Biosynthesis of ecdysones in isolated prothoracic glands and oenocytes of Tenebrio molitor in vitro

Isolated prothoracic glands from Tenebrio larvae synthesize in vitro α-ecdysone, but not β-ecdysone from 4-¹⁴C-cholesterol. Isolated abdominal oenocytes from the larvae synthesize mainly β-ecdysone, but only little α-ecdysone. When prothoracic glands and oenocytes are cultured together, the α-ecdysone derived from the prothoracic glands is oxidized by the oenocytes to β-ecdysone. The newly synthesized hormones are not stored in the cells, but are secreted into the medium if sufficient amounts of non-labelled hormones are present. If no unlabelled hormones are added to the culture medium, the newly formed hormones are converted to a large extent into polar conjugates.     

Presence of sialic acid in prothoracic glands of Galleria mellonella (Lepidoptera)

The presence of sialic acid (SA) in prothoracic glands (PGs) of Galleria mellonella was determined by the methods of electron microscopy (EM), histochemistry, spectrophotometry (SP) and electronic ionization (EI)-mass spectroscopy. Histochemical observations were carried out by the cationic dye ruthenium red (RR), staining with and without neuraminidase digestion in the larval stage. Neuraminidase-sensitive SA was demonstrated by the decrease in the amount of RR-binding following neuraminidase digestion. The total amount of SA was found to be 0.09016 mg g(-1) in dry tissue by spectrophotometric determination. EI-mass spectroscopy results confirmed the EM and SP observations. The fragmentation scheme derived from EI-mass analysis exhibited the presence of the lactonized form of Neu5Gc7, 9Ac(2). On the basis of the various pieces of evidence described above, it was firmly concluded that Neu5Gc7, 9Ac(2) molecules were present in PGs of G. mellonella.     

Larval growth and allometry in the cabbage butterfly Pieris brassicae (Lepidoptera: Pieridae)

By adopting a longitudinal study design and through geometric morphometrics methods, we investigated individual and ontogenetic variation in size, shape and timing during larval development of the cabbage butterfly Pieris brassicae under laboratory conditions. We found that ontogenetic size progression departs modestly, but significantly, from growth at a constant rate and that size at hatching contributes considerably to determine the size of the individual at all subsequent stages. As for the shape, ontogenetic allometry is much more conspicuous than static allometry, the latter in many cases being close to isometry. Analysis of developmental timing revealed a stage of apparently more effective developmental control at stage 3, supported by both the relatively small variance in cumulative developmental time up to stage 3 and by the pattern of correlation between duration of single stages. While presenting detailed quantitative aspects of growth in P. brassicae, in particular with respect to individual variation, this study and the associated dataset can provide a basis for further explorations of the post‐embryonic development in this insect and contribute to the ongoing investigations on growth regulation and control in insects.     

Juvenile hormone regulates the titer of a hemolymph factor that enhances ecdysone synthesis by insect (Manduca sexta) prothoracic glands in vitro

The hemolymph of last instar Manduca sexta larvae contains a protein factor that enhances ecdysone synthesis by prothoracic glands in vitro. The titer of the factor fluctuates during development in a pattern that suggests that it is regulated by juvenile hormone (JH). In untreated control larvae, the titer drops from 2.17 U ml^?1 on day 1 to 0.27 U ml^?1 on day 3. When larvae were treated with (7S)-hydroprene (a JH analog), the titer remained elevated (2.09 U ml^?1 on day 3). JH I, however, was ineffective in preventing the precommitment drop in the titer of the factor. After pupal commitment, the titer of the factor increases in untreated larvae from 0.84 U ml^?1 on day 5 to 1.62 U ml^?1 on day 7. This increase was blocked when the sources of JH (the corpora allata) were removed on day 5 by head ligation. When head-ligated day 5 larvae were treated with either (7S)-hydroprene or JH I, the titer of the factor was driven to a level (1.88 U ml^?1 and 2.05 U ml^?1, respectively) that was not significantly different from that found in untreated day 7 larvae (1.62 U ml^?1). The combined results indicate the titer of the hemolymph factor is regulated by JH.     

In vitro stimulation of the prothoracic gland of the insect as a test system (prothoracic gland test)

In vitro cultures of prothoracic glands of larvae of Periplaneta americana and of some Lepidoptera as biological tests are described. Incorporation of ³H-5-uridine in the RNA of the prothoracic glands represented the measure of the cellular activity of the glands.Activation factor I separated from extracts of corpora cardiaca of the cockroach Periplaneta americana by means of gel filtration techniques caused significant stimulation of RNA synthesis of the glands.     

