, final instar larvae of Trichoplusia ni was analyzed for the presence of basic forms of normally positively charged storage proteins. Basic forms of arylphorin and a normally acidic juvenile hormone suppressible protein were identified. For each of these two proteins, variation was observed in the immunoreactivity of forms with different basic charges, where the antisera had been generated against acidic forms of each protein. A basic protein of high molecular size (ca. M, 150,000) was identified that cross-reacted specifically with an antiserum raised against a normally basic, M 74,000 juvenile hormone suppressible protein in the hemocyanin superfamily. © 1993 Wiley-Liss, Inc.     

Evidences for a stage-specific juvenile hormone binding protein in the hemolymph of the silkworm,Bombyx mori L.: Identification and characterization by photoaffinity labeling and immunological analyses

Two molecular forms of juvenile hormone binding proteins were identified in the larval hemolymph of Bombyx mori by photoaffinity labeling. One form having an Mr of 33 kDa was present constantly in the hemolymph of the third to the fifth instar larvae while the other form having an Mr of 35 kDa was detected in the hemolymph until in the early fifth instar larvae but not in the prewandering larvae and prepupae. A 33 kDa binding protein was purified by hydrophobic interaction chromatography, gel filtration, and native PAGE. Antiserum against 33 kDa binding protein cross-reacted with 35 kDa binding protein on Western blots, suggesting that these binding proteins shared the same epitopes. From the results of saturation binding assays, it was inferred that 33 and 35 kDa binding proteins had a similar binding affinity for JH 1. It was revealed that one of these binding proteins, 35 kDa binding protein, was produced in the fat body in a stage-specific manner: fat body of the early fifth instar larvae synthesized both 33 and 35 kDa binding proteins while that of prewandering larvae synthesized only 33 kDa binding protein. © 1996 Wiley-Liss, Inc.     

Photoaffinity labeling and biochemical characterization of binding proteins for pheromones,juvenile hormones,and peptides

Radiolabeled photoaffinity analogs can be used to purify and characterize proteins involved in pheromone perception, juvenile hormone (JH) action, and neuropeptide reception. Several photoaffinity analogs and purification strategies are described for each of these physiological targets. First, a diazoacetate photoaffinity label is used to selectively modify the pheromone binding protein of the gypsy moth, Lymantria dispar. Reverse-phase HPLC is then employed to fractionate the male antennal proteins. Second, a tandem procedure involving preparative isoelectric focusing (IEF) and ion-exchange (IEX) HPLC is employed for the purification of the Manduca sexta hemolymph juvenile hormone binding protein (JHBP), which has now been cloned and sequenced. A separate application of this strategy for the purification of the 29 kDa JH I/methoprene receptor proteins from epidermal nuclei of M. sexta larvae is outlined. A new photolabel, farnesyl diazoketone, has been employed for the characterization of crustacean hemolymph methyl farnesoate binding proteins. Third, the development of neuropeptide photoaffinity labels is described for two systems: (1) the red pigment concentrating hormone (RPCH) of shrimp and (2) the allatostatins isolated from the brain of the cockroach Diploptera punctata.     

Identification of specific interaction of juvenile hormone binding protein with isocitrate dehydrogenase

Juvenile hormone (JH) is essential for multiple physiological processes: it controls larval development, metamorphosis and adult reproduction. In insect hemolymph more than 99 % of JH is bound to juvenile hormone binding protein (JHBP), which protects JH from degradation by nonspecific hydrolases and serves as a carrier to supply the hormone to the target tissues. In Galleria mellonella hemolymph, JHBP is found in a complex with lipid-binding high molecular weight proteins (HMWP) and this interaction is enhanced in the presence of JH. In this report, we present studies on the interaction of JHBP with low molecular weight proteins (LMWP) in the hemolymph. Using ligand blotting we found that JHBP interacts with a protein of about 44 kDa. To identify the protein that preferentially binds JHBP, a LMWP fraction was applied to a Sepharose-bound JHBP and, after washing, the column was eluted with free JHBP acting as a specific competitor or with carbonic anhydrase as a negative control. The eluted proteins were separated by SDS/PAGE and analyzed by mass spectrometry. Isocitrate dehydrogenase was identified as a component of the supramolecular complex of JHBP with hemolymph proteins.     

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.     

A zymogen form of masquerade-like serine proteinase homologue is cleaved during pro-phenoloxidase activation by Ca2+ in coleopteran and Tenebrio molitor larvae.

