Mamiko Ozaki

An Inhibitory Sex Pheromone Tastes Bitter for Drosophila Males

Sexual behavior requires animals to distinguish between the sexes and to respond appropriately to each of them. In Drosophila melanogaster, as in many insects, cuticular hydrocarbons are thought to be involved in sex recognition and in mating behavior, but there is no direct neuronal evidence of their pheromonal effect. Using behavioral and electrophysiological measures of responses to natural and synthetic compounds, we show that Z-7-tricosene, a Drosophila male cuticular hydrocarbon, acts as a sex pheromone and inhibits male-male courtship. These data provide the first direct demonstration that an insect cuticular hydrocarbon is detected as a sex pheromone. Intriguingly, we show that a particular type of gustatory neurons of the labial palps respond both to Z-7-tricosene and to bitter stimuli. Cross-adaptation between Z-7-tricosene and bitter stimuli further indicates that these two very different substances are processed by the same neural pathways. Furthermore, the two substances induced similar behavioral responses both in courtship and feeding tests. We conclude that the inhibitory pheromone tastes bitter to the fly.

Characteristics of genes up-regulated and down-regulated after 24 h starvation in the head of Drosophila

Starvation is a common experience under fluctuating food conditions in nature, and response to it is vital for many organisms. Many studies have investigated the response at physiological and behavioral level, whereas the studies on starvation-induced transcriptional changes in the brain and the surrounding tissues are still limited. We here investigated global changes in transcript abundance in the head after 24 h starvation by microarray expression profiling of 2 wild-derived inbred strains of Drosophila melanogaster, and identified a core set of 65 up-regulated and 48 down-regulated genes upon starvation. Among these up-regulated genes, 22 genes were circadian oscillating genes previously identified in the head of Drosophila. Interestingly, most (86%) of these circadian genes show their expression peak in a narrow time range of ZT7.0-12.0, when flies are relatively restless and less feeding in the normal condition. Among the down-regulated genes, 2 genes with highest fold-differences, fit and CG8147, are known to have female-biased expression in the head, and 1 gene, Obp99b, is known to be male-biased. Together with the realtime qPCR experiments on female and male transcripts, our data suggest that these sex-specific genes are candidate genes mediating a possible trade-off between starvation resistance and reproduction. Eleven down-regulated genes are known to be involved in the immune response. These changes in head transcriptome upon starvation reflect modulation of expression in some normally oscillating rhythmic genes and reduction in the resource allocation toward sexual activity and immunity.

Effects of cyclic GMP on the sugar taste receptor cell of the fly Phormia regina

Abstract In some sensory receptors of vertebrates, cyclic GMP (cGMP) and/or cyclic AMP (cAMP) is demonstrated to mediate information from the receptor molecule to the ion channel. The labellar chemosensillum of the fly, Phormia regina, is a hair-like organ which contains four taste cells, i.e. the sugar, the salt, the water receptor cells and the fifth cell. They generate spike potentials of different sizes which are easily discriminated from each other. When a membrane-permeable cGMP analogue, dibutyryl cyclic GMP, was applied to the tip of sensillum, large spikes were evoked. Membrane-impermeable nucleotides also evoked the same spikes but their stimulating effect was less than that of dibutyryl cyclic GMP. Judging from the size of the spikes and feeding behaviour of flies to the nucleotides, the spikes were identified as those from the sugar receptor cells. When the mixture of dibutyryl cyclic GMP and phosphodiesterase inhibitor, both of which are membrane-permeable, was repeatedly applied to the sensillum tip, “remaining spikes” were observed after the removal of the mixture. Furthermore, application of dibutyryl cyclic GMP to the sensillum induced extremely slow adaptation of the sugar receptor cell while adaptation of the cell by sugar stimulation is usually rapid. Based upon these results, we discuss the possibility that cGMP works as a second messenger for the sugar receptor excitation of the fly.

