Soluble proteins of chemical communication in the social wasp <Emphasis Type="Italic">Polistes dominulus</Emphasis>

Members of the odorant-binding protein (OBP) and chemosensory protein (CSP) families were identified and characterised in the sensory tissues of the social wasp Polistes dominulus (Hymenoptera: Vespidae). Unlike most insects so far investigated, OBPs were detected in antennae, legs and wings, while CSPs appeared to be preferentially expressed in the antennae. The OBP is very different from the homologous proteins of other Hymenopteran species, with around 20% of identical residues, while the CSP appears to be much better conserved. Both OBP and CSP, not showing other post-translational modifications apart from disulphide bridges, were expressed with high yields in a bacterial system. Cysteine pairing in the recombinant and native proteins follows the classical arrangements described for other members of these classes of proteins. OBPs isolated from the wings were found to be associated with a number of long-chain aliphatic amides and other small organic molecules. Binding of these ligands and other related compounds was measured for both recombinant OBP and CSP.Received 14 May 2003; received after revision 8 June 2003; accepted 12 June 2003     

Cooperative interactions between odorant-binding proteins of Anopheles gambiae

To understand olfactory discrimination in Anopheles gambiae, we made six purified recombinant OBPs and investigated their ligand-binding properties. All OBPs were expressed in bacteria with additional production of OBP47 in the yeast Kluveromyces lactis. Ligand-binding experiments, performed with a diverse set of organic compounds, revealed marked differences between the OBPs. Using the fluorescent probe N-phenyl-1-naphthylamine, we also measured the binding curves for binary mixtures of OBPs and obtained, in some cases, unexpected behaviour, which could only be explained by the OBPs forming heterodimers with binding characteristics different from those of the component proteins. This shows that OBPs in mosquitoes can form complexes with novel ligand specificities, thus amplifying the repertoire of OBPs and the number of semiochemicals that can be discriminated. Confirmation of the likely role of heterodimers was demonstrated by in situ hybridisation, suggesting that OBP1 and OBP4 are co-expressed in some antennal sensilla of A. gambiae.     

<Emphasis Type="Italic">Anopheles gambiae</Emphasis> odorant binding protein crystal complex with the synthetic repellent DEET: implications for structure-based design of novel mosquito repellents

Insect odorant binding proteins (OBPs) are the first components of the olfactory system to encounter and bind attractant and repellent odors emanating from various sources for presentation to olfactory receptors, which trigger relevant signal transduction cascades culminating in specific physiological and behavioral responses. For disease vectors, particularly hematophagous mosquitoes, repellents represent important defenses against parasitic diseases because they effect a reduction in the rate of contact between the vectors and humans. OBPs are targets for structure-based rational approaches for the discovery of new repellent or other olfaction inhibitory compounds with desirable features. Thus, a study was conducted to characterize the high resolution crystal structure of an OBP of Anopheles gambiae, the African malaria mosquito vector, in complex with N,N-diethyl-m-toluamide (DEET), one of the most effective repellents that has been in worldwide use for six decades. We found that DEET binds at the edge of a long hydrophobic tunnel by exploiting numerous non-polar interactions and one hydrogen bond, which is perceived to be critical for DEET’s recognition. Based on the experimentally determined affinity of AgamOBP1 for DEET (K _d of 31.3 μΜ) and our structural data, we modeled the interactions for this protein with 29 promising leads reported in the literature to have significant repellent activities, and carried out fluorescence binding studies with four highly ranked ligands. Our experimental results confirmed the modeling predictions indicating that structure-based modeling could facilitate the design of novel repellents with enhanced binding affinity and selectivity.     

Expression and immunolocalisation of odorant-binding and chemosensory proteins in locusts

We have identified, cloned and expressed a new chemosensory protein (CSP) in the desert locust Schistocerca gregaria belonging to a third sub-class of these polypeptides. Polyclonal antibodies stained a band of 14 kDa, as expected, in the extracts of antennae and palps of the adults, but not in the 4th and 5th instars. In the related species Locusta migratoria, instead, the same antibodies cross-reacted only with a band of apparent molecular mass of 35 kDa in the extract of 1st–5th instars, but not in the adults. The recombinant protein binds the fluorescent probe N-phenyl-1-naphthylamine, but none of the compounds so far reported as pheromones for S. gregaria. The expression of the odorant-binding protein (OBP) and of CSPs of sub-classes I and II was also monitored in antennae, tarsi, palpi, wings and other organs of solitary and gregarious locusts in their nymphal and adult stages. OBP was found to be antenna specific, where it is expressed at least from the 3rd instar in both solitary and gregarious locusts. CSPs, instead, appear to be more ubiquitous, with different expression patterns, according to the sub-class. Immunocytochemistry experiments revealed that OBP is present in the sensillum lymph of sensilla trichodea and basiconica, while CSP-I and CSP-III were found in the outer sensillum lymph of sensilla chaetica and in the sub-cuticular space between epidermis and cuticle of the antenna. Sensilla chaetica on other parts of the body showed the same expression of CSP-I as those on the antenna.Received 11 Janury 2005; received after revision 21 February 2005; accepted 18 March 2005X. Jin and A. Brandazza contributed equally to this work.     