Development of the parasitoid,Cotesia rubecula (Hymenoptera: Braconidae) in Pieris rapae and Pieris brassicae (Lepidoptera: Pieridae): evidence for host regulation

Several recent models examining the developmental strategies of parasitoids attacking hosts which continue feeding and growing after parasitism (=koinobiont parasitoids) assume that host quality is a non-linear function of host size at oviposition. We tested this assumption by comparing the growth and development of males of the solitary koinobiont endoparasitoid, Cotesia rubecula, in first (L1) to third (L3) larval instars of its preferred host, Pieris rapae and in a less preferred host, Pieris brassicae. Beginning 3 days after parasitism, hosts were dissected daily, and both host and parasitoid dry mass was determined. Using data on parasitoid dry mass, we measured the mean relative growth rate of C. rubecula, and compared the trajectories of larval growth of the parasitoid during the larval and pupal stages using non-linear equations. Parasitoids generally survived better, completed development faster, and grew larger in earlier than in later instars of both host species, and adult wasps emerging from P. rapae were significantly larger than wasps emerging from all corresponding instars of P. brassicae. During their early larval stages, parasitoids grew most slowly in L1 P. rapae, whereas in all other host classes of both host species growth to pupation proceeded fairly uniformly. The growth of both host species was markedly reduced after parasitism compared with controls, with the development of P. brassicae arrested at an earlier stage, and at a smaller body mass, than P. rapae. Our results suggest that C. rubecula regulates certain biochemical processes more effectively in P. rapae than in P. brassicae, in accordance with its own nutritional and physiological requirements. Furthermore, we propose that, for parasitoids such as C. rubecula, which do not consume all host tissues prior to pupation, that parasitoid size and host quality may vary independently of host size at oviposition and at larval parasitoid egression.     

BAC selection of the large white butterfly,Pieris brassicae (Lepidoptera: Pieridae) containing orthologs of the silkworm,Bombyx mori (Lepidoptera: Bombycidae)

Applied Entomology and Zoology - We constructed a bacterial artificial chromosome (BAC) library from the large white butterfly, Pieris brassicae (L.) (Lepidoptera: Pieridae), which...     

Conversion of 3-dehydroecdysone by a ketoreductase in post-diapause, pre-hatch eggs of the gypsy moth Lymantria dispar

The prothoracic glands (PGs) of Lymantria dispar (day-5 female, last-stage larvae) produce both ecdysone and an ecdysteroid which has the same retention time on reverse-phase liquid chromatography (RPLC) as a known standard of 3-dehydroecdysone. The latter ecdysteroid can be converted by a heat-labile factor in extracts of post-diapause, pre-hatch L. dispar eggs to an ecdysteroid which has the same retention time on RPLC as ecdysone. Purified 3-dehydroecdysone, similarly treated with egg extract, also gives the same retention time on RPLC as ecdysone. Taken together, these data suggest that, like Manduca sexta, a major product of the PGs in L. dispar is 3-dehydroecdysone. Furthermore, these data suggest that L. dispar eggs, which contain mature embryos, possess ecdysteroid ketoreductase activity capable of converting 3-dehydroecdysone to ecdysone. This is the first report of ecdysteroid ketoreductase activity in embryonated eggs.     

Aestivation in Pieris brassicae (L.) affects the parasitoid load caused by Cotesia glomerata (L.)

The large white butterfly Pieris brassicae (L.) (Lepidoptera: Pieridae) undergoes a unique aestivation in southern Spain. Its pupae remain dormant for three months in summer, emerging in early September. Its main parasitoid, Cotesia glomerata (L.) (Hymenoptera: Braconidae), shows no such diapause behavior and is thus deprived of its main host for this period and has to switch to less suitable host species. To study the effect the aestivation has on the parasitoids, caterpillars were collected from the region in May and September and levels of parasitization were determined. Results show that parasitoid attacks decrease clearly after the summer diapause. The number of parasitized butterfly clutches in September was only one third as the number in May and the infestation rate of an attacked clutch decreased by 55% after aestivation. The distribution of brood sizes of C. glomerata showed clear signs of superparasitism before but not after the diapause. Therefore, the butterfly generation after summer diapause has to deal with distinctly diminished numbers of parasitoids. This increases the survival rate of the caterpillar and thus improves the fitness of the butterfly.     

