Proteins of the fat body of non-diapausing and diapausing larvae of the southwestern corn borer, Diatraea grandiosella: Effect of juvenile hormone

The proteins of the fat body of non-diapausing, pre-diapausing, and newly-diapaused larvae of the southwestern corn borer, Diatraea grandiosella, were examined. Since a low titre of juvenile hormone (JH) is present in the haemolymph throughout the final instar of non-diapausing larvae, the hormone does not appear to stimulate the pre-metamorphic synthesis of proteins. In contrast, the high titre of JH in the haemolymph during the final instar of pre-diapausing larvae appears to stimulate the synthesis of selected proteins. For example, pre-diapausing larvae store in their fat body a low molecular weight protein which has been named the ‘diapause-associated protein’. When non-diapausing larvae were treated topically with C₁₇-JH or a JH mimic, from 50 to 70% entered a diapause-like state as fully grown larvae. These hormone-treated larvae accumulated the diapause-associated protein and a high molecular weight protein in their fat bodies. Both of these proteins were shown to be released from the fat body of newly-diapaused larvae in vitro, and may function in the haemolymph during diapause. The high molecular weight protein, isolated from the haemolymph, was shown to contain neutral and polar lipids, including biochromes. Its storage in the fat body and release into the haemolymph may be essential for the transport of lipids during diapause. The fat body proteins of newly-diapaused larvae of the southern cornstalk borer, Diatraea crambidiodes, were also examined electrophoretically. They were found to contain a similar protein pattern to that of D. grandiosella, including the presence of a diapause-associated protein.     

Protein release from the larval fat body of the southwestern corn borer, Diatraea grandiosella: An in vitro study

The release of protein from the perivisceral fat body of non-diapausing, pre-diapausing and diapausing larvae of the southwestern corn borer, Diatraea grandiosella, was examined in vitro. Time course studies showed a selective release of proteins into macromolecule-free Grace's medium. The rate of release of individual proteins differed. The release of some proteins was partially inhibited by the incorporation of potassium cyanide (10^?2 M) and ouabain (5 × 10^?3 M) into the medium. During a 5 min incubation a single major high molecular weight protein fraction was released at a high rate from the fat body of both non-diapausing and diapausing larvae. A low molecular weight protein (the diapause-associated protein) was also released readily from the fat body of diapausing larvae. Although most proteins released from the fat body in vitro appeared to be present in the haemolymph in vivo, one notable exception was the absence of the diapause-associated protein from the haemolymph. The method holds promise for facilitating further studies of protein release from insect fat body.     

An immunochemical study of the diapause-associated protein of the southwestern corn borer,Diatraea grandiosella

The distribution and titre of the diapause-associated protein (DAP), which accumulates in the fat body of diapausing larvae of the southwestern corn borer, Diatraea grandiosella, were determined using an antibody raised to the purified protein. Immunoblotting and immunodiffusion showed that the highest concentration of DAP was present in the fat body. Small amounts of the protein were present in the haemolymph and even lower levels were detected in other tissues. Rocket immunoelectrophoresis showed that DAP accumulated in the fat body at the beginning of diapause, reached a plateau, and gradually declined towards the end of diapause. Similarly, the titre of DAP in last instar non-diapausing larvae treated with a JH analogue increased, reached a plateau and then declined. Double immunodiffusion and immunoblotting using the DAP-antibody and extracts of the fat body of D. grandiosella and the southern cornstalk borer, Diatraea crambidoides, and the sugarcane borer, Diatraea saccharalis, revealed the partial immunochemical identity of the DAP of D. grandiosella and a protein present in these two species. Denaturing electrophoresis with immunoblotting showed that the DAP-related proteins of D. crambidoides and D. saccharalis have apparent molecular weights of 33,000 and 36,000, respectively, as compared to a molecular weight of 35,000 for DAP of D. grandiosella. A dot blot analysis showed that no cross reaction occurred between the “diapause proteins” present in the haemolymph of adults of the Colorado potato beetle, Leptinotarsa decemlineata, and the DAP of D. grandiosella.     

