mRNA changes during imaginal determination of the epidermal cells in the pupa ofGalleria mellonella L

Summary Changes in the mRNA population of the mesonotal epidermal cells were investigated inGalleria mellonella during the first 48 h after pupation. Total RNA was extracted and assayed by in vitro translation. The translational products were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by autoradiography. The changing banding pattern of the in vitro synthesized proteins indicates changes in the cellular pattern of mRNAs, most of which occur between 6 h and 18 h after pupal ecdysis. These changes mostly consist in the decrease or disappearance of bands. The injection of juvenile hormone (JH) immediately after pupal ecdysis does not qualitatively influence mRNA changes, but does alter their time course, for they are postponed for 6–12 h. After the injection of 20-hydroxyecdysone (20HE) the same changes can again be seen, but they are greatly accelerated. A comparison of these results with known data on the time course of reprogramming and ecdysteroid titre leads to the conclusion that the mRNA changes in the epidermal cells are a prerequisite for the renewed expression of a developmental programme. This is independent of whether, in the absence of JH, a new programme is determined or whether, under the influence of JH, the previous programme is restored. 20HE does not have any effect on the change in the developmental programme. The change seems to occur as an active and autonomous process in the epidermal cells, in accordance with a genetically fixed developmental programme.     

Ecdysone 20-hydroxylation in imaginal wing discs of Pieris brassicae (lepidoptera): Correlations with ecdysone and 20-hydroxyecdysone titers in pupae

Ecdysone 20-hydroxylase activity has been detected in pupal wing discs of Pieris brassicae. This activity is due to an enzyme system located in microsomal fractions. Its apparent Km is 58 nM for ecdysone. The enzyme is inhibited by the reaction product 20-hydroxyecdysone with an apparent Ki of 2.6 μM. Its activity varied during pupal-adult development with a maximum on day 4, when ecdysone levels are the highest in the animal. Although low, the peak activity is sufficient to assure 25% of the conversion of endogenous ecdysone into 20-hydroxyecdysone in pupae. Ecdysone and 20-hydroxyecdysone levels were measured in hemolymph and whole animals; ecdysone appears to be mainly located in hemolymph, whereas 20-hydroxyecdysone seems to be equally distributed between hemolymph and tissues. All these findings are discussed in relation to the roles of ecdysone and 20-hydroxyecdysone during pupal-adult development.     

Puromycin enhances 20-hydroxyecdysone-induced protein synthesis in wing discs of Drosophila melanogaster

The effect of cycloheximide and puromycin on 20-hydroxyecdysone-induced protein synthesis in wing discs of Drosophila melanogaster has been studied by one-dimensional and two-dimensional SDS polyacrylamide electrophoresis. It is found that puromycin, but not cycloheximide, when applied simultaneously with the hormone enhanced the hormone-induced synthesis of the early and late proteins. However, when puromycin was applied after hormone treatment, only the late proteins were induced. The possible implication of these observations is discussed.     

Identification of three Wx proteins in wheat (Triticum aestivum L.)

Nullisomic analysis of waxy (Wx) protein of hexaploid wheat (Triticum aestivum L.) cv. “Chinese Spring” using two-dimensional polyacrylamide gel electrophoresis revealed that threeWx loci,Wx-A1, Wx-B1, andWx-D1, located on chromosome arms 7AS, 4AL, and 7DS, produce three distinct Wx subunit groups, subunit group-A (SGA), SGB, and SGD, respectively. SGA has a higher molecular weight and a more basic isoelectric point (pI) than the other two. SGB and SGD have the same molecular weight but a slightly different pI range. Owing to the detection of these three subunit groups, we were able to identify the expression of three waxy genes in wheat endosperm and to find two types of mutants among Japanese wheat cultivars, one lacking SGA and the others SGB. These results suggest the possibility of breeding a waxy wheat.     

