The instantly released Drosophila immune proteome is infection-specific

In this study, we analyzed the hemolymph proteome of Drosophila third instar larvae, which were induced with a suspension of Gram-positive bacteria or yeast. Profiling of the hemolymph proteins of infected versus non-infected larvae was performed by two-dimensional difference gel electrophoresis. Infection with Micrococcus luteus or Saccharomyces cerevisiae induced, respectively, 20 and 19 differential protein spots. The majority of the spots are specifically regulated by one pathogen, whereas only a few spots correspond to proteins altered in all cases of challenging (including after challenge with lipopolysaccharides). All of the upregulated proteins can be assigned to specific aspects of the immune system, as they did not increase in the hemolymph of sterile pricked larvae. Next to known immune proteins, unannotated proteins were identified such as CG4306 protein, which has homologues with unknown function in all metazoan genome databases available today.     

Locustatachykinin III and IV: two additional insect neuropeptides with homology to peptides of the vertebrate tachykinin family

Two myotropic peptides termed locustatachykinin III and IV were isolated from 9000 brain-corpora cardiaca-corpora allata-suboesophageal ganglion extracts of the locust, Locusta migratoria. The primary structures of Lom-TK III and IV were established as amidated decapeptides: Ala-Pro-Gln-Ala-Gly-Phe-Tyr-Gly-Val-Arg-NH2 (Lom-TK III) and Ala-Pro-Ser-Leu-Gly-Phe-His-Gly-Val-Arg-NH2 (Lom-TK IV). The locustatachykinins were synthesized and shown to have chromatographic and biological properties identical with those of the native materials. They stimulate visceral muscle contractions of the oviduct and the foregut of Locusta migratoria and of the hindgut of Leucophaea maderae. Both peptides exhibit sequence homologies with the vertebrate tachykinins. Sequence similarity is greater with the fish and amphibian tachykinins (up to 40%) than with the mammalian tachykinins. In addition, the intestinal and oviducal myotropic activity of the locustatachykinins is analogous to that of vertebrate tachykinins. Both chemical and biological similarities of vertebrate and insect tachykinins substantiates the evidence for a long evolutionary history of the tachykinin peptide family.     

Locustakinin, a novel myotropic peptide from Locusta migratoria, isolation, primary structure and synthesis.

The isolated hindgut of the cockroach, Leucophaea maderae is a very efficient bioassay tool for the monitoring of certain structural types of insect myotropic peptides during HPLC purification. Using this detection system, a six residue peptide has been isolated from an extract of 9000 brain corpora cardiaca-corpora allata suboesophageal ganglion complexes of Locusta migratoria. Amino acid composition and sequence analysis combined with enzymatic digestion data established the structure of the novel peptide as Ala-Phe-Ser-Ser-Trp-Gly-amide. The chromatographic and biological properties of the synthetic peptide were the same as those of the native peptide, thus confirming structural analysis. The carboxy-terminal pentamer sequence is the active core of leucokinins II, V and VII and of achetakinin III (myotropic neuropeptides isolated from Leucophaea m. and from Acheta domesticus; Holman et al., 1990). Furthermore, the octapeptide leucokinin VII contains the novel sequence as its carboxy-terminal hexamer and Achetakinin V (AFHSWGamide) differs from it by one residue. This new peptide designated as locustakinin I (locusts) may therefore represent an evolutionary molecular link between leucokinin VII (cockroaches) and achetakinin V (crickets). Using synthetic locustakinin, physiological studies will be performed in the locust. In view of the known effects of leucokinins, locustakinin may be important in the stimulation of ion transport and inhibition of diuretic activity in Malpighian tubules. This study indicates that the AFXSWGamide sequence appears to have been well conserved and that members of this peptide family may be widely distributed among insects and posses a number of functions.     

Three dimensional typological studies using scanning electron microscopy for characterization of Termitomyces pellets obtained from submerged growth conditions

There are gaps in existing understanding of fungal pellet growth dynamics. We used scanning electron microscopy (SEM) for morphological characterization of the biomass organization of Termitomyces pellets for seven species: T. microcarpus (TMI1), T. albuminosus (TAL1, TAL2), T. striatus (TSTR), T. aurantiacus (TAUR), T. heimii (THE1, THE2), T. globulus (TGLO) and T. clypeatus (TCL1, TCL2, TCL3, TCL4, TCL5). We assessed the utility of SEM for morphological and structural characterization of Termitomyces spp. in three dimensional (3D) pellet form to identify ideal pellet morphology for industrial use. Typological classification of Termitomyces species was based on furrows, isotropy, total motifs and fractal dimensions. The pellets formed were entangled and exhibited highly compacted mycelial mass with microheterogeneity and microporosity. The mean density of furrows of Termitomyces species was between 10,000 and 11,300 cm/cm², percentage isotropy was 30?80 and total motifs varied from 300 to 2500. TGLO exhibited the highest furrow mean density, 11243 cm/cm², which indicated a compact, cerebroid structure with complex ridges and furrows, whereas TAL2 exhibited the lowest furrow density. TMI1a exhibited a high percentage isotropic value, 74.6, TSTR exhibited the lowest, 30.9. Total motif number also was used as a typological classification parameter. Fractal values were 2.64?2.78 for various submerged conditions of Termitomyces species. TAL1 exhibited the highest fractal dimension and TAL2 the lowest, which indicates the complexity of branching patterns. Three-dimensional SEM image analysis can provide insight into pellet micromorphology and is a powerful tool for exploring topographical details of pellets.     

