High molecular transport proteins for JH-III in insect hemolymph

Summary Using two independent techniques, ultracentrifugation in a KBr-gradient, and native pore polyacrylamide gel electrophoresis in combination with [³H]-epoxyfarnesyldiazoacetate photoaffinity labeling, we showed that in the hemolymph ofPeriplaneta americana, and probably also inLeptinotarsa decemlineata JH-III binds to the lipophorin, whereas inLocusta migratoria JH-III binds to a different protein.     

Immunochemical similarities among the hemolymph juvenile hormone binding proteins of moths

The hemolymph from various species of moths was analyzed for cross-reactivity with a panel of six monoclonal antibodies made against the hemolymph juvenile hormone binding protein ofManduca sexta. With the exception of one antibody, the immunoreactivity was limited to the sphingid family. One monoclonal antibody cross-reacted with a number of lepidopteran species; however, families such as Noctuidae and Pyralidae, known to have high affinity, low molecular weight juvenile hormone binding proteins, did not cross-react. Immunological cross-reactivity withManduca sexta juvenile hormone binding protein in several primitive moth families supports the current model of phylogenetic relationships in the order Lepidoptera.     

Juvenile hormone titre and regulation in the cockroachDiploptera punctata

Summary Titres of juvenile hormone (JH) have been determined in both hemolymph and whole body extracts of femaleDiploptera punctata during the first gonotrophic cycle using a method employing gas chromatography/mass spectrometry for qualitative and quantitative analysis. JH III is the sole JH found in both adult and last instarD. punctata. Maximum values of 1500 ng/ml (6M) were observed at the middle of the gonotrophic cycle, when basal oocyte growth rate was greatest. Changes in rates of JH release in vitro by corpora allata paralleled closely the changes in JH titre, suggesting that biosynthesis is a major regulator of titre. JH levels per animal were calculated from observed JH titres, and at certain time points in the gonotrophic cycle JH levels obtained from analysis of whole bodies were significantly greater than those predicted from hemolymph titres. These results suggest the existence of a nonhemolymph JH pool inD. punctata. Decay in JH titre after allatectomy of 5 day females has also been studied. Following a rapid initial decline, the rate of decay slowed appreciably 4 h post-operation. Thus, use of a first-order rate constant to estimate half-life of JH significantly underestimated the longevity of the hormone. The apparent persistence of JH following allatectomy may be due to the existence of a nonhemolymph JH pool.     

Transesterification of juvenile hormone occurs in vivo in locust when injected in alcoholic solvents

Catabolism of juvenile hormone was studied in vivo in the African locust by injection of the labelled natural enantiomer (10R) (12-³H) JH-III. Due to the poor solubility of JH in aqueous solution, it was injected in a water-miscible solvent. Ethanol was chosen for its apparently low toxicity towards the locust. In these experimental conditions, reverse phase liquid chromatography procedure (RP-HPLC) coupled with on line radiodetection, revealed an apolar metabolite of JH-III. This compound was found both in adult females and in fifth stadium larvae. We demonstrate that this metabolite resulted from substitution of the carboxyl methyl group of JH-III by some hydrophobic moiety. This compound co-migrates in our RP-HPLC system with the JH analog epoxy-ethyl farnesoate (JH-III ethyl ester) obtained by KCN-catalysed transesterification of JH-III in ethanol. Both JH-III ethyl ester of chemical origin and biological compounds extracted from locusts give the same spectra when analyzed by gas chromatography-mass spectroscopy (GC-MS). Transesterification of JH-III was not observed with locust tissues incubated in vitro but occurred in vivo even if JH was injected in other alcoholic solvents such as propanol. Our data suggest that transesterification of JH-III occurred in vivo and underline the role of injecting solvent in in vivo studies.     

