High-performance liquid chromatography of ecdysone metabolites applied to the cabbage butterfly, Pieris brassicae L

HPLC allowed separation of twelve major labeled compounds after injection of ³H-ecdysone into Pieris pharate pupae. These compounds were identified as six pairs of metabolites (3α and 3β epimers), comprising ecdysone, 20-hydroxyecdysone, 26-hydroxyecdysone, 20,26-dihydroxy-ecdysone and the polar metabolites P and 20-hydroxy-P. These last two products could not be enzymatically split by any hydrolase tested and are weak acids arising respectively from 26-hydroxyecdysone and 20,26-dihydroxyecdysone. They might be 26-oic compounds.Epimerization appears as a fundamental inactivation process in Pieris and could well be a general characteristic of closed systems (eggs and pupae). No significant amounts of hydrolyzable conjugates were detected in our biological system (pharate pupae and pupae).     

Diuresis in the cabbage white butterfly, Pieris brassicae: Fluid secretion by the malpighian tubules

The sudden weight loss at the pupal-adult ecdysis of the cabbage white butterfly, Peiris brassicae, is caused by a diuresis which greatly reduces the blood volume after emergence. Study of the Malpighian tubules as isolated preparations shows that only those of the adult insect possess the ability to secrete fluid at extremely fast rates when stimulated. In contrast to the fast-secreting tubules of bloodsucking insects, the mechanism of fluid secretion is not unusual, probably depending on the active transport of potassium ions.     

Biosynthesis and inactivation of ecdysone during the pupal-adult development of the cabbage butterfly, Pieris brassicae L.

Injection of labelled ecdysone and 20-hydroxyecdysone into Pieris pupae showed that their catabolism proceeds through 26-hydroxylation followed by conversion into acidic steroids assumed to be 26-oic compounds. This biological system is characterized by the lack of conjugation reactions and by rather long-lived hormones.In vivo biosynthesis of ecdysteroids was investigated by 24 hr [³H]cholesterol labelling, followed by HPLC analysis of the resulting [³H]ecdysone and 20-hydroxyecdysone. Active conversion (up to 0.07% in 24 hours) was observed between 48 hr and 120 hr following pupal ecdysis, a result in good agreement with the variations observed in hormone contentLong-term [³H]cholesterol incorporation experiments made it possible to monitor ecdysteroid dynamics during pupal development. Three periods were observed, corresponding to the successive accumulation of ecdysone, 20-hydroxyecdysone and an acidic metabolite. Comparison of these results with those of the experiments involving labelled ecdysone injection shows that the catabolism of injected hormones is not the same as that of endogenous hormones.     

Diuresis in the cabbage white butterfly, Pieris brassicae: Water and ion regulation and the rôle of the hindgut

The significance of diuresis in the water and ion balance of newly emerged Pieris was examined by comparing the composition of haemolymph and urine during diuresis. The high potassium content of the urine results in a marked increase in the Na/K ratio of the haemolymph. The haemolymph osmolarity is well regulated, in spite of the very hypo-osmotic urine. By means of an isolated preparation of the ileum, it was shown that rapid resorption of potassium ions by this part of the hindgut is responsible for the low osmolarity of the urine.     

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.     

Diapause induction and clock mechanism in the cabbage butterfly Pieris melete Ménétriés

Photoperiodic control of diapause induction was systematically investigated in the cabbage butterfly, Pieris melete, which enters summer and winter diapause as a pupa. Summer and winter diapause are induced principally by short and long scotophases, respectively; the intermediate scotophases (11-12 h) permit pupae to develop without diapause. Photoperiodic responses under 24-h light-dark cycles at 16.9, 18, 20 and 22 °C showed that the hibernation response was temperature compensated, whereas aestivation response was strongly temperature-dependent. The incidence of diapause for both aestivation and hibernation showed a decline at the ultra-short and ultra-long scotophases. Experiments using non-24-h light-dark cycles showed that the length of the scotophase played an essential role in the determination of diapause. The highest photosensitivity differed under hibernation and aestivation conditions. With a 3 × LD 12:12 interruption, a maximal inhibition of aestivation occurred in the L3/2 stage, and of hibernation it occurred in the L4/0 stage. A long-night of LD 10:14 induced hibernation diapause but inhibited aestivation diapause and, conversely, a short-night of LD 14:10 inhibited hibernation diapause but induced aestivation diapause. With a 1-h light pulse at LD 11:13, a maximal inhibition of hibernation occurred 3 h before lights-on (late scotophase), whereas, with a 1-h light pulse at LD 12.5:11.5, a maximal induction of aestivation occurred 2-3 h after the onset of darkness (early scotophase). Nanda-Hamner and Bünsow experiments failed to reveal the involvement of a circadian system, suggesting that the photoperiodic time measurement for diapause induction in this butterfly resembles an hourglass-like timer or a damped circadian oscillator.     