To elucidate the biochemical activation mechanism of the insect pro-phenoloxidase (pro-PO) system, we purified a 45-kDa protein to homogeneity from the hemolymph of Tenebrio molitor (mealworm) larvae, and cloned its cDNA. The overall structure of the 45-kDa protein is similar to Drosophila masquerade serine proteinase homologue, which is an essential component in Drosophila muscle development. This Tenebrio masquerade-like serine proteinase homologue (Tm-mas) contains a trypsin-like serine proteinase domain in the C-terminal region, except for the substitution of Ser to Gly at the active site triad, and a disulfide-knotted domain at the amino-terminal region. When the purified 45-kDa Tm-mas was incubated with CM-Toyopearl eluate solution containing pro-PO and other pro-PO activating factors, the resulting phenoloxidase (PO) activity was shown to be independent of Ca2+. This suggests that the purified 45-kDa Tm-mas is an activated form of pro-PO activating factor. The55-kDa zymogen form of Tm-mas was detected in the hemolymph when PO activity was not evident. However, when Tenebrio hemolymph was incubated with Ca2+, a 79-kDa Tenebrio pro-PO and the 55-kDa zymogen Tm-mas converted to 76-kDa PO and 45-kDa Tm-mas, respectively, with detectable PO activity. Furthermore, when Tenebrio hemolymph was incubated with Ca2+ and beta-1,3-glucan, the conversion of pro-PO to PO and the 55-kDa zymogen Tm-mas to the 45-kDa protein, was faster than in the presence of Ca2+ only. These results suggest that the cleavage of the 55-kDa zymogen of Tm-mas by a limited proteolysis is necessary for PO activity, and the Tm-mas is a pro-PO activating cofactor.     

Development and partial characterization of monoclonal antibodies to the hemolymph juvenile hormone binding protein of Manduca sexta

Murine monoclonal antibodies were made against the hemolymph juvenile hormone binding protein (JHBP) of Manduca sexta. Binding studies in conjunction with Western blot analysis of native and sodium dodecyl sulfate gels confirmed that antibodies from 10 hybridoma lines interacted with the juvenile hormone binding protein. The pattern of cross-reactivity among the hybridoma lines suggests that different epitopes are recognized. The cross-reactivity pattern for monoclonal antibody 9 suggested a common epitope in three different hemolymph proteins: JHBP, insecticyanin and a 40–45 kDa protein. Western blot analysis of a two-dimensional gel using monoclonal antibody 6 revealed interaction with JHBP and with several proteins that may be precursors or degradation products of the binding protein. An enzyme-immunoassay was developed that detects JHBP in the hemolymph at nanogram levels.     

Cloning,partial purification and in vivo developmental profile of expression of the juvenile hormone epoxide hydrolase of Ctenocephalides felis

cDNAs encoding two different epoxide hydrolases (nCfEH1 and nCfEH2) were cloned from a cDNA library prepared from the wandering larval stage of the cat flea, Ctenocephalides felis. Predicted translations of the open reading frames indicated the clones encoded proteins of 464 (CfEH1) and 465 (CfEH2) amino acids. These proteins have a predicted molecular weight of 53 kDa and a putative 22 amino acid N-terminal hydrophobic membrane anchor. The amino acid sequences are 77% identical, and both are homologous to previously isolated epoxide hydrolases from Manduca sexta, Trichoplusia ni, and Rattus norvegicus. Purification of native juvenile hormone epoxide hydrolase (JHEH) from unfed adult cat fleas generated a partially pure protein that hydrolyzed juvenile hormone III to juvenile hormone III-diol. The amino terminal sequence of this;50-kDa protein is identical to the deduced amino terminus of the protein encoded by the nCfEH1 clone. Affinity-purified rabbit polyclonal antibodies raised against Escherichia coli-expressed HisCfEH1 recognized a approximately 50-kDa protein present in the partially purified fraction containing JHEH activity. Immunohistochemistry experiments using the same affinity-purified rabbit polyclonal antibodies localized the epoxide hydrolase in developing oocytes, fat body, and midgut epithelium of the adult flea. The presence of JHEH in various flea life stages and tissues was assessed by Northern blot and enzymatic activity assays. JHEH mRNA expression remained relatively constant throughout the different flea larval stages and was slightly elevated in the unfed adult flea. JHEH enzymatic activity was highest in the late larval, pupal, and adult stages. In all stages and tissues examined, JHEH activity was significantly lower than juvenile hormone esterase (JHE) activity, the other enzyme responsible for JH catalysis.     

Juvenile hormone esterase is a biochemical anti-juvenile hormone agent

Juvenile hormone esterase, purified by affinity chromatography from the larval hemolymph of Manduca sexta in the fifth stadium, was injected into larvae of the same species in the earlier stadia resulting in a blackening of the cuticle following ecdysis to the next larval stadium. This anti-juvenile hormone response was dose-dependent for an injection in the second, third or fourth stadium. Cuticular blackening was prevented by treating larvae with the juvenoid epofenonane. Larval response to injected juvenile hormone esterase also varied with the time of injection within a single stadium, having a maximum effect for injections at the time of head capsule slippage. Juvenile hormone esterase activity measured from the hemolymph after injection of larvae in the second stadium decreased over an 11 h time-course. Because the anti-juvenile hormone effects resulting from a single injection of juvenile hormone esterase were dependent on the time of injection, it appears that when juvenile hormone biosynthesis is active in the insect, the duration of enzyme activity limits the anti-juvenile effects that can be induced.     