Effect of the juvenoid methoprene on the hemolymph composition of the cabbage maggotDelia radicum (Diptera: Anthomyiidae)

Summary Treatment of post-feeding larvae of the cabbage maggotDelia radicum with methoprene did not affect the capacity of the insect to pupate, but suppressed eclosion to the adult stage. The concentration of hemolymph trehalose was significantly decreased by methoprene treatment, although hemolymph protein and amino nitrogen levels were unaffected.19 December 1986The authors are grateful to D. C. Read of the Research Centre, Agriculture Canada, Charlottetown, P.E.I., Canada for supplying eggs ofD. radicum for the stock colony. TheD. radicum colony was maintained at the Agriculture Canada Research Station, St. John's West, Newfoundland, Canada. The sample of methoprene was donated by Zoecon Corp., Palo Alto, California, U.S.A. This research was supported by a grant from the Natural Sciences and Engineering Research Council of Canada (Operating grant A6679).     

Specificity of binding of juvenile hormone III to hemolymph proteins ofLeptinotarsa decemlineata andLocusta migratoria

Summary Binding specificity of juvenile hormone (JH) III enantiomers and analogs to hemolymph proteins ofLeptinotarsa decemlineata andLocusta migratoria was investigated by competitive displacement tests. The order of binding affinity was 10R-JH-III>10R, 10S-JH-III10S JH-III> methylfarnesoate for analogs of the epoxide group and diazo-JHA-IV>EFDA for analogs of the methylester. Both the epoxide and ester groups are important for the interaction of JH-III with its binding protein.18 November 1986     

Juvenile hormone titre and regulation in the cockroach Diploptera punctata

Titres of juvenile hormone (JH) have been determined in both hemolymph and whole body extracts of female Diploptera punctata during the first gonotrophic cycle using a method employing gas chromatography/mass spectrometry for qualitative and quantitative analysis. JH III is the sole JH found in both adult and last instar D. punctata. Maximum values of approximately 1500 ng/ml (approximately 6 microM) were observed at the middle of the gonotrophic cycle, when basal oocyte growth rate was greatest. Changes in rates of JH release in vitro by corpora allata paralleled closely the changes in JH titre, suggesting that biosynthesis is a major regulator of titre. JH levels per animal were calculated from observed JH titres, and at certain time points in the gonotrophic cycle JH obtained from analysis of whole bodies were significantly greater than those predicted from hemolymph titres. These results suggest the existence of a nonhemolymph JH pool in D. punctata. Decay in JH titre after allatectomy of 5 day females has also been studied. Following a rapid initial decline, the rate of decay slowed appreciably 4 h post-operation. Thus, use of a first-order rate constant to estimate half-life of JH significantly underestimated the longevity of the hormone. The apparent persistence of JH following allatectomy may be due to the existence of a nonhemolymph JH pool.     

Juvenile hormones inThermobia domestica females: Identification and quantification during biological cycles and after precocene application

Summary JH I and JH III immunoreactive substances were detected in the hemolymph of imaginal females of the primitive insectThermobia domestica. Periodic changes in the levels of these hormones were investigated in correlation with molting and reproductive cycles in inseminated, virgin and precocene-treated females. The presumed influence of JH III on the various phases of vitellogenesis is discussed, also taking into account the periodic changes of the hemolymphatic ecdysteroid levels.     

Degradation of pheromone biosynthesis-activating neuropeptide (PBAN) by hemolymph enzymes of the tobacco hornworm,Manduca sexta,and the corn earworm,Helicoverpa zea

The tritium-labeled bis-norleucine analog ofHelicoverpa zea pheromone biosynthesis-activating neuropeptide ([³H]NLPBAN) was incubated in vitro with hemolymph fromManduca sexta orH. zea adult females. The incubations resulted in the formation of several tritium-labeled degradation products. At a [³H]NLPBAN concentration of 0.9 μM the degradation proceeded at a very slow but physiologically plausible rate (2–10 fmol/min/μl hemolymph). The primary [³H]NLPBAN degradation reaction inM. sexta hemolymph was not inhibited by 20 μM leupeptin, 0.1 mM amastatin, 1 mM EDTA, 1 mM EGTA, 1 mM 1,10-phenanthroline, or 2 mM 4-(2-aminoethyl)benzenesulfonyl fluoride; but secondary reactions may have been affected, as some of the inhibitors changed the radio-HPLC profile of the degradation products. It is concluded that hemolymph ofM. sexta andH. zea contains peptidase(s) capable of inactivating circulating PBAN.     