Supradian and infradian cycles in oxygen uptake of diapausing pupae of Pieris brassicae

The general characteristics of diapause respiration in P. brassicae are described, together with an examination of short-term (supradian) and long-term (infradian) variation in oxygen uptake. Supradian cycles occur approximately every 3 hr at 10°C and are shown by closed box analyses to be initiated by carbon dioxide bursts. Maximal rates of oxygen uptake occur shortly after the burst in carbon dioxide release, not at the start of the burst as recorded in other diapausing species. The frequency of supradian cycles is directly related to temperature and metabolism in accordance with the characteristics of discontinuous carbon dioxide release.Infradian cycles of between 3 and 7 days duration are recorded for both oxygen uptake and net exchange rates. Peaks in oxygen demand occur on average every 4 days at 10°C, and are related in frequency to the level of metabolism of individual pupae. Just before post-diapause development, oxygen demands fall to about half their normal levels; these changes are associated with appropriate changes in the frequency of supradian and infradian cycles.     

Anti-quorum sensing and antibiofilm potential of Alternaria alternata, a foliar endophyte of Carica papaya, evidenced by QS assays and in-silico analysis

In the present study, secondary metabolites from an endophytic fungus, Alternaria alternata, colonizing Carica papaya, demonstrated antiquorum sensing properties against Pseudomonas aeruginosa. This study reports the antagonistic effects of fungal crude extract of A. alternata against the various quorum sensing (QS) associated virulent factors such as percentage decrease in production of pyocyanin, alginate, chitinase and rhamnolipid; significant decrease in proteases activity such as LasA protease activity, staphylolytic activity, Las B elastase; and a marked decrease in biofilm formation and associated factors such as exopolysaccharide (EPS) production and cell surface hydrophobicity (CSH). Further, motility pattern i.e., swimming and swarming was also found to be inhibited. This down regulation of QS and associated factors are further supported by in-silico analysis of interaction between QS receptor LasR and bioactive molecules viz., sulfurous acid, 2-propyl tridecyl ester and 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester present in fungal crude extract, found based on GCMS analysis, sketches the modulating ability of QS expression. This is the first report on an endophytic fungus of C. papaya having a role in QS inhibition against P. aeruginosa and lays a platform to explore further the endophytes for potent therapeutic agents in QS.     

Optimal defense strategy against herbivory in plants: Conditions selecting for induced defense, constitutive defense, and no-defense

To examine the conditions selecting for induced defense, constitutive defense, and no-defense, we developed a model of plant defense strategy against herbivory. In the model, a plant consists of two modules between which signal inducing defense compounds can be translocated. We assume three strategies: plants produce defense compounds responding to herbivory (induced defense), they have the compounds beforehand (constitutive defense), and they never produce the compounds (no-defense). We found that no-defense is optimal if the amount of biomass lost due to herbivory is small because of the growth cost of having defense compounds. The constitutive defense is optimal if the amount of biomass lost is not so small and the probability of herbivory is high. If the biomass loss is not so small but the probability of herbivory is low, the induced defense or no-defense is optimal. When the induced defense is optimal, the probability of herbivory necessarily increases in plants once herbivory has occurred. If the probability stays the same, no-defense is optimal. Thus, the behavior of herbivores, i.e., whether they remain around a plant and attack it repeatedly, affects the evolution of the induced defense.     

Some factors affecting spore replication of Nosema algerae (Microspora,Nosematidae) in Pieris brassicae (Lepidoptera)

The production of Nosema algerae spores was examined in Pieris brassicae. Spore replication in the insect host followed a logistic pattern of development. The factors studied which affected spore production and replication were dose level (5 × 10², 5 × 10³, and 5 × 10⁴ spores per insect), larval instar (fourth and fifth), and cool pretreatment of the insects at 20°C prior to inoculation compared with a constant temperature of 26°C. A three-way analysis showed the interactions between these factors. The logistic pattern of spore replication was used to explain the results.     

The effects of actinomycin D,cycloheximide and puromycin on the 20-hydroxyecdysone induced autophagocytosis in larval fat body cells of Pieris brassicae

The effects of 20-hydroxyecdysone (5 μg/g body weight), cycloheximide (5 μg/g body weight), puromycin (30 μg/g body weight) and actinomycin D (30 μg/g body weight) were investigated on the larval fat body cells of 48–70-hours-old last instar larvae of Pieris brassicae.20-Hydroxyecdysone was found to induce the formation of autophagic vacuoles 3 hr after its administration, but this was, however, prevented by simultaneous cycloheximide treatment in a parallel experiment. On the contrary, puromycin proved to induce autophagocytosis. These diverse effects of the two translational inhibitors on hormone-induced autophagocytosis may be explained by differences in their modes of action.Actinomycin D, when administered 21 hr prior to the hormone, exerted a preventive effect on induced autophagocytosis, but was ineffective when applied 3 hr before the injection of 20-hydroxyecdysone.It was concluded that the presence of RNA synthesized several hours prior to the hormone treatment was a prerequisite for induction of autophagocytosis by 20-hydroxyecdysone.     

