Haemolymph ecdysone concentrations in Hyalophora cecropia pupae,dauer pupae and adults

Haemolymph ecdysone concentrations were determined by radioimmunoassay in diapausing pupae, pharate adults, adults, and chilled dauer pupae. The concentration in diapausing pupae after 6 months chilling (5.35 pg/μl) increased dramatically after 3 days at 27°C (>200 pg/μl) and then decreased to low levels in adult females (1.63 pg/μl). In adult males ecdysone was undetectable in all except one animal. Dauer pupae showed a decrease from 6.1 to 1.7 pg/μl 1 day after being transferred from 6 to 27°C. Over a 3-day period the value increased to 3.19 pg/μl and remained constant for more than a year. These results suggest that diapausing pupae with and without brain neurosecretory cells maintain a low concentration of ecdysone in the haemolymph.     

Characterization of the DNA of Hyalophora cecropia

DNA was isolated from the pupae and various tissues of pharate adults of the silkmoth Hyalophora cecropia. CsCl equilibrium density analysis showed the presence of one major band at 1.693 and two minor satellites, 1.705 and 1.709, comprising 3 and 6% respectively of the total DNA. We could detect no difference in the renaturation kinetics of DNA prepared from pupal or pharate adult tissues. The genome appears to be composed of 30% redundant and 70% unique sequences. The haploid information content of the unique sequences is 11.4 × 10¹⁰ daltons.     

The goblet cavity matrix in the larval midgut of Hyalophora cecropia

The larval midgut of the silkmoth Hyalophora cecropia was examined using scanning electron microscopy. Goblet cells were observed to contain within their cavities a matrix plug. This matrix material was extruded onto the lumen side of the epithelium when the tissue was stretched. The rôle of this matrix material in maintenance of the capacity of the midgut to transport ions in vivo and in vitro is discussed.     

Regulation of glycogen phosphorylase in fat body ofCecropia silkmoth pupae

Summary Glycogen phosphorylase of pupal fat body of the silkmoth,Hyalophora cecropia, and its activation by different stimuli have been studied. Spectrophotometric assay in the direction of glycogenolysis, used in most of the experiments, indicated higher amounts of phosphorylasea than assay by release of P_i from glucose-1-phosphate; both assays, however, estimated changes in proportion of phosphorylasea equally. TheK _ms for P_i were estimated as 5 mM for phosphorylasea in the absence of AMP and 18 mM for phosphorylaseb with 2 mM AMP.When diapausing pupae were held at 4°C, fat body phosphorylase was quickly activated by conversion to thea form up to about 50% of the total, and then declined again after 30 days, when glycerol had accumulated in the hemolymph. Cold activation in vivo was quickly reversed at 25°C. Removal of the brain did not prevent cold activation. After storage at 15°C, sensitivity to cold activation was diminished. Locusts and crickets also showed activation of phosphorylase after chilling.Exposure of fat body to air, transfer to Ringer solution, or physical agitation, caused activation of phosphorylase which is classed as shock activation. After about 1 h incubation in Ringer at 25°C, this effect reversed spontaneously. Activation also occurred in fat body in vitro after transfer to 0°C (cold activation), and was reversed at 25°C. The previously reported inhibition of activation by glycerol, however, could not be consistently reproduced.In fat body homogenates, phosphorylaseb was converted to phosphorylasea by incubation with ATP and Mg²⁺, which indicates activity of phosphorylase kinase. In preparations treated with Sephadex G-25 and then incubated, the reverse conversion took place, which was inhibited by fluoride, and indicates activity of phosphorylase phosphatase.Cyclic AMP added to fat body in vitro, or theophylline either in vivo or in vitro, stimulated the activation of phosphorylase. In fat body in vitro, shock activation was paralleled by elevation of tissue cyclic AMP, whereas cold activation was not. Cyclic GMP did not stimulate activation, and showed no significant changes in tissue levels.It is concluded that the conversion of silkmoth pupal fat body phosphorylaseb to phosphorylasea can be stimulated by a shock-initiated mechanism involving cyclic AMP and a distinct cold-initiated mechanism independent of cyclic AMP.Abbreviations DTT dithiothreitol - cyclic AMP 3,5-cyclic adenosine monophosphate - cyclic GMP 3,5-cyclic guanosine monophosphate - P _i inorganic phosphate This investigation was begun in the Department of Biology, Yale University, New Haven, Connecticut, USA     

Differential synthesis of bacteria-induced proteins of Manduca sexta larvae and pupae

The range of inducible antibacterial and other associated haemolymph proteins in Manduca sexta larvae and pupae was examined by high resolution two-dimensional (2D) isoelectric focusing-polyacrylamide gel electrophoresis. Twenty-two major inducible proteins were consistently resolved on gels of haemolymph from bacteria-injected larvae. Haemolymph from bacteria-injected pupae showed a different pattern of induced proteins. The proteins of the two stages include those which (i) are induced in both stages, (ii) those which are exclusively induced in either larvae or pupae, (iii) those which are inducible in larvae, but consititutively present in pupae, and, (iv) those which are induced in larvae, and which are present at intermediate levels but may be induced to higher levels in pupae.The antibacterial activity of the haemolymph from larvae and pupae was compared on acidpolyacrylamide gels, and the apparent M_r and pI of the inducible proteins determined. Certain of the inducible proteins appear to resemble the cecropin and attacin proteins of Hylophora cecropia.     