Effect of 20-hydroxyecdysone and diflubenzuron on chitin production by a cockroach cell line

Summary Twelve insect cell lines were tested for their ability to synthesize chitin. Three of the 7 lepidopteran lines tested (UMN-PIE, IAL-PID2, MRRL-CH) produced a hyamine-resistant material, that was digested by chitinase. The material did not withstand a more rigorous base digestion, and 20-hydroxyecdysone and diflubenzuron had no consistent effect on the production of the, material. One of the 5 cockroach cell lines (UMBGE-4), which is an ecdysone-producing vesicle line, produced a material resistant to a strong base which was degraded to chitobiose andN-acetylglucosamine by chitinase. Production of this material by the cells is increased by 20-hydroxyecdysone and inhibited by diflubenzuron. The UMBGE-4 vesicles are covered with microvilli which bear membrane plaques and produce extra cellular material that resembles insect cuticle. Published with the approval of the Director of the North Dakota Agricultural Experiment Station as journal article No. 1622. Mention of a company name or propiertary product does not imply endorsement, by the U.S. Department of Agriculture.     

Regenerants from Ajuga hairy roots with high productivity of 20-hydroxyecdysone

Summary Regenerants derived from hairy roots of Ajuga reptans var. atropurpurea induced by infection with Agrobacterium rhizogenes MAFF03-01724 harboring pRi revealed a dwarfing response, i.e. decrease in leaf size, reduction in internode distance, and increase in leaf number. These morphogenic alterations were accompanied by an increase in root mass and lack of floral differentiation. In the pRi-transformed regenerants, the proportion of root mass to whole plant mass was higher than that of the untransformed ones, although both kinds of regenerants were comparable on a fresh weight basis. High capacity of rooting and 20-hydroxyecdysone production associated with the original hairy root line were stably maintained in clonal regenerants.     

Susceptibility of Larvae of Galleria mellonella to Infection by Aspergillus fumigatus is Dependent upon Stage of Conidial Germination

The ability of conidia of the human pathogenic fungus Aspergillus fumigatus to kill larvae of the insect Galleria mellonella was investigated. Conidia at different stages of the germination process displayed variations in their virulence as measured using the Galleria infection model. Non-germinating (‘resting’) conidia were avirulent except when an inoculation density of 1 × 10⁷ conidia per insect was used. Conidia that had been induced to commence the germination process by pre-culturing in growth medium for 3 h were capable of killing larvae at densities of 1 × 10⁶ and 1 × 10⁷ per insect. An inoculation density of 1 × 10⁵ conidia per insect remained avirulent. Conidia in the outgrowth phase of germination (characterised as the formation of a germ tube) were the most virulent and were capable of killing 100% of larvae after 5 or 24 h when 1 × 10⁷ or 1 × 10⁶ conidia, that had been allowed to germinate for 24 h, were used. Examination of the response of insect haemocytes to conidia at different stages of the germination process established that haemocytes could engulf non-germinating conidia and those in the early stages of the germination process but that conidia, which had reached the outgrowth stages of germination were not phagocytosed. The results presented here indicate that haemocytes of G. mellonella are capable of phagocytosing A. fumigatus conidia less than 3.0 μm in diameter but that conidia greater than this are too large to be engulfed. The virulence of A. fumigatus in G. mellonella larvae can be ascertained within 60–90 h if infection densities of 1 × 10⁶ or 1 × 10⁷ activated conidia (pre-incubated for 2–3 h) per insect are employed.     

Electron microscopical-immunocytochemical evidence of ecdysteroids in the prothoracic gland of Galleria mellonella

Summary Fixation of prothoracic glands of Galleria mellonella with a solution containing saponin permits immunocytochemical staining of the entire gland. By this means ecdysteroids were demonstrated electron microscopically to be present in the hyaloplasm and microtubules.Supported by Sächsische Akademie der Wissenschaften zu Leipzig     

Genetic transformation ofAjuga multiflora bunge withAgrobacterium rhizogenes and 20-hydroxyecdysone production in hairy roots

An efficient transformation system forAjuga multiflora Bunge was established by usingAgrobacterium rhizogenes strain A4. After inoculation with the bacteria, we obtained a number of hairy-root clones from micro-calli of the explant petioles. One fast-growing line showed the highest production of 20-hydroxyecdysone (20-HE). PCR amplification of rooting locus (rol) genes revealed that the left hand-transferred DNA of the root-inducing plasmid was inserted into the genome of our transformedAjuga hairy roots. This integration was further confirmed by DNA-DNA hybridization. The 20-HE content in hairy roots was 10 times higher than that measured in the wild type.     