Synthesis and release of proteins from cultured larval fat body of the southwestern corn borer,Diatraea grandiosella

The larval fat body of the southwestern corn borer, Diatraea grandiosella, was cultured in vitro to examine the relationship between proteins present in the fat body, those released into the medium, and those present in the haemolymph. While the incorporation of [³H]leucine into fat body proteins was high in last instar pre-diapausing and non-diapausing larvae, it fell in early diapausing larvae to about 11% of that found in prediapausing larvae. Incorporation of [³H]leucine into the diapause-associated protein of the fat body increased gradually in pre-diapausing larvae and reached a maximum in newly-diapaused larvae at a time when the incorporation of [³H]leucine into other proteins of the fat body had declined. The proteins released from the cultured fat body showed identical electrophoretic properties and close immunochemical relationships to most of those present in the haemolymph. Small amounts of the diapause-associated protein were released in vitro from the fat body of larvae of different ages in diapause. Lipophorin was also released in vitro from the fat body of non-diapausing and diapausing larvae, and shown to be immunochemically identical to the lipophorin present in the haemolymph.     

Organization of the retrocerebral gland system in lepidopterous larvae of the family pyralidae

The organization of the retrocerebral gland system in larvae of six species of Lepidoptera belonging to the family Pyralidae was compared using light and electron microscopy. We have demonstrated for the first time the presence of separate corpora cardiaca and corpora allata in the following economically important borers: the southwestern corn borer, Diatraea grandiosella, the sugar cane borer, Diatraea saccharalis, the European corn borer, Ostrinia nubilalis, and the rice stalk borer, Chilo plejadellus. In these species a long nervus corporis allati (ca. 300 μm) runs from the corpus cardiacum to the corpus allatum which is attached to the duct of the mandibular gland.The identity of the corpora allata of D. grandiosella was confirmed by transplantation. Corpora allata removed from pre-diapausing larvae and implanted into the haemocoele of early last stage non-diapausing larvae led to a high incidence of supernumerary larval rather than pupal ecdyses.     

Comparison of some properties of the high-affinity juvenile hormone binding protein from the larval hemolymph of pyralid moths

The properties of the high-affinity low molecular weight juvenile hormone (JH) binding protein present in the hemolymph of larvae of five species of pyralid moths, a noctuid moth, and a sphingid moth were compared. The pyralid moths exhibit a facultative diapause as last-instar larvae. The species employed were the southwestern corn borer, Diatraea grandiosella, the southern cornstalk borer, Diatraea crambidoides, the sugarcane borer, Diatraea saccharalis, the European corn borer, Ostrinia nubilalis, the sunflower moth, Homoeosoma electellum, the cabbage looper, Trichoplusia ni, and the tobacco hornworm, Manduca sexta. The binding characteristics of the proteins were determined using saturation binding assays and competitive binding assays. The dissociation constants of JH I, JH II, and JH III for the binding protein of all the species varied from 0.8 x 10^?7 M to 2.8 x 10^?7 M. Calibrated gel filtration showed that the binding protein of all the species had apparent molecular weights ranging from 29,000 to 31,000. Electrophoresis in 7% acrylamide gels revealed that the relative mobilities of the binding proteins ranged from 0.33 to 0.43. Isoelectric focusing showed that the binding proteins had isoelectric points between 4.4 and 5.0.     

Larval esterases of the southwestern corn borer, Diatraea grandiosella: Temporal changes and specificity

Disc electrophoresis was used to examine and characterize the esterases present in the fat body, haemolymph, and midgut of last stage larvae of the southwestern corn borer, Diatraea grandiosella. Significant temporal changes were observed in the pattern of the 4 major esterases of the fat body and 3 major esterases of the haemolymph. These changing profiles presumably relate, in part, to a requirement for the degradation of juvenile hormone (JH) in preparation for metamorphosis.The binding capacity of esterases present in the larval midgut towards JH I and three JH mimics (alkyl-3,7,11-trimethyl-2,4-dodecadienoates) was also examined. The midgut of last stage nondiapausing larvae was shown to contain a carboxylesterase which bound all three JH mimics. Another esterase which bound JH I, but not the mimics, was also present. An esterase with a similar electrophoretic mobility was detected in the haemolymph and integument. Since the JH I binding esterase did not bind the JH mimics, the mimics do not appear to synergize JH by inhibiting its ester hydrolysis.     