Structure,development and death of sensory cells and neurons in the pupal labial palp of the butterflies Pieris rapae L. and Pieris brassicae L. (Insecta,Lepidoptera)

Summary The development of neurons possibly related to the outgrowth of axons from the labial palp-pit organ was studied in Pieris rapae. Serial sections of six successive stages between pupation and emergence of the imago were examined with the electron microscope. At pupation the palp contains an apical scolopidial organ (ASO) and cellular strands connected to it. The ASO consists of three type-1 scolopidia, which are characterized by the presence of a ciliary 9 × 2 + 0 pattern throughout the dendritic outer segment and a ciliary dilation beneath the cap. The scolopidia show two special features: (i) the dendritic outer segments reach beyond the cap, and (ii) an intricate junctional complex develops between the dendritic inner segments and the scolopale cells. The cellular strands comprise two types of cells: (1) bipolar cells regarded as neurons due to their cytological features, and (2) enveloping cells, which are wrapped around the bipolar cells. The strands degenerate about 10 h after pupation. The sensory cells of the ASO degenerate consecutively between 28 h and 130 h after pupation. However, their enveloping cells survive and endure in the imago, which emerges about 160 h after pupation. An ASO similarly lacking sensory cells was observed in imagines of Pieris brassicae. It is hypothesized that the ASO and the bipolar neurons of the strands play a role in pathfinding of the axons of the labial palp-pit organ.Supported by the Deutsche Forschungsgemeinschaft (SFB 4/G1)     

Electrophysiological responses of gustatory sensilla of Mamestra brassicae (Lepidoptera,Noctuidae) larvae to three ecdysteroids: ecdysone, 20-hydroxyecdysone and ponasterone A

Specialised phytophagous Lepidoptera such as Bombyx mori and Pieris brassicae have contact chemoreceptors that perceive ecdysteroids at very low concentrations. This sensory perception allows them to feed on substrates with a high content of phytoecdysteroids. We have evaluated if a polyphagous insect like Mamestra brassicae does possess contact chemoreceptor cells that are sensitive to these molecules. Electrophysiological recordings were performed from contact chemoreceptors located on the maxilla. These receptors were stimulated with some sugars, amino acids and salts and with three ecdysteroids. Our results demonstrate that a specific cell within the lateral sensilla responds to 20-hydroxyecdysone and ponasterone A but not to ecdysone.     

In vitro metabolism of 3beta-hydroxy-, and 3beta,14alpha-dihydroxy-[3alpha-3H]-5beta-cholest-7-en-6-one by the prothoracic glands of Manduca sexta.

alpha-Ecdysone (2beta,3beta,14alpha,22R,25-pentahydroxy-5beta-cholest-7-en-6-one) has been identified as the metabolism product of 3beta,14alpha-dihydroxy-5beta-cholest-7-en-6-one in isolated prothoracic glands of the tobacco hornworm, Manduca sexta. In contrast, 3beta-hydroxy-5beta-cholest-7-en-6-one is metabolized to 14-deoxy-alpha-ecdysone and a variety of intermediates all lacking the 14-hydroxy group. The results suggest that either the normal precursor for the synthesis of alpha-ecdysone by prothoracic glands is a sterol more highly oxygenated than cholesterol or that hydroxylation of a minimally oxygenated precursor at C-14 must precede introduction of the C-6 ketone and/or delta7 bond. The data further suggest that several alternative hydroxylation routes may exist for the latter steps of alpha-ecdysone biosynthesis.     

Molecular characterization of a defensin in the IZD-MB-0503 cell line derived from immunocytes of the insect Mamestra brassicae (Lepidoptera)

The induction of anti-microbial peptides against Gram positive and negative bacteria in the IZD-MB-0503 cell line from the lepidopteran Mamestra brassicae is demonstrated, while no anti-fungal activity is detected. The identification of a defensin-like molecule active against Gram positive bacteria is described for the first time in Lepidoptera. This molecule shows between 43% and 59% homology with group A defensins from other dipteran and hymenopteran species.     

Egg-mimics of Streptanthus (Cruciferae) deter oviposition by Pieris sisymbrii (Lepidoptera: Pieridae)

Summary Streptanthus breweri, a serpentine-soil annual mustard, produces pigmented callosities on its upper leaves which are thought to mimic the eggs of the Pierid butterfly Pieris sisymbrii. P. sisymbrii is one of several inflorescence-infructescence-feeding Pierids which assess egg load visually on individual host plants prior to ovipositing. Removal of the egg-mimics from S. breweri plants in situ significantly increases the probability of an oviposition relative to similar, intact plants.     

Presence of IL-1- and TNF-like molecules in Galleria mellonella (Lepidoptera) haemocytes and in an insect cell line Fromestigmene acraea (Lepidoptera).

In this study the authors give immunocytochemical evidence for the presence of interleukin (IL)-1alpha- and tumour necrosis factor (TNF) alpha-like molecules in the haemocytes of last instar larvae from the greater wax moth Galleria mellonella. Similar results are demonstrated in a continuous haemocyte line (BTI-EA-1174-A) from the salt marsh caterpillar Estigmene acraea. In Galleria mellonella larvae granular cells show a strong positive reaction with both primary antibodies, whereas plasmatocytes are stained to a lesser extent. Cell line haemocytes also react positively with both antibodies. After activating the cells with lipopolysaccharide (LPS) staining of Estigmene acraea cells is decreased, whereas Galleria mellonella haemocytes show no visible reaction in comparison to non-activated cells.