Dehydroepiandrosterone sulfate-binding sites in plasma membrane from human uterine cervical fibroblasts

Dehydroepiandrosterone sulfate (DHA-S) plays a critical role in cervical dilation at labor. Incubation of cervical fibroblasts with [³H]DHA-S caused a rapid and saturable increase in cellular radioactivity: an apparent equilibrium was reached by 2 min. There was no detectable conversion of DHA-S into DHA or oestradiol. When the fibroblasts loaded with [³H]DHA-S were homogenized and fractionated, the specific radioactivity in the plasma membrane fraction was enriched approximately 8- to 9-fold compared with the whole homogenate; only low amounts of radioactivity were observed in the other subcellular fractions. The binding of DHA-S to plasma membrane preparations showed saturation kinetics with an apparent equilibrium dissociation constant (K _d) of 12 nM, and the binding capacity (B _max) was calculated to be 1.25 fmol/mg protein. Neither DHA nor oestrone sulfate affected [³H]DHA-S binding to the plasma membrane. The plasma membranes of skin fibroblasts did not show specific binding sites for DHA-S. These findings demonstrate the presence of specific binding sites for DHA-S in the plasma membrane of cervical stroma cells. The fetal adrenal steroid may exert its action on cervical ripening at least in part through membrane-associated binding sites, or receptors.     

Carboxylesterases of high molecular weight in the hemolymph ofLocusta migratoria

Summary The main carboxylesterase in the hemolymph of the migratory locust,Locusta migratoria, is a protein of high molecular weight; about 700–750 kDa. This esterase hydrolyzes juvenile hormone III, -naphthylacetate and -naphthylacetate. The carboxylesterase dissociates to give an esterase of molecular weight 148 kDa after treatment of the hemolymph with mercaptoethanol.     

Crystal cell pattern modification in a melanotic tumor strain ofDrosophila melanogaster

Summary 3rd instar larvae of the melanotic tumortu-pb strain ofDrosophila melanogaster hold a lower number of free-circulating crystal cells in their hemolymph than the wild type ones. This pattern could result from an abnormal retention of mature crystal cells in the hematopoietic organs, as the strong hemocyte melanization inside the lymph glands of heat-treatedtu-pb larvae seems to demonstrate. Melanotic tumor formation and modification of the crystal cell pattern may be related.     

A specific protein in the genusRhynchosciara (Diptera,Sciaridae)

Summary Proteins immunologically related toRhynchosciara americana larval protein 10 occur in the hemolymph and ovaries of five different fly species of the genusRhynchosciara. Electrophoretic analyses showed these proteins to have a mol.wt similar to that of theR. americana protein 10 (43,000), e.g. theR. hollanderi protein 44,300, theR. milleri protein 45,500.     

Blockade by zinc ions of K+-induced contraction and calcium in guinea pigTaenia coli

Preincubation with 0.3 mM Zn²⁺ markedly inhibited both the tonic response and Ca²⁺ binding at low affinity sites induced by K⁺ (60 mM), with smaller effects on the phasic response and the high affinity Ca²⁺ sites, inTaenia coli. However, when the muscle was kept in Zn²⁺-containing medium following the first stimulation with the K⁺, the phasic response and the high affinity Ca²⁺ sites were more severely inhibited during the second stimulation with K⁺. This probably indicates that Zn²⁺ reduced the tonic tension response to K⁺ mainly by inhibiting Ca²⁺ influx at the cell membranes ofTaenia coli. However, when Zn²⁺ is continuously present, Ca²⁺ is not supplied at the storage sites and is not available for the phasic response to a second stimulation with K⁺.     

Endogenous suppression of pheromone production in virgin female moths

Summary Sexual receptivity generally is reduced in moths after mating. We found that even in virginHeliothis zea females the titer of pheromone declines after the third night of adult life, although the number of eggs laid increases. Reduction in pheromone titer is not due to reduced amounts of pheromone biosynthesis activating neuropeptide. We have discovered that a substance present in the bursa, ovaries and hemolymph of senescing virgins suppresses pheromone production. A similar factor was found in 2-day-old mated females indicating that both virgin and mated females use this factor to suppress pheromone production.     