Identification of peptide hormones of the amphipathic helix class using the helical hydrophobic moment algorithm

Eisenberg's helical hydrophobic moment (less than mu H greater than) algorithm was applied to the analysis of the primary structure of amphipathic alpha-helical peptide hormones and an optimal method for identifying other peptides of this class determined. We quantitate and compare known amphipathic helical peptide hormones with a second group of peptides with proven nonamphipathic properties and determine the best method of distinguishing between them. The respective means of the maximum 11 residue less than mu H greater than for the amphipathic helical and control peptides were 0.46 (+/-/-0.07) and 0.33 (0.07) (P + 0.004). To better reflect the amphipathic potential of the entire peptide, the percent of 11 residue segments in each peptide above a particular less than mu H greater than was plotted vs less than mu H greater than. The resulting curves are referred to as HM-C. The mean HM-C (of the two groups) was highly significantly different such that the HM-C method was superior to others in its ability to distinguish amphipathic from nonamphipathic peptides. Several potential new members of this structural class were identified using this approach. Molecular modeling of a portion of one of these, prolactin inhibitory factor, reveals a strongly amphipathic alpha helix at residues 4-21. This computer-based method may enable rapid identification of peptides of the amphipathic alpha-helix class.     

Effect of abscisic acid on vitellogenesis in Sarcophaga bullata

In the carnivorous dipteran Sarcophaga bullata Parker, vitellogenesis was partially inhibited by injection of two doses of 12 g abscisic acid (ABA). There was no significant difference between injections on day 2 and 4, or on day 4 and 6. Higher and lower doses were less effective. The mixture of isomers inhibited vitellogenesis more than the cis-trans isomer. ABA had no effect on the total lipid concentration of the haemolymph but it inhibited the sharp increase in total protein concentration of the haemolymph and particularly that of vitellogenin, which normally occurred within 24 hr following liver feeding on day 4. Since vitellogenin synthesis is under the control of moulting hormone (MH), the MH activity was measured by radioimmunoassay to find out whether ABA might interact with vitellogenin synthesis via its hormonal inductor. Eight hours after liver feeding, there was a MH peak in the control groups while following ABA treatment this peak occurred after 18 hr. The inhibitory effect of ABA on vitellogenesis could be overruled by feeding sugar impregnated with ecdysterone (5 mg/g), not by topical application of JH. Our results suggest that ABA might interact with a mechanism which phytophagous and non-phytophagous insects share in common. If in phytophagous insects the same amount of ABA per gram weight, as was effective in Sarcophaga (about 200 g/g), is needed to reduce fecundity, it is not probable that this plant hormone plays a role in the seasonal synchronisation of the growth and reproduction of insects with the senescence of their host plants.

---

Résumé La vitellogenèse du diptère carnivore, Sarcophaga bullata a été partiellement inhibée par injection de deux doses de 12 g d'acide absisique (ABA). Il n'y avait pas de différence significative entre les injections du deuxième et quatrième jour, ou du quatrième et sixième jour. La mixture d'isomères a plus inhibé la vitellogenèse que le cis-trans isomère.ABA n'a pas eu d'effect sur la concentration totale des lipides de l'hémolymphe, mais il a inhibé l'augmentation brutale de la concentration protéique totale de l'hémolymphe, et particulièrement celle de la vitellogénine qui se produit normalement dans les 24 h après consommation de foie le 4ème jour. La synthèse de la vitellogénine étant sous le contrôle de l'hormone de mue (MH), l'activité MH a été mesurée par radio-immunologie, pour découvrir si l'ABA agissait sur la synthèse de la vitellogénine par intermédiaire de son inducteur hormonal. Huit heures après alimentation sur foie, le taux de MH chez les témoins était élevé tandis qu'après traitement avec ABA ce pic n'apparaissait qu'après 18 h. L'effet inhibiteur de l'ABA sur la vitellogénine a pu être neutralisé en fournissant aux mouches du sucre impregné d'ecdystérone (5mg/g), mais non par application locale d'hormone juvénile. Nos résultats suggèrent qu'ABA agit probablement sur un mécanisme commun aux insectes phytophages et non phytophages.Il est peu probable que l'acide absisique joue un rôle dans la synchronisation saisonnière de la croissance et de la reproduction des insectes avec la sénescence des plantes-hôtes.