Eclosion hormone activity in haemolymph of eclosing silkmoth, Bombyx mori

The presence of eclosion hormone in the haemolymph of eclosing adults of the silkworm, Bombyx mori, was demonstrated to provide evidence that the hormone is involved in the induction of eclosion of this insect.The eclosion of the adult occurred within 55–75 min after light-on, when the pupae had been kept under conditions of 16 h light and 8 h dark during adult development. The hormone became detectable in the haemolymph 10–20 min after light-on, followed by eclosion 40 min later. The hormonal activity increased sharply to a maximal level (30 units/ml haemolymph) at 30 min prior to the eclosion and then fell rapidly: detectable eclosion hormone was present in the haemolymph for only 15–20 min. The hormone was partially purified from the haemolymph of eclosing animals and a 400-fold purification was achieved. The preparation was rather stable against heat treatment, and the molecular weight was estimated to be 5,000–10,000 by gel-filtration on a Sephadex G-50 (superfine). The origin of eclosion hormone in the haemolymph is discussed.     

Arrest of adult development in debrained pupae of the silkworm, Bombyx mori

The occurrence of developmental arrest after brain removal in pupae of Bombyx mori was examined using a racial hybrid Chinese No. 115 × Japanese No. 122. The results are as follows: (1) Adult development was blocked for a long period in most insects debrained shortly after larval-pupal ecdysis; (2) the earlier the brain removal, the more arrested pupae were obtained; (3) the critical period of brain hormone secretion for adult development was earlier in the female than in the male; (4) the developmental arrest which had been induced in female pupae tended to terminate earlier than that in males; (5) the developmental arrest which was induced by extremely early brain removal terminated earlier than that obtained by later brain removal.     

Ecdysteroids and diapause in pupae of Heliothis punctiger

Most pupae of H. punctiger enter diapause when reared at 19°C, 12L:12D. When pharate pupae were treated for only 12 hr at 28°C about 50% developed at 19°C. The proportion of non-diapausing pupae increased as the temperature at which the pharate pupal stage was spent increased.The quantity of injected 20-hydroxyecdysone necessary to promote development in diapausing pupae varied from about 1 μg g^?1 soon after pupation to about 4 μg g^?1 after 50 days. It fell somewhat after 150 days.Removing brains from non-diapausing pupae showed that the brain secreted its hormone at the time of pupation (or just before). However, if the pupae were kept at 19°C development did not occur unless the brain remained in situ for at least 20 hr at 28°C. Implanting brains from non-diapausing pupae into diapausing ones had no measurable effect.These results may be explained by postulating that the prothoracic gland is ‘activated’ by exposure to high temperature, but that it reverts to inactivity over a period at 19°C. The ‘active’ gland must then be stimulated by brain hormone for a long period to trigger secretion of its hormone, which results in development. Diapause is thus the result of the failure of the prothoracic gland to secrete.     

Choline and β-methylcholine acetylation in pupae of the moth, Celerio euphorbiae

Acetylation of choline and β-methylcholine in the tissues of Celerio euphorbiae was investigated during development after larval-pupal ecdysis in diapausing and non-diapausing generations. β-Methylcholine was acetylated more efficiently than choline. The stage- and sex-dependent changes in acetylating activity have been observed, their patterns being similar for both substrates. K⁺ and Na⁺ ions do not influence the specificity of the acetylation process.     

Wax secretion in non-diapausing and diapausing pupae of the tobacco hornworm, Manduca sexta

Scanning electron microscopy of the epicuticle of the tobacco hornworm, Manduca sexta, showed that pupae in diapause possess thicker wax layers than non-diapausing pupae. Extractions of the epicuticle with CHCl₃ established that diapausing pupae secreted over three times as much wax as non-diapausing pupae, and that the total period of wax secretion in diapausing pupae was two to three times longer than that of non-diapausing pupae. Apparently, the extra thickness of the wax layer of diapausing pupae protects the insect from desiccation during diapause. The deposition of additional wax may result from hormonal changes accompanying entry into diapause.     

Patterns of protein synthesis following heat shock in pupae of Drosophila melanogaster

Pupae of Drosophila melanogaster were heat-shocked under conditions required to induce phenocopies in more than 90% of the flies that subsequently emerge. The effects of these treatments on protein synthesis in two tissues (thoracic epithelium and brain) were followed for several hours after the heat treatments. Results from pulse-labeling and protein separations on sodium dodecylsulfate (SDS) acrylamide gels showed a virtually complete cessation of protein synthesis immediately after the shock, followed by a noncoordinate resumption of the starting pattern. Similar experiments following double heat shocks demonstrated a more rapid resumption of synthesis of heat shock proteins after two successive heat treatments than after a single one.     