Biochemical and immunochemical evidence for the origin of the spermatophore material inGlossina morsitans morsitans Westwood

Summary Protein patterns in secretions from fully differentiated male accessory reproductive glands (ARG), spermatophore (Sp) and testes (Te) of the tsetse,Glossina morsitans morsitans Westwood, were determined by isoelectrofocusing. Isoelectrofocusing patterns of total ARG proteins and those of Sp were remarkably similar. At least 27 bands were detected in ARG and Sp. Out of these, 13 were major protein bands and isoelectrofocused in the pl range of 4 and 6.55. About 10 of these 13 were found to be acidic. Ouchterlony immunodiffusion and straight line immunoelectrophoresis showed that male accessory reproductive gland secretory proteins and spermatophore share common immunological characteristics which are different from those of the testes.     

Sterility in tsetse flies (Glossina morsitans Westwood) caused by loss of symbionts

Summary Tsetse flies fed on blood containing oxytetracycline, sulphaquinoxaline or lysozyme do not reproduce. It could be proved that primarily the symbionts in flies are damaged, which secondarily leads to sterility.I thank Dr.A. M. Jordan, Tsetse Research Laboratory at Bristol, for providing me with pupae ofG. morsitans, Dr.H. Wetzel, International Atomic Energy Authority, Vienna, for providing antibiotictreated flies and the Gesellschaft für Strahlen und Umweltforschung, München, for financial support.     

The role of bHLH genes in ear development and evolution: revisiting a 10-year-old hypothesis

In mouse ear development, two bHLH genes, Atoh1 and Neurog1, are essential for hair cell and sensory neuron differentiation. Evolution converted the original simple atonal-dependent neurosensory cell formation program of diploblasts into the derived developmental program of vertebrates that generates two neurosensory cell types, the sensory neuron and the sensory hair cell. This transformation was achieved through gene multiplication in ancestral triploblasts resulting in the expansion of the atonal bHLH gene family. Novel genes of the Neurogenin and NeuroD families are upregulated prior to the expression of Atoh1. Recent data suggest that NeuroD and Neurogenin were lost or their function in neuronal specification reduced in flies, thus changing our perception of the evolution of these genes. This sequence of expression changes was accompanied by modification of the E-box binding sites of these genes to regulate different downstream genes and to form inhibitory loops among each other, thus fine-tuning expression transitions.     

Ethanol as a ‘food’ forDrosophila melanogaster: Influence of theEbony gene

Summary The survival time of adultDrosophila melanogaster flies without food is greater in the presence of ethanol, especially for flies of strains or lines with a higher alcohol dehydrogenase activity. It seems that theebony gene can act in some populations as a selective factor favoring the ADH^F allele, as well as the minor genes enhancing the alcohol dehydrogenase activity level.Chargé de Recherches au F. N. R. S.     

Symbionts in the female tsetse flyGlossina morsitans morsitans

Summary A rickettsia-like symbiont, located in the ovaries ofG. m. morsitans is maternally transmitted to the offspring via the egg. It is suggested that they may be essential for normal ovarian development.

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Résumé Un symbionte de type rickettsien localisé dans les ovaires deG. m. morsitans est transmis maternellement par l'uf à la progéniture. Le symbionte est probablement indispensable au développement normal des ovaires.

     

SET domain proteins modulate chromatin domains in eu- and heterochromatin

The SET domain is a 130-amino acid, evolutionarily conserved sequence motif present in chromosomal proteins that function in modulating gene activities from yeast to mammals. Initially identified as members of the Polycomb- and trithorax-group (Pc-G and trx-G) gene families, which are required to maintain expression boundaries of homeotic selector (HOM-C) genes, SET domain proteins are also involved in position-effect-variegation (PEV), telomeric and centromeric gene silencing, and possibly in determining chromosome architecture. These observations implicate SET domain proteins as multifunctional chromatin regulators with activities in both eu- and heterochromatin – a role consistent with their modular structure, which combines the SET domain with additional sequence motifs of either a cysteine-rich region/zinc-finger type or the chromo domain. Multiple functions for chromatin regulators are not restricted to the SET protein family, since many trx-G (but only very few Pc-G) genes are also modifiers of PEV. Together, these data establish a model in which the modulation of chromatin domains is mechanistically linked with the regulation of key developmental loci (e.g. HOM-C).