Roy D. Speirs

Immunochemical evidence for a gastrin-like peptide in insect neuroendocrine system

Manduca sexta (L.), contains a peptide that is gastrin-like in its antigenicity, size, and susceptibility to degradative enzymes is presented. No immunoreactive gastrin was found in gut extracts from four species of insects. These results suggest the existence of a peptide in insect nervous tissue having structural similarities to vertebrate gastrin. There is much interest in the origin and evolution of polypeptides in vertebrates that may be common to both nerve cells of the brain and gastrointestinal endocrinelike cells (Pearse, 1976). These peptides include gastrin (Vanderhaegen et al., 1975), somatostatin (Brazeau et al., 1973; Arimura et al., 1975), vasoactive intestinal peptide (Said and Rosenburg, 1976), and substance P (Hokfelt et al., 1975; Pearse and Polak, 1975). For a more precise picture of the molecular evolution of these materials, a search for their presence in the invertebrate phyla is essential. Straus ef al. (1975) have recently demonstrated the presence of gastrin-like immunoreactive peptides in the blood and gastrointestinal tissues from two molluscan species. We have conducted a survey for immunoreactive gastrin in insect tissues and report here the identification of a gastrin-like polypeptide in the neuroendocrine system (brain, corpus cardiacum, and corpus allatum) of the tobacco hornworm,

Insulin-like hypoglycemic and immunological activities in honeybee royal jelly.

Abstract An aqueous extract of royal jelly from Apis mellifera produced hypoglycemia when injected into larvae of Manduca sexta . Application of specific radioimmunoassay to the partially-purified extract showed that royal jelly contains several insulin-like peptides, the major immunoreactive component of which has an apparent mol. wt similar to that of bovine insulin. These results suggest the existence of a peptide in the honeybee having both biological and structural similarities to vertebrate insulin.

A Method for Separation of Trehalose from Insect Hemolymph

A method was developed for the determination of trehalose levels in insect hemolymph. The disaccharide is first purified by gel-permeation chromatography and then quantitated by the anthrone calorimetric procedure. The concentration of trehalose in hemolymph from eleven species is reported. The method is applicable to determinations in other tissues and organisms as well. J In many insect species, trehalose (a-o-glucopyranosyl-a-o-glucopyranoside) is the predominant circulating saccharide that plays a central role in carbohydrate metabolism (18). In order to understand the physiological interactions of this metabolite with regulatory agents, precise determinations of trehalose levels in tissues are essential. The presence of other saccharides makes a direct in situ analysis virtually impossible and has led several laboratories to use indirect methods to measure trehalose. A common procedure involves the use of trehalase to convert the disaccharide to glucose, which is then measured by the specific glucose oxidase enzymatic assay (5-7). The difficulty here is that trehalase and glucose oxidase regulate trehalose, glucose, and glycogen levels in vivo (4) and are subject to inhibition by other substances found in blood or tissue extracts such as hormones, ions, and metabolites (10, 12- 14). Another method requires the use of a gas-liquid chromatograph after the sample has been deproteinized, extracted, and silylated (2). The present study was undertaken to develop a simple, direct, and quantitative spectrophotometric method for trehalose determination that would not be subject to interference by other components in tissues and that could be set up rather inexpensively. Our procedure consists of purifying trehalose by gel-permeation chromatography, followed by direct quantitation using the anthrone reagent.

Toxicity of purothionin and its homologues to the tobacco hornworm, Manduca sexta (L.) (lepidoptera: sphingidae)

Purothionin from wheat or homologues from barley and rye were injected into the hemocoel of larvae of Manduca sexta and caused 50% mortality at doses of 17–46 μg/g. Applications at 2–50 μg purothionin to a Manduca moth nerve-flight muscle preparation produced a rapid, dose-dependent depolarization of the muscle-fiber membrane. The α-isopeptides were two- to threefold more toxic than the β-forms. Alkylation of the cysteine residues of the peptides abolished both activities.

N-β-alanyldopamine and N-acetyldopamine occurrence and synthesis in the central nervous system of Manduca sexta (L.)

Abstract N - β -Alanyldopamine (NBAD), N -acetyldopamine (NADA), dopamine (DA), and 3,4-dihydroxyphenylalanine (DOPA) were detected in the brain and ganglia of the central nervous system of larval and adult tobacco hornworms, Manduca sexta , by reversed phase HPLC with electrochemical detection. NBAD predominated in larval neural tissue during development of the fifth instar and increased to peak concentrations of 936, 650 and 263 nmol g −1 in the abdominal ganglia, subesophageal plus thoracic ganglia and brain, respectively, at the wandering stage of development. Concentrations of all catecholamines decreased in the pharate pupa and were generally lowest in the adult nerve cord. DA was the second most abundant catecholamine in larval ganglia, but the primary catecholamine in adult ganglia. Relatively high levels of DOPA also occurred in the ganglia of wandering larvae but not at other times. NADA was detected only in the abdominal ganglia of day 3 larvae. N -Acyltransferases that catalyze synthesis of NBAD and NADA from DA also were present in abdominal ganglia, as demonstrated by analysis of in vitro cultures in which exogenous DA stimulated synthesis of both N -acylated catecholamines. Maximal NBAD synthesis occurred in ganglia removed from wandering stage larvae (9.3 nmol g −1 day −1 ), whereas NADA synthesis was highest in ganglia from pharate pupae (7.3 nmol g −1 day −1 ). Thus, N - β -alanylation and N -acetylation are competing metabolic reactions for DA in the hornworms nervous system. The role played by the N -acylated catecholamines in M. sexta neurophysiology is unknown, but these compounds may be storage or inactive forms of the putative neurotransmitter DA.