     

Effects of synthetic cecropia juvenile hormone and of a mimic substance, applied to different stages of the cabbage maggot, Hylemyia brassicae

Both synthetic cecropia juvenile hormone (I) and the juvenile hormone mimic substance 1-(4-ethylphenoxy)-6,7-epoxy-3,7-dimethyl-2-octene (II), possess ovicidal activity against freshly laid eggs of the cabbage maggot, Hylemyia brassicae. Topical application of both products to last-instar larvae and young pupae prevents the adults escaping from the puparium. Mixing the hormone substances with the liquid food of the adults, or topical application to adults, even in very high concentrations, does not influence fecundity or fertility.

---

Résumé L'hormone juvénile synthétique (I) (3, 11-diméthyl 7-éthyl 10-époxy 2,6-tridécadiénoate de méthyle, isomère E, E, Z) et le mimétique de l'hormone juvénile (II) (1-(4-éthylphénoxy)-6,7-époxy-3,7-diméthyl-2-octene) possèdent une action ovicide sur les ufs fraîchement pondus de la mouche du chou (H. brassicae (Bouché)). Le dernier produit semble avoir une action ovicide plus prononcée. Les deux produits semblent également avoir un effet répulsif vu que le nombre moyen d'ufs pondus par femelle diminue quand la concentration augmente.Une application topique sur les larves du dernier stade et sur les pupes, inhibe l'émergence des imagines qui n'accusent pourtant aucune malformation. Cinq g (II) par larve empêchent l'éclosion de 90% des imagines; dix g (II) par pupe inhibent totalement l'émergence.Le mélange du produit (II) à l'alimentation ou une application directe sur la mouche adulte n'influence ni la fécondité ni la fertilité.

     

Immunocytochemical demonstration of proopiomelanocortin- and other opioid-related substances and a CRF-like peptide in the gut of the american cockroach,Periplaneta americana L.

Summary Using the peroxidase-antiperoxidase technique, we showed the presence of peptides which are immunologically resembling mammalian corticotropin releasing hormone (CRF)-, adrenocorticotropic hormone (ACTH)-, -endorphin (-END)-, -melanocyte stimulating hormone (-MSH)-, methionine-enkephaline (met-ENK)- and leucine enkephaline (leu-ENK)- like immunoreactivity in hundreds to thousands of endocrine cells and nerve fibers in the midgut of the American cockroach Periplaneta americana.In the cockroach hindgut no immunoreactive cell bodies could be observed, although nerve fibers were clearly noticed to be recognized by antisera to CRF, ACTH_1–24, ACTH_11–24 and -END.Nothing is exactly known as to the function(s) of the demonstrated materials, but one can speculate that these numerous immunoreactive cells, might have important paracrine and/or endocrine functions in the insect physiology.     

Functional differentiation of amphiphilic helices of the apolipoproteins by hydrophobic moment analysis

The amphiphilic character of different plasma apolipoproteins was investigated by a combination of established hydrophobicity analysis methods. These methods proved to be powerful in the detection of amphiphilic phospholipid-binding domains. Within this class of lipid-binding domains, lecithin-cholesterol acyltransferase activating and non-activating helices could be differentiated by calculating hydrophobic moments at different angles. We conclude that the hydrophobic characteristics of the different helices determined the mode of lipid binding and the substrate properties of these phospholipid-protein complexes for the lecithin-cholesterol acyltransferase reaction.     

Immunohistochemical localisation of the hypertrehalosaemic hormone II (Cam-HrTH-II) and related peptides in the nervous system of Carausius morosus and Sarcophaga bullata

Summary A polyclonal antiserum was prepared against an N-terminal modified Cam-HrTH-II (Leu-Asn-Phe-...), one of the members of the large AKH/RPCH peptide family, first isolated from Carausius morosus. The localisation of this peptide was performed by means of immunocytochemical methods in the brain and corpora cardiaca-corpora allata complex of the stick insect, Carausius morosus and the grey fleshfly, Sarcophaga bullata. The distribution patterns of molecules reactive to the Cam-HrTH-II and the LomAKH-I antisera in both insect species were compared. In Carausius, both antisera reacted in the same cell bodies. In Sarcophaga, some neurons were stained by both, others only by one of the two antisera. By combining two different antisera, we demonstrated that there are no Lom-AKH-I-like molecules present in Carausius and that there must occur at least three different AKH-like molecules in the brain of Sarcophaga. One is similar to Cam-HrTH-II, the second to Lom-AKH-I and the third is an AKH/RPCH-like peptide, different from Lom-AKH-I and Cam-HrTH-II.