Diel oxygen uptake rhythms in diapausing pupae of Pieris brassicae and Papilio machaon

Diel rhythms of oxygen uptake are described for P. brassicae and P. machaon. The rhythms are bimodal in both species at 10°C, with a main midday peak, a smaller peak in the afternoon or early evening and low nocturnal uptake rates under natural and artificial (LD 9:15) light regimes. In P. brassicae, the rhythm of oxygen uptake is linked with a diel rhythm of the incidence of short-term oxygen uptake cycles. Summated batch curves for both species contain significant elements of individual variation. In P. machaon, the timing of daily peaks in oxygen uptake is related directly to the level of metabolism.     

Muscle growth and protein synthesis in the puparia of Calliphora vomitoria

The effects of cycloheximide on the development of the dorsal longitudinal flight muscle of 3- to 5-day-old puparia of Calliphora vomitoria have been investigated. One μg of cycloheximide injected into the puparia reduced the incorporation of ¹⁴C phenylalanine and lysine into protein to 5 and 8 per cent of their normal levels. The cycloheximide was found to have produced its maximum effect within 2 hr of its injection and increasing the concentration did not further depress the amount of amino acid incorporation. The sixth dorsal longitudinal muscles continued to increase in length after the injection of cycloheximide and the elongation of the muscle fibres was accompanied by an increase in protein content in normal and cycloheximide-treated animals. An injection of colchicine (which is believed to disrupt microtubules) immediately halted muscle growth. Electron microscopy of the muscle fibre revealed that fibres from cycloheximide-treated animals contained myofilaments, although there were some differences in myofilament structure between normal and treated animals. The formation of the muscle fibres in the absence of protein synthesis is discussed.     

Morphological and electrophysiological differences in tarsal chemosensilla between the wild silkmoth Bombyx mandarina and the domesticated species Bombyx mori

Gustatory and olfactory senses of phytophagous insects play important roles in the recognition of host plants. In the domestic silkmoth Bombyx mori and its wild species Bombyx mandarina, the morphologies and responses of adult olfactory organs (antennae) have been intensely investigated. However, little is known about these features of adult gustatory organs and the influence of domestication on the gustatory sense. Here we revealed that both species have two types of sensilla (thick [T] and slim [S] types) on the fifth tarsomeres of the adult legs. In both species, females have 3.6–6.9 times more T-sensilla than males. Therefore, T-sensilla seem to play more important roles in females than in males. Moreover, gustatory cells of T-sensilla of B. mandarina females responded intensely to mulberry leaf extract in electrophysiological experiments, while T-sensilla of B. mori females (N4 strain) hardly responded to mulberry leaf extract. These results suggest that T-sensilla of B. mandarina females are involved in the recognition of oviposition sites. We also observed that, in three B. mori strains (N4, p50T, and Kinshu × Showa), the densities of sensilla on the fifth tarsomeres were much lower than in B. mandarina. These results indicate that domestication has influenced the tarsal gustatory system of B. mori.     

Bacteria-induced haemolymph proteins of Manduca sexta pupae and larvae

The haemolymph of Manduca sexta larvae and pupae has been analyzed for proteins potentially associated with the bacterial defence response of the insect. Five proteins, M13, M18, M20, M23, and M24 in pupae, and M4, M11, M13, M18 and M23 in larvae, are induced by the injection of bacteria into the haemolymph. Proteins M4 and M11 are always present at high levels in uninjected pupae. Proteins M20 and M24 could not be induced in larvae. These proteins, as well as those not showing a response to bacterial challenge or injury, were also analyzed for presence of disulphide bonds and carbohydrate moieties, and their apparent molecular weights determined.     

Virus DNA replication and protein synthesis in Amsacta moorei entomopoxvirus-infected Estigmene acrea cells

Amsacta moorei entomopoxvirus DNA synthesis was detected in Estigmene acrea cells by [³H]thymidine incorporation 12 hr after virus inoculation. Hybridization of ³²P-labeled Amsacta entomopoxvirus DNA to the DNA from virus-infected cells indicated that viral-specific DNA synthesis was initiated between 6 and 12 hr after virus inoculation. A rapid increase in the rate of virus DNA synthesis was detected from 12 to 24 hr after virus inoculation. Amsacta entomopoxvirus protein biosynthesis in E. acrea cells was studied by [su35S]methionine incorporation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Extracellular virus and virus-containing occlusion bodies were first detected in virus-infected cell cultures 18 hr after virus inoculation. Thirty-seven virus structural proteins, ranging in molecular weight from 13,000 to 208,000 were detected in both occluded and nonoccluded forms of the virus. The biosynthesis of virus structural proteins increased rapidly from 18 to 34 hr after infection. A major viral-induced protein corresponding in molecular weight to viral occlusion body protein (110,000) was detected approximately 24 hr after virus inoculation.