Identification of the novel bioactive peptides dRYamide-1 and dRYamide-2, ligands for a neuropeptide Y-like receptor in Drosophila

A number of bioactive peptides are involved in regulating a wide range of animal behaviors, including food consumption. Vertebrate neuropeptide Y (NPY) is a potent stimulator of appetitive behavior. Recently, Drosophila neuropeptide F (dNPF) and short NPF (sNPF), the Drosophila homologs of the vertebrate NPY, were identified to characterize the functions of NPFs in the feeding behaviors of this insect. Dm-NPFR1 and NPFR76F are the receptors for dNPF and sNPF, respectively; both receptors are G protein-coupled receptors (GPCRs). Another GPCR (CG5811; NepYR) was indentified in Drosophila as a neuropeptide Y-like receptor. Here, we identified 2 ligands of CG5811, dRYamide-1 and dRYamide-2. Both peptides are derived from the same precursor (CG40733) and have no significant structural similarities to known bioactive peptides. The C-terminal sequence RYamide of dRYamides is identical to that of NPY family peptides; on the other hand, dNPF and sNPF have C-terminal RFamide. When administered to blowflies, dRYamide-1 suppressed feeding motivation. We propose that dRYamides are related to the NPY family in vertebrates, similar to dNPF and sNPF.

Sexual Dimorphism in the Antennal Lobe of the Ant Camponotus japonicus

Abstract The carpenter ant, a social hymenopteran, has a highly elaborated antennal chemosensory system that is used for chemical communication in social life. The glomeruli in the antennal lobe are the first relay stations where sensory neurons synapse onto interneurons. The system is functionally and structurally similar to the olfactory bulbs of vertebrates. Using three-dimensional reconstruction of glomeruli and subsequent morphometric analyses, we found sexual dimorphism of the antennal lobe glomeruli in carpenter ants, Camponotus japonicus. Female workers and unmated queens had about 430 glomeruli, the highest number reported so far in ants. Males had a sexually dimorphic macroglomerulus and about 215 ordinary glomeruli. This appeared to result from a greatly reduced number of glomeruli in the postero-medial region of the antennal lobe compared with that in females. On the other hand, sexually isomorphic glomeruli were identifiable in the dorsal region of the antennal lobe. For example, large, uniquely shaped glomeruli located at the dorso-central margin of the antennal lobe were detected in all society members. The great sexual dimorphism seen in the ordinary glomeruli of the antennal lobe may reflect gender-specific tasks in chemical communications rather than different reproductive roles.

Chemical disguise as particular caste of host ants in the ant inquiline parasite Niphanda fusca (Lepidoptera: Lycaenidae)

The exploitation of parental care is common in avian and insect ‘cuckoos’ and these species engage in a coevolutionary arms race. Caterpillars of the lycaenid butterfly Niphanda fusca develop as parasites inside the nests of host ants (Camponotus japonicus) where they grow by feeding on the worker trophallaxis. We hypothesized that N. fusca caterpillars chemically mimic host larvae, or some particular castes of the host ant, so that the caterpillars are accepted and cared for by the host workers. Behaviourally, it was observed that the host workers enthusiastically tended glass dummies coated with the cuticular chemicals of larvae or males and those of N. fusca caterpillars living together. Cuticular chemical analyses revealed that N. fusca caterpillars grown in a host ant nest acquired a colony-specific blend of cuticular hydrocarbons (CHCs). Furthermore, the CHC profiles of the N. fusca caterpillars were particularly close to those of the males rather than those of the host larvae and the others. We suggest that N. fusca caterpillars exploit worker care by matching their cuticular profile to that of the host males, since the males are fed by trophallaxis with workers in their natal nests for approximately ten months.

Isolation of the bioactive peptides CCHamide-1 and CCHamide-2 from Drosophila and their putative role in appetite regulation as ligands for G protein-coupled receptors

There are many orphan G protein-coupled receptors (GPCRs) for which ligands have not yet been identified. One such GPCR is the bombesin receptor subtype 3 (BRS-3). BRS-3 plays a role in the onset of diabetes and obesity. GPCRs in invertebrates are similar to those in vertebrates. Two Drosophila GPCRs (CG30106 and CG14593) belong to the BRS-3 phylogenetic subgroup. Here, we succeeded to biochemically purify the endogenous ligands of Drosophila CG30106 and CG14593 from whole Drosophila homogenates using functional assays with the reverse pharmacological technique, and identified their primary amino acid sequences. The purified ligands had been termed CCHamide-1 and CCHamide-2, although structurally identical to the peptides recently predicted from the genomic sequence searching. In addition, our biochemical characterization demonstrated two N-terminal extended forms of CCHamide-2. When administered to blowflies, CCHamide-2 increased their feeding motivation. Our results demonstrated these peptides actually present as the major components to activate these receptors in living Drosophila. Studies on the effects of CCHamides will facilitate the search for BRS-3 ligands.