N-β-Alanyldopamine levels and synthesis in integument and other tissues of Manduca sexta (L.) during the larval-pupal transformation

Abstract N - β -Alanyldopamine (NBAD) and other diphenols in tissues of the fifth larval instar of the tobacco hornworm, Manduca sexta (L.), were analyzed by HPLC with electrochemical detection. NBAD accumulated in the integument during the intermolt feeding period, with maximal levels in the wandering stage (6 mmol/g). It then declined to a low level during apolysis and endocuticle digestion, while hemolymph NBAD increased during the same interval to a peak concentration (3 mM) shortly before pupal ecdysis. Trachea and foregut contained lesser amounts of NBAD (0.5 mmol/g), perhaps associated with cuticle, whereas fat body, muscle, midgut and hindgut had 0.1 mmol/g or less. Dopamine (DA), N -acetyldopamine and 3,4-dihydroxyphenylalanine (DOPA) were at least 10-fold less abundant than NBAD in the integument. NBAD synthase, which catalyzes the formation of NBAD from DA and β-alanine, was assayed in both integument and fat body. Highest activity was detected in the integument, where two peaks were observed, one at day 3 near the end of larval feeding and the other at day 9 as pupal cuticle tanning was initiated. Fat body enzyme was substantially less and was detected only in the pharate pupa. Maximal NBAD synthesis by integument cultured in vitro was dependent upon DA supplementation of at least 1.4 mM. 20-Hydroxyecdysone did not alter NBAD synthesis in vitro in either the integument or the fat body, even though injection of this hormone into isolated larval abdomens induced synthesis and/or transport of integumental NBAD back into the hemolymph. The rate-limiting steps in the NBAD biosynthetic pathway appear to be the production of DOPA and DA, because β-alanine occurs in the hemolymph at relatively high levels throughout larval-pupal development.

Immunocytochemical evidence for the occurrence of insulin in the frontal ganglion of a lepidopteran insect, the tobacco hornworm moth, Manduca sexta L

The frontal ganglion of the adult forms of the tobacco hornworm, Manduca sexta, was investigated immunocytochemically for the occurrence of the gastro-entero-pancreatic (GEP) neurohormonal peptides, namely insulin, nerve growth factor, epidermal growth factor, insulin C-peptide, somatostatin, glucagon, glicentin, pancreatic polypeptide (PP), polypeptide YY (PYY), secretin, vasoactive intestinal peptide (VIP), gastric inhibitory peptide (GIP), gastrin, cholecystokinin (CCK), enkephalin, alpha- and beta-endorphins, substance P, neurotensin, bombesin, motilin, ACTH, serotonin, and calcitonin. Among all the antisera tested, positive immunostaining was obtained with anti-insulin B-chain serum only. The insulin B-chain immunoreactivity was localized in 4-6 large (30-40 microns) neurons, in the neuropile, and in the recurrent nerve. It is speculated that the insulin-like immunoreactive material may be transported to the neurohaemal organ (corpora cardiaca) through the nervi cardiaco-somatogastrici.

Immunological relationships between β-N-acetylglucosaminidases from the tobacco hornworm, Manduca sexta (L.)☆

Abstract Antisera were raised in rabbits directed against three purified β-N-acetylglucosaminidases from the tobacco hornworm, Manduca sexta (L.). Gamma globulin fractions were prepared by ammonium sulphate precipitation and ion-exchange chromatography. Ouchterlony diffusion and quantitative precipitation reactions demonstrated that the antibodies recognized specific types of β-N-acetylglucosaminidases: one precipitated exochitinase, another exo-β-N-acetylglucosaminidase and the third endochitinase. These results are in accord with the enzyme classifications proposed previously based upon substrate specificity and kinetic properties.

Comparative biochemistry of mycophagous and non-mycophagous grain beetles. Chitinolytic activities of foreign and sawtoothed grain beetles

Abstract 1. 1. The chitinolytic enzymes in a mycophagous insect, the foreign grain beetle, Ahasverus advena , and a non-mycophagous insect, the sawtoothed grain beetle, Oryzaephilus surinamensis , were measured. 2. 2. Larvae and adults of A. advena exhibit three-fold higher chitinase levels than do the same stages of O. surinamensis . 3. 3. The foreign grain beetle adult contains more than two-fold higher β - N -acetylglucosaminidase than the sawtoothed grain beetle adult. 4. 4. The results suggest that feeding stages of the mycophagous beetle have elevated levels of chitinolytic enzymes so that fungal chitin can be utilized as a food source.

Effect of neutral red on carbohydrate levels in the tobacco hornworm, Manduca sexta (L.) (Lepidoptera: Sphingidae)☆

Abstract 1. 1. The effect of acute hemocoel injection (100 mg/kg) of neutral red on hemolymph trehalose and fat body glycogen has been examined in the tobacco hornworm, Manduca sexta (L.). 2. 2. Neutral red caused a 70% increase in hemolymph sugar 6 hr after injection. 3. 3. Hypertrehalosemia persisted for 18 hr and was followed by hypotrehalosemia. 4. 4. Fat body glycogen was unchanged up to 12 hr. 5. 5. A 60% glycogenolytic response was observed 48 hr after injection.