Transfer of N, N'-diacetylchitobiose from dolichyl diphosphate into a gray matter membrane glycoprotein

Gray matter and white matter membranes catalyze the transfer of label from UDP-N-acetyl-[${}^{14}\text{C}$] glucosamine into N-acetyl[${}^{14}\text{C}$]glucosaminyl-pyrophosphoryl-dolichol, N,N′-diacetyl [${}^{14}\text{C}$]chitobiosyl-pyrophosphoryl-dolichol, and N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycoprotein. Gel filtration of the Pronase digests of gray matter N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycoprotein reveals two N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycopeptide fractions. One fraction (A) contains approximately eight glycose units. All of the radioactivity is at nonreducing termini and can be released by treatment with an exo-β-N-acetylglucosaminidase. A smaller N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycopeptide (B) is recovered in the elution volume expected for an asparaginyl disaccharide. Structural studies show that the labeled saccharide unit in glycopeptide B is N,N′-diacetyl[${}^{14}\text{C}$]chitobiose. The linkage between the ${}^{14}\text{C}$-labeled disaccharide and the polypeptide has the properties of an N-glycosidic attachment to asparagine. Only the larger N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycopeptide (A) is found in Pronase digests of white matter membrane N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycoprotein after incubation with UDP-N-acetyl[${}^{14}\text{C}$]glucosamine. When gray matter membranes are incubated with UDP-N-acetyl[${}^{14}\text{C}$]glucosamine in the presence of tunicamycin or UMP, the labeling of glycolipid and the asparaginyl disaccharide is inhibited. UMP and tunicamycin have no effect on the transfer of N-acetyl[${}^{14}\text{C}$]glucosamine to external acceptor sites of the larger glycopeptide (A). The transfer of N,N′-diacetyl[${}^{14}\text{C}$]-chitobiose from carrier lipid to protein is observed when extensively washed membranes containing endogenous, prelabeled ${}^{14}\text{C}$-labeled glycolipids are incubated in the presence or absence of unlabeled GDP-mannose. UMP treatment of the prelabeled membranes selectively discharged over 80% of the label from N-acetyl[${}^{14}\text{C}$]glucosaminyl-pyrophosphoryl-dolichol, but had no effect on the transfer of the ${}^{14}\text{C}$-labeled disaccharide to protein. All of these results are concordant with transfer of N,N′-diacetylchitobiose from dolichyl diphosphate to gray matter glycoprotein. The major membrane glycoprotein labeled by the lipid-mediated [${}^{14}\text{C}$]disaccharide transfer reaction has an apparent molecular weight of 24,000. Tunicamycin prevents the enzymatic labeling of the gray matter glycoprotein having an apparent molecular weight of 24,000.     

Juvenile hormone and the induction of larval polymorphism and the diapause of the southwestern corn borer, Diatraea grandiosella

The southwestern corn borer, Diatraea grandiosella, enters diapause as an immaculate mature larva which is a polymorphic variant of the spotted non-diapause larva. Because dormant immaculate larvae could be obtained by treating last stage non-diapause larvae with a juvenile hormone mimic (JHM), experiments were conducted to determine whether these hormonally induced immaculate larvae (HIL) were physiologically comparable to the normal environmentally induced immaculate diapause larvae (EIL). Comparative data obtained about pupation rates, response to JHM, metabolic reserves, oxygen consumption, and the state of spermatogenesis of HIL and EIL led to the conclusion that the HIL were physiologically similar to the EIL. The results demonstrate that the developmental programme of non-diapause larvae could be switched and ‘diapause’ induced solely by the topical application of JHM. We believe that the data further support our hypothesis that the larval diapause of D. grandiosella is initiated and maintained by the juvenile hormone.     

Ultrastructure and respiration of the fat body of diapausing and non-diapausing larvae of the corn borer,Diatraea grandiosella

A comparative study of the fat body of diapausing and non-diapausing larvae of the corn borer, Diatraea grandiosella, was undertaken using the electron microscope and the oxygen electrode. The electron microscopic results showed a shift from a synthetic to a storage function taking place in a 1 to 2 day period during the final instar of non-diapausing larvae, and in a 4 to 8 day period in that of pre-diapausing larvae. This transition was characterized by a decrease in the number of mitochondria and amount of rough endoplasmic reticulum, and by an increase in the number of proteinaceous granules and lysosomes. In vitro measurements using the oxygen electrode showed that the fat body is a normal aerobic respiratory tissue. The tissue reacted in a predictable manner to inhibitors of oxidative metabolism, including malonate, rotenone, oligomycin, and antimycin, and to the uncoupler, dinitrophenol. During the last instar the observed decrease in the respiratory rate of the fat body coincided with the observed ultrastructural changes in its cells. The fat body of 75 day old environmentally induced and juvenile hormone induced diapausing larvae consumed 90% and 78% less oxygen, respectively than that of 14 day old non-diapausing larvae.     