Cholinergic control of extracardiac pulsations in insects

Summary The autonomic nerve system which controls extracardiac pulsations of hemolymph pressure appears to be quite susceptible to cholinesterase inhibitors (physostigmine, organophosphorus insecticides). In pupae ofTenebrio molitor L. andGalleria mellonella L., the treatment induced large peaks in hemolymph pressure (over 4 kPa). The peaks were repeated at more-or-less constant, 30-s intervals for several hours or days. Removal of the pupal brain neither affected extracardiac pulsations nor influenced the specific responses to the investigated drugs or insecticides.     

Zinc ions block the intracellular calcium release induced by caffeine in guinea-pig taenia caeci

Zn²⁺ in low concentrations (0.005–0.1 mM) inhibited the transient contractions in response to caffeine (25 mM) in a dose-dependent manner in smooth muscle of intact guinea-pig taenia caeci. At Zn²⁺ concentrations higher than 0.1 mM, caffeine did not elicit any response. After saponin-treatment of the fibres, which leaves the Ca²⁺ storage sites intact, caffeine contraction was completely inhibited by Zn²⁺ at a relatively low concentration (0.03 mM). However, in Triton-X-100-treated fibres, in which the Ca²⁺ release sites are destroyed, the contraction could be induced in the presence of Zn²⁺ by an increase in Ca²⁺. In conclusion, Zn²⁺ can block the intracellular Ca²⁺ release from caffeine-sensitive release sites in taenia caeci.     

Hormonal levels and protein variations during sexual maturation ofSchistocerca gregaria; effect of rearing temperature

Summary As a result of the decrease of diurnal rearing temperature from 33 to 28°C the following phenomena were induced in adults ofSchistocerca gregaria: a) In females, a delay in the appearance of maximal levels of JH III and ecdysteroids in the hemolymph, a slowing down of oocyte growth, and an accumulation of hemolymph proteins; b) in males, a decrease of hemolymphatic JH III levels without changes in protein levels.     

Why isPyrrhocoris apterus insensitive to precocene II?

Summary Precocene II (P II) was applied to the adult females of 2 Pyrrhocorid bugs —Pyrrhocoris apterus (insensitive to P II) andDysdercus cingulatus (sensitive to P II)-subjected to allatectomy and intraspecific or interspecific reimplantations of corpus allatum (CA). The failure of P II to inhibit ovarian development inP. apterus appears to be caused by both a low sensitivity to P II of the CA itself and unknown anti-precocene mechanisms outside the CA.Precocene II was kindly supplied by Zoecon.     

The enteric neural receptor for 5-hydroxytryptamine

Summary An enteric neural receptor for serotonin (5-HT) has been characterized. This receptor was assayed, using³H-5-HT as a radiologand, by rapid filtration of isolated enteric membranes and by radioautography. In addition, intracellular recordings were made from ganglion cells of the myenteric plexus. High affinity, saturable, reversible, and specific binding of³H-5-HT was demonstrated both to membranes of the dissected longitudinal muscle with adherent myenteric plexus and the mucosa-submucosa. Radioautographs showed these³H-5-HT binding sites to be in myenteric ganglia and in a broad unresolved band at the mucosal-submucosal interface. Antagonists active at receptors for other neurotransmitters than 5-HT, at either of the two known types of CNS 5-HT receptor, and at 5-HT uptake sites on serotonergic neurons failed to inhibit binding of³H-5-HT. The structural requirements of analogues for binding to the enteric 5-HT receptor matched the known pharmacology of M or neural 5-HT receptors. A novel 5-HT antagonist was found. This compound, N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide (5-HTP-DP), antagonized the action of 5-HT on type II/AH cells of the myenteric plexus but did not affect the release or actions of acetylcholine (nicotinic or muscarinic) or substance P. 5-HTP-DP was also an equally potent displacer of³H-5-HT from its binding sites on enteric membranes. It is concluded that the sites responsible for specific binding of³H-5-HT are enteric M or neural 5-HT receptors. These receptors differ from those now known to be present in the CNS.