Correlation between Gliotoxin Production and Virulence of Aspergillus fumigatus in Galleria mellonella

Thanatephorus cucumeris is a ubiquitous fungus responsible for many types of plant diseases worldwide. All isolates from infected Hevea brasiliensis trees secreted pectolytic enzymes; polygalacturonase (PG), pectin lyase (PL) and cellulolytic enzymes; beta-glucosidase and cellobiase in culture. The extracts of the rubber tree leaf tissues, inoculated with T. cucumeris did not show any PG activity. However, PL activity was detected in tissue with the establishment of the infection. The levels of beta-glucosidase, an inherent enzyme in Hevea spp. increased rapidly following infection. However, cellobiase was detected only with the initiation of infection. Molecular weights of PG in all isolates were similar and in the range of 53,000 to 58,000. PL also followed the same pattern showing a molecular weight around 39,000.     

Primary structure of apolipophorin-III from the greater wax moth,Galleria mellonella

The complete amino acid sequence of apolipophorin-III (apoLp-III), a lipid-binding hemolymph protein from the greater wax moth,Galleria mellonella, was determined by protein sequencing. The mature protein consists of 163 amino acid residues forming a protein of 18,075.5 Da. Its sequence is similar to apoLp-III from other Lepidopteran species, but remarkably different from the apoLp-IIIs of insects from other orders. As shown by mass spectrometric analysis, the protein carries no modifications. Thus, all of its known physiological functions, including its recently discovered immune response-stimulating activity, must reside in the protein itself.     

Effects of 2,4-D removal on the synthesis of specific proteins by Catharanthus roseus cell cultures

Total and neosynthesized proteins of periwinkle cell suspensions (Catharanthus roseus) were first investigated in cells grown in a 2,4-D-containing medium. Analysis of total (silver-stained) proteins by two-dimensional gel electrophoresis revealed that the levels of seventeen polypeptides were altered during the growth cycle of the cells. Analysis of in vivo [³⁵S]-methionine labeled polypeptides revealed differences in the synthesis of at least 35 polypeptides. Three polypeptides with molecular masses of 30, 35 and 39 kDa appeared to be specific markers of the early stationary phase. In a second sequence of experiments, cells were grown in a 2,4-D-free medium. Alterations in protein synthesis were observed: several polypeptides were expressed earlier in the 2,4-D-starved cells than in control cells; the synthesis of at least two specific polypeptides was increased in cells grown in 2,4-D-free medium, whereas the synthesis of three other polypeptides (molecular masses 33, 34 and 52.5 kDa) was switched on in these cells. As previous studies showed that 2,4-D depletion increased the alkaloid production in C. roseus cells, the present results may suggest that these polypeptides are implicated in the regulation of the alkaloid pathway.     

Toxicity of mycotoxins of Fusarium sambucinum for feeding in Galleria mellonella

Fusarium sambucinum Fuckel showed antifeedant activity towards larvae of Galleria mellonella L. when incorporated into insect diet. The activity appeared mostly due to the concentration of trichothecenes present in the fungal extracts. Diacetoxyscirpenol and neosolaniol showed similar levels of activity and were significant potent antifeedants against larvae at 50 and 100 ppm. On the contrary, enniatin B showed no activity up to 100 ppm.     