Some observations on a new type of point average molecular weight

A new type of (reduced) point average molecular weight A¹, is described. Several interesting properties are developed: (i) $\text{A}{}^1\text{=}\text{reduced}$ weight average molecular weight over the whole cell, $\text{A}{}_{\mathrm{<mtext>w</mtext>}}{}^{\mathrm{o}}\text{A}{}^1\text{(}\text{meniscus}\text{) = A}{}_{\mathrm{<mtext>w</mtext>}}\text{(}\text{meniscus}\text{); (iii) A}{}^1\text{(}\text{zero concentration}\text{) =}\text{reduced}$ number average molecular weight, A_n (meniscus). In addition, its usefulness in extracting the meniscus concentration, J(a), and in examining heterogeneous systems such as mucus glycoproteins, are discussed. The evaluation and application of $\text{A}{}^1$ requires only simple computational facilities, without the use for large-scale multiple data acquisition and recycling techniques.     

A study of the diffusion of compact particles in polymer solutions using quasi-elastic light scattering

This paper describes an investigation, using quasi-elastic light scattering, of the diffusion of polystyrene spheres through solutions of dextran. The diffusion coefficient, D, of the spheres is shown to vary inversely with the volume fraction, φ, of dextran according to $\text{D =}\text{D}{}_0\text{(1 + νφ + κφ}{}^2\text{)}$. Changes in the molecular weight of dextran are shown to reflect changes in the macromolecular shape parameter, ν, and the interaction parameter, κ. This result differs from previous studies which suggested an $\text{exp}\text{(?}\text{B}\text{φ}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{)}$ dependence and no molecular weight dependence [8].     

The partial purification of growth-inhibiting factor of the brachypodism-H mouse embryos

The extract of 13-day-old $\text{bp}{}^{\mathrm{H}}\text{bp}{}^{\mathrm{H}}$ embryos contains the factor causing inhibition of the growth of the long bone rudiments of normal embryos in vitro. A 400-fold purification of the factor has been achieved. The molecular weight of the main protein component of the preparation obtained is 76,000. The synthesis of this protein appears to be controlled by bp^H gene.     

Tyrosine metabolism for cuticle tanning in the tobacco hornworm, Manduca sexta (L.) and other Lepidoptera: identification of beta-D-glucopyranosyl-O-L-tyrosine and other metabolites

When [${}^{14}\text{C}$]tyrosine and [${}^{14}\text{C}$]glucose were fed or injected into feeding fifth-instar larvae of the tobacco hornworm, Manduca sexta (L.), they were incorporated into a conjugate identified in hemolymph and carcass extracts as β-d-glucopyranosyl-O-l-tyrosine. In wandering larvae and pupae, the conjugate was hydrolyzed, and tyrosine was hydroxylated and decarboxylated to dihydroxyphenylalanine and 2-(dihydroxyphenyl)ethylamine. None of these metabolites were formed in fourth-instar larvae or in adults. [${}^{14}\text{C}$]Phenylalanine was hydroxylated to tyrosine in all stages of insect development. β-d-Glucopyranosyl-O-l-tyrosine was also detected in 18 other species of Lepidoptera but not in species from other insect orders. This conjugate appears to be the major tyrosine storage metabolite for production of tanning diphenol substrates in Lepidoptera.     

Structure and action of heteronemertine polypeptide toxins Binding of cerebratulus lacteus toxin B-IV to axon membrane vesicles