Factors controlling in vitro hatching of Vairimorpha plodiae (Microspora) spores and their infectivity to Plodia interpunctella,Heliothis virescens, and Pieris brassicae

Factors which influence the hatching of spores and proliferation of stages of the microsporidium Vairimorpha plodiae in two susceptible insects, Plodia interpunctella and Heliothis virescens, and one nonsusceptible insect, Pieris brassicae, were investigated. Spores hatched in 0.1 and 1 m KCl solutions when subjected to a change in pH, from pH 11 to pH 8. K⁺ was essential for hatching; NaCl solutions were not effective. Ca²⁺ and Mg²⁺ inhibited hatching, and calcium and magnesium chelating agents enhanced it. All three insect species had alkaline midgut contents and smooth, fragile peritrophic membranes. Spores hatched inside the midguts of all three insect species (P. interpunctella: maximum rate, 92.5%; H. virescens, 91.5%; P. brassicae, 82%). Sporoplasms were observed in the midgut epithelial and associated tracheole cells of P. brassicae. Both H. virescens and P. brassicae became infected when injected intrahemocoelically with spores.     

Ommochrome formation and kynurenine excretion in Pieris brassicae: Relation to tryptophan supply on an artificial diet

The brown-red pigment in the larval epidermis and in the testis of Pieris brassicae was identified as xanthommatin on the basis of solubility, redox behaviour, chromatography, degradation, visible and infrared spectra. In the epidermis, this pigment accumulates during the larval feeding period and disappears rapidly in the wandering stage. Larvae fed an artificial diet produce about half the amount of xanthommatin as larvae fed cabbage. This effect is caused by a lack of dietary tryptophan. Xanthommatin formation is increased by the addition of tryptophan which also increases body weight. At a tryptophan concentration of 0.2 mg per g, however, weight increase is lower than in controls and high mortality is observed. Pieris larvae excrete kynurenine in relation to dietary tryptophan. No measurable amounts are excreted in the last instar on the non-supplement diet. After feeding different quantities of tryptophan, different amounts of kynurenine are excreted only on the day following ecdysis.     

Effects of elicitation treatment and genotypic variation on induced resistance in Populus: impacts on gypsy moth (Lepidoptera: Lymantriidae) development and feeding behavior

We examined the effects of various wounding treatments and genotypic variation on induced resistance in Populus (Salicales: Salicaceae) against herbivory by the gypsy moth, Lymantria dispar L. (Lepidoptera: Lymantriidae). Second-instar larvae grew and consumed less on leaves from induced than non-induced trees. Likewise, larvae preferred leaf disks from non-induced trees. Among induction treatments, gypsy moth feeding had the strongest and most consistent effect in behavioral choice tests. Mechanical wounding of leaves and mechanical wounding plus application of gypsy moth regurgitant had intermediate effects, while application of jasmonic acid had the weakest overall effect. Under no-choice conditions, there were no consistent trends across clones in the ability of various treatments to elicit plant responses affecting the herbivore. Levels of constitutive and inducible resistance to herbivory varied significantly among 12 Populus clones. Larvae grew up to 30-fold more, and consumed up to 250-fold more on the most suitable than the least suitable clone. Prior feeding by gypsy moths reduced larval feeding up to 71.4% on the most highly inducible clone, but it had little or no effect for the least inducible clones. There was no evidence for a relationship between levels of inducible and constitutive resistance, or between inducible resistance and phylogenetic relatedness among clones. We discuss implications for the ecology and evolution of plant-insect interactions and the management of insect pests. Received: 12 October 1998 / Accepted: 22 March 1999     

Single cell responses in female Pieris brassicae (Lepidoptera: Pieridae) to plant volatiles and conspecific egg odours

Action potentials from individual olfactory cells and receptor potentials were recorded from antennae of female cabbage white butterflies, Pieris brassicae, using a new ‘surface-contact’ recording technique. Stimuli used were plant volatiles and conspecific egg odours. Most cells were activated by some stimuli and inhibited by others. Hence, the ‘odour spectra’ of most cells included activating as well as inhibiting substances. In addition, the ‘response profile’ of a given stimulus contained both positively and negatively responding cells.Cluster analysis of the odour spectra revealed the existence of moderately separated clusters of olfactory receptors. No groups of cells specifically tuned to odours of eggs or cabbage leaves were found. Analysis of the response profiles did not show a clear clustering of stimuli. The results suggested that the membranes of different receptor cells contain several types of acceptor sites in various proportions.No correlation between EAG and receptor potential amplitude or spike frequency was found. It was concluded that the EAG may be useful in comparing the relative receptor sensitivities only when chemically related compounds are involved.     