Purification and characterization of biliverdin-binding vitellogenin from the hemolymph of the common cutworm,Spodoptera litura

Biliverdin-binding vitellogenin (Vg) was purified from adult female hemolymph of the common cutworm, Spodoptera litura, by using gel filtration and ion exchange chromatographies. The molecular mass of the protein was 490 kDa and it was composed of two 188-kDa subunits. Three internal amino acid sequences obtained by digestion of the protein with lysylendopeptidase showed high similarity to those of Bombyx mori Vg, supporting the purified blue protein to be vitellogenin. latroscan analyses demonstrated the presence of biliverdin in Vg that occupied 2.4% of total lipid components. Among the lipids of Vg (9.5 micrograms total lipids per 100 micrograms protein), diacylglycerol was the most predominant, followed by phospholipid, hydrocarbons, and then triacylglycerol, while in biliverdin-binding proteins (BPs) purified from larval hemolymph (3.1 micrograms total lipids per 100 micrograms protein), phospholipid was the most abundant lipid followed by diacylglycerol; hydrocarbons and triacylglycerol were minor components. Vg was first detected in the hemolymph of female pupae one day before eclosion, but injection of 5 micrograms of methoprene into a 3-day-old pupa induced Vg in the hemolymph 4 days earlier than in the control. Methoprene also induced a faster decline in BP-A and BP-B titers in the hemolymph with a corresponding increase of the Vg titer. These results suggest that juvenile hormone (JH) induces not only vitellogenesis but also the uptake of these proteins by stimulating the metamorphosis of fat body during the pupal stage.     

Lipophorin of female Blattella germanica (L.): characterization and relation to hemolymph titers of juvenile hormone and hydrocarbons

High density lipophorin (HDLp) from the hemolymph of the German cockroach, Blattella germanica (L.) (Family Blattellidae), has an apparent molecular weight of 670kDa, with an isoelectric point of 7.0 and a density of 1.109g/ml. It is composed of two subunits, apolipoprotein-I (212kDa) and apolipoprotein-II (80kDa), and consists of 51.4% lipid, 46.2% protein and 2.4% carbohydrate. Hydrocarbons constitute 42.2% of the total lipids which also contain diacylglycerol, cholesterol and phospholipid. Lipophorin is rich in the amino acids glutamic acid, aspartic acid, lysine, valine, and leucine. Specificity of a polyclonal antibody was demonstrated by Western blotting and Ouchterlony immunodiffusion: the antiserum recognized native HDLp and apolipoprotein-I, but not apolipoprotein-II, purified vitellin, or other hemolymph proteins. It also recognized a protein in the hemolymph of Supella longipalpa (Blattellidae) but did not cross-react with hemolymph proteins from Periplaneta americana (Blattidae) or Diploptera punctata (Blaberidae). An enzyme-linked immunosorbent assay was developed to measure the HDLp titer in the hemolymph of adult females. The titer of HDLp, a juvenile hormone binding protein, exhibited no clear relationship to the changing titer of juvenile hormone in hemolymph. The hemolymph titer of hydrocarbon, which is also carried by HDLp, showed some functional relation to the concentration of HDLp in the hemolymph. Because it concurrently serves multiple functions in insect development and reproduction, lipophorin titer might covary with the titers of lipid ligands that occur at high concentrations and require extensive shuttling through the hemolymph.     

Expression of a parasitism-specific protein in lepidopteran hosts of Chelonus sp.

The hemolymph of each noctuid species successfully parasitized by Chelonus near curvimaculatus possessed a parasitism-specific protein (PSP) previously identified in host T. ni (Insect Biochem. 19:445; 21:845). Expression of PSP occurred in a stage-specific manner in the stadium during which the host undergoes precocious metamorphosis. The appearance of the protein was not due to nutritional stress associated with parasitism of hosts, since starved nonparasitized larvae did not produce the protein, or to low juvenile hormone titers occurring in precociously metamorphosing hosts, but rather was dependent on the presence of the endoparasite larva. Results of in vivo incorporation experiments with [³⁵S]-methionine showed that synthesis and subsequent appearance of the protein in the hemolymph of parasitized hosts was abrogated by prior surgical removal of endoparasite. Immunoprecipitation analysis of proteins from C. near curvimaculatus larvae cultured in vitro using antibodies specific to PSP indicated that the source of the protein was the endoparasite. Synthesis of PSP by the endoparasitic larvae with its subsequent secretion into the hemocoel of hosts was specific to the advanced stages of parasite development prior to its egression from the host. © 1993 Wiley-Liss, Inc.