The ultrastructure and ultracytochemistry of the basement membrane of the Galleria mellonella fat body

Summary The fat body lobes of Galleria mellonella are surrounded by basement membrane — a fine granular layer of connective tissue. This membrane has an affinity for ruthenium red. The results obtained after treatment of the fat body with neuraminidase, hyaluronidase, phospholipase C and proteolytic enzymes suggest that glycoproteins and phospholipoproteins are constituents of this basement membrane. The basement membrane also has the ability to bind concanavalin A-peroxidase, which is associated with the presence of mannoside residues.The preliminary results of these studies were presented at the IX Conference of Electron Microscopy, Gdask, Poland (Dutkowski, 1975)I am greatly indebted to Professor A. Przececka for her encouragement to undertake this study. The excellent technical assistance of Mrs. K. Mroziska, Mrs. Z. Kamiska and the engineering staff of the Laboratory is gratefully acknowledged     

Separation of oviposition-stimulating peptides and myotropic factors from head extracts of Galleria mellonella L.: Comparative effects of myotropic and non-myotropic factors on egg laying

Methanolic extracts from heads of the wax moth, Galleria mellonella L. contain several factors that stimulate oviposition of virgin females in vivo and spontaneous contractions of the oviduct in vitro of the cricket, Gryllus bimaculatus. The myotropic and egglaying activities behaved distinctly during all steps of purification, including fractionation on reverse-phase high-performance liquid chromatography. A novel neuropeptide was isolated from Galleria mellonella. The peptide does not increase the motility of isolated cricket oviducts. Among 13 other myotropic or non-myotropic factors studied, none were found to be as potent as the new peptide in stimulating egg-laying activity. The dose-response curves of myotropic and non-myotropic factors indicate that (i) proctolin, l-glutamate, octopamine, leucokinin-VI, leucopyrokinin, ecdysone and 20-hydroxy-ecdysone, or equivalent structures, might be involved in Galleria mellonella oviposition control, and (ii) that only the novel wax moth neuropeptide has the ability to trigger egg laying in that insect. The significance of these findings is discussed.Abbreviations AcN acetonitrile - E head equivalent - 20-HE 20-hydroxyccdysone - JH 20-hydroxyecdysone, juvenile hormone - Lem-K VI leucokinin-VI - Lem-K I leucokinin-I - Lem MS leucomyosupressin - Lem PK leukopyrokinin - TFA trifluoroacetic acid     

Effect of pre-incubation temperature on susceptibility of Galleria mellonella larvae to infection by Candida albicans

The use of insects for evaluating the virulence of microbial pathogens and for determining the efficacy of antimicrobial drugs is increasing. When larvae of the greater wax moth Galleria mellonella were incubated at 4 or 37°C for 24 h. prior to infection, they manifested increased resistance to infection by the yeast Candida albicans compared to larvae that had been pre-incubated for 24 h at 30°C. Incubation at 4 or 37°C led to an increase in haemocyte density and the expression of genes coding for gallerimycin, transferrin, an inducible metalloproteinase inhibitor (IMPI) and galiomicin. Peak expression of these genes was recorded at approximately 24 h after the commencement of the 4 or 37°C incubation. These results indicate that exposure of larvae to mild thermal shock conditions induces a protective cellular and humoral immune response mediated by increased numbers of haemocytes and elevated expression of antimicrobial peptides.     

Two-dimensional gel electrophoresis of proteins for genetic studies in douglas fir (Pseudotsuga menziesii)

A method of two-dimensional gel electrophoresis of proteins from Douglas fir needles is described. Extraction in the presence of sodium dodecyl sulfate (SDS) and 2-mercaptoethanol followed by heating at 100°C produces reliable two-dimensional gels which are convenient for genetic studies. Three genotypes from different geographical origins have been compared: among 225 loci expressed, 22 display regulatory variations and 7 show allelic variations. Thus it is now possible to undertake the genetic study of Douglas fir using this powerful technique.This work was supported in part by Grant ATP PIRDES 508 444 from the INRA-CNRS.     

Use of SDS-PAGE of cell-wall proteins for rapid differentiation of Lactobacillus delbrueckii subsp. lactis and Lactobacillus helveticus

Cell-wall protein profiles of different strains of Lactobacillus helveticus and L. delbrueckii subsp. lactis isolated from regional cheeses were studied by SDS-PAGE. The patterns were highly reproducible and the presence of numerous bands with molecular weight ranging from 14 to 160 kDa allowed L. delbrueckii subsp. lactis to be differentiated from L. helveticus. The method is a reliable and rapid way to identify thermophilic lactobacilli.