Chemosensory basis of behavioural plasticity in response to deterrent plant chemicals in the larva of the Small Cabbage White butterfly Pieris rapae

Behavioural and electrophysiological responsiveness to three chemically different secondary plant substances was studied in larvae of Pieris rapae L. (Lepidoptera: Pieridae). Three groups of caterpillars were studied that during their larval development were exposed to different rearing diets: an artificial diet or one of two host-plants, cabbage, Brassica oleracea, or nasturtium, Tropaeolum majus. In dual-choice leaf disc assays, caterpillars reared on cabbage were strongly deterred by the phenolic chlorogenic acid, the flavonol glycoside naringin and the alkaloid strychnine. However, behavioural plasticity was found in caterpillars reared on nasturtium or artificial diet in that these did not discriminate against chlorogenic acid. Caterpillars reared on the artificial diet were also significantly less sensitive to naringin and strychnine in the behavioural assay. Electrophysiological studies of the maxillary sensilla styloconica revealed that the deterrent neuron in the medial sensillum, but not in the lateral sensillum, of cabbage-reared caterpillars was more sensitive than the same neuron type of caterpillars reared on nasturtium or artificial diet. We conclude that (1) the diet-induced behavioural habituation to deterrents can at least partly be explained by chemosensory desensitisation of a generalist type of maxillary deterrent neuron; (2) behavioural cross-habituation to the three structurally diverse deterrent compounds can be traced back to cross-sensitivity for these compounds in the same gustatory neuron.     

Constituents of Musa balbisiana seeds and their activity against Cryptolestes pusillus

The chloroform and acetone extracts of the seeds of Musa balbisiana Colla (Musaceae) and four of its constituents were assayed in the diet for toxicity against the flat grain beetle (Cryptolestes pusillus Schöherr). A mixture of fatty esters of phytol and the flavan (+)-epiafzelechin showed a significant growth inhibition and toxicity against this economically important pest of stored cereals. The LD₅₀ was 6.3% for the most active compound, (+)-epiafzelechin.     

Lichen chemistry is concordant with multilocus gene genealogy in the genus Cetrelia (Parmeliaceae,Ascomycota)

The lichen genus Cetrelia represents a taxonomically interesting case where morphologically almost uniform populations differ considerably from each other chemically. Similar variation is not uncommon among lichenized fungi, but it is disputable whether such populations should be considered entities at the species level. Species boundaries in Cetrelia are traditionally delimited either as solely based on morphology or as combinations of morpho- and chemotypes. A dataset of four nuclear markers (ITS, IGS, Mcm7, RPB1) from 62 specimens, representing ten Cetrelia species, was analysed within Bayesian and maximum likelihood frameworks. Analyses recovered a well-resolved phylogeny where the traditional species generally were monophyletic, with the exception of Cetrelia chicitae and Cetrelia pseudolivetorum. Species delimitation analyses supported the distinction of 15 groups within the studied Cetrelia taxa, dividing three traditionally identified species into some species candidates. Chemotypes, distinguished according to the major medullary substance, clearly correlated with clades recovered within Cetrelia, while samples with the same reproductive mode were dispersed throughout the phylogenetic tree. Consequently, delimiting Cetrelia species based only on reproductive morphology is not supported phylogenetically. Character analyses suggest that chemical characters have been more consistent compared to reproductive mode and indicate that metabolite evolution in Cetrelia towards more complex substances is probable.     

Characterization of cell clusters in larval hemolymph of the cabbage armyworm Mamestra brassicae and their role in maintenance of hemocyte populations

Maintenance of hemocyte populations is critical for both development and immune responses. In insects, the maintenance of hemocyte populations is regulated by mitotic division of circulating hemocytes and by discharge from hematopoietic organs. We found cell clusters in the hemolymph of Mamestra brassicae larvae that are composed of small, spherical cells. Microscopic observations revealed that the cells in these clusters are similar to immature or precursor cells present in hematopoietic organs. The results of bromodeoxyuridine (BrdU) incorporation experiments demonstrate that these cells are mitotically active. Furthermore, these cells maintain their immature state and proliferate until late in the last larval instar. The results of in vitro experiments showed that most of the cells changed their morphology to one consistent with plasmatocytes or granulocytes, and that the change was promoted by addition of larval hemolymph to the culture medium, in particular when hemolymph was collected at a prepupal stage. Taken together, our results suggested that cells in clusters may be an additional source of hemocytes during larval development.