The binding of the crustacean selective protein neurotoxin, toxin B-IV, from the nemertine Cerebratulus lacteus to lobster axonal vesicles has been studied. A highly radioactive, pharmacologically active derivative of toxin B-IV has been prepared by reaction with Bolton-Hunter reagent. Saturation binding and competition of ¹²⁵I-labeled toxin B-IV by native toxin B-IV have shown specific binding of ¹²⁵I-labeled toxin B-IV to a single class of binding sites with a dissociation constant of 5–20 nM and a binding site capacity, corrected for vesicle sidedness, of 6–9 pmol per mg membrane protein. This compares to a value of 3.8 pmol [³H]saxitoxin bound per mg in the same tissue. Analysis of the kinetics of toxin B-IV association ($\text{k}{}_{\mathrm{+1}}\text{=7.3·10}{}^5\text{M}{}^{\mathrm{?1}}\text{·s}{}^{\mathrm{?1}}$) and dissociation ($\text{k}{}_{\mathrm{?\; 1}}\text{=2·10}{}^{\mathrm{?3}}\text{s}{}^{\mathrm{?1}}$) shows a nearly identical $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ of about 3 nM. There is no competition of toxin B-IV binding by purified toxin from Leiurus quinquestriatus venom while Centruroides sculpturatus Ewing toxin I appears to cause a small enhancement of toxin B-IV binding.     

Reconstitution of dopamine-sensitive adenylate cyclase from dissociated components in canine caudate nucleus

The catalytic component of adenylate cyclase and [${}^3\text{H}$]dopamine binding protein were solubilized with 2% Lubrol PX in the presence of NaF from the synaptic membranes of canine caudate nucleus and were separated into distinct fractions by gel exclusion chromatography on a Sephadex G-200 column. The dissociated adenylate cyclase was no longer responsive to dopamine but was considerably stimulated by 10 mm NaF. Dissociated [${}^3\text{H}$]-dopamine binding protein possessed the apparent dissociation constant of 3.2 μm for dopamine, almost identical to that of the particulate preparations. The affinities of [${}^3\text{H}$]-dopamine binding protein to catecholamines and neuroleptics were also very similar to those of particulate preparations. After the adenylate cyclase and [${}^3\text{H}$]dopamine binding protein were preincubated together at 4 °C for 30 min, the cyclase activity displayed a dose-dependent increase by dopamine with the K_a of 1.6 μm, the concentration of dopamine to stimulate half-maximally. Stimulation of the reconstituted adenylate cyclase by dopamine was maximally 2.7-fold and was strongly inhibited by neuroleptics such as chlorpromazine and haloperidol. These results suggest that [${}^3\text{H}$]dopamine binding protein is identical to the regulatory subunit of dopamine-sensitive adenylate cyclase in the synaptic membranes of canine caudate nucleus.     

Free amino acids of the haemolymph of the southwestern corn borer and the European corn borer in relation to their diapause

The titres of free amino acids present in the haemolymph of diapausing larvae of the southwestern corn borer, Diatraea grandiosella and the European corn borer, Ostrinia nubilalis, were examined. High titres of serine were found in the haemolymph of both species. Serine may serve as a storage form of compounds that are required for the synthesis of uric acid and other purines. The high titres of proline found in the haemolymph of O. nubilalis during the fall and winter may contribute to the freezing tolerance of this species. Alanine accumulated in the haemolymph of both species during the winter.     

Characterization of the parathyrin receptor in renal plasma membranes by labelled hormones and labelled antibody binding techniques

The parathyrin receptor in renal cortex has been investigated by studying the binding of ¹²⁵I-labelled parathyrin, or of unlabelled parathyrin detected with ¹²⁵I-labelled antibodies, to a partially purified plasma membrane fraction. The kinetics of hormone uptake demonstrated a biphasic response in both systems at 22 °C but this phenomenon was not detectable at 37 °C. Specific displacement of lactoperoxidase labelled ¹²⁵I-labelled parathyrin occurred with 8 ng unlabelled bovine parathyrin. The apparent affinity constant was $\text{2.3 · 10}{}^8\text{M}{}^{\mathrm{?1}}$ and the apparent binding capacity of the membranes 1.25 pmol/mg protein. Using the labelled antibody technique the receptor showed maximal binding at pH 7.0–7.5. As little as 80 pg bovine parathyrin produced a significant increase in binding of labelled anti-bovine parathyrin antibody and saturation of binding sites was demonstrated at 2.5 pmol/mg protein. Oxidized hormone showed undetectable binding. Treatment of membranes with phospholipases A or D, or Trypsin greatly reduced subsequent hormone binding. Prior incubation of membranes with 1–34 synthetic parathyrin decreased the binding of intact hormone whereas gastrin, insulin and glueagon had no effect. Growth hormone and calcitonin slightly increased parathyrin binding.