Sugar receptor response of the food-canal taste sensilla in a nectar-feeding swallowtail butterfly, Papilio xuthus.

The feeding behavior in nectar-feeding insects is triggered by a sugar-receptor response in contact chemosensilla. The contact chemosensilla are distributed not only on tarsi and the outside of the proboscis but also on the inside of the food canal in Lepidoptera. Although the chemosensilla inside the food canal are assumed to detect sweet taste during the passage of nectar through the food canal, their electrophysiological function has received little attention. In the nectar-feeding Asian swallowtail butterfly, Papilio xuthus (Lepidoptera: Papilionidae), we found 15- to 30-μm-long sensilla neatly lined up along the inside galea wall, which forms the food canal in the proboscis. The receptor neurons of these sensilla responded to sucrose. We hypothesized that starch and sucrose compete with each other for a taste receptor site on the sensilla. When we added starch and sucrose to the food-canal sensilla, the electrophysiological responses of food-canal sensilla were inhibited in parallel with the food-sucking behavior of the butterflies. These results suggest that the food-canal sensilla are involved in the behavioral control of nectar-sucking in this butterfly species.

A novel Takeout-like protein expressed in the taste and olfactory organs of the blowfly, Phormia regina

In insects, the functional molecules responsible for the taste system are still obscure. The gene for a 28.5 kDa protein purified from taste sensilla of the blowfly Phormia regina belongs to a gene family that includes takeout of Drosophila melanogaster. Molecular phylogenetic analysis revealed that the Phormia Takeout‐like protein is most similar to the protein encoded by a member of the Drosophila takeout gene family, CG14661, whose expression and function have not been identified yet. Western blot analyses revealed that Phormia Takeout‐like protein was exclusively expressed in antennae and labellum of the adult blowfly in both sexes. Immunohistochemical experiments demonstrated that Takeout‐like protein was localized around the lamella structure of the auxiliary cells and in the sensillar lymph of the labellar taste sensillum. In antennae, Takeout‐like protein was distributed at the base of the olfactory sensilla as well. No significant differences in Takeout‐like protein expression were found between the sexes. Our results suggest that Phormia Takeout‐like protein is involved in some early events concerned with chemoreception in both the taste and olfactory systems.

Nucleotide receptor-site on the labellar sugar receptor cell of the blowfly Phormia regina

Abstract Previously we reported that, among nucleotides applied extracellularly to the chemosensillum of the blowfly Phormia regina, a membrane-permeable cyclic GMP analogue, dibutyryl cyclic GMP, was particularly effective, inducing much impulse-discharge. From the results obtained, we discussed the possibility that a nucleotide site exists on the sugar receptor cell. Here we report results of our electrophysiological experiments to investigate this putative nucleotide receptor-site. As previously reported, when dibutyryl cyclic GMP was applied with sucrose, the impulse-discharge was depressed. Cyclic GMP or another membrane-permeable cyclic GMP analogue, 8-bromo cyclic GMP, enhanced the impulse frequency when applied to a chemosensillum with sucrose. However, 8-bromo cyclic GMP partly depressed the impulse-discharge evoked by dibutyryl cyclic GMP. If we hypothesize that the sugar receptor-site has a nucleotide receptor-site separated from the receptor site for sucrose (P-site) on the membrane and that the cell uses cyclic GMP as an intrinsic intracellular messenger mediating the taste transduction, then, these results can be explained as follows. Dibutyryl cyclic GMP inhibits the P-site with its butyryl groups. On the other hand, it binds the nucleotide receptor-site and stimulates the cell like other nucleotides. However, 8-bromo cyclic GMP, which does not affect the P-site, binds the nucleotide receptor-site like a competitive inhibitor. Thus, the impulses induced by 8-bromo cyclic GMP are due to the 8-bromo cyclic GMP acting in place of cyclic GMP as the intracellular messenger. As for the responses induced by dibutyryl cyclic GMP, a part which can be reduced by 8-bromo cyclic GMP is due to the dibutyryl cyclic GMP binding the nucleotide receptor-site, and other part is due to the dibutyryl cyclic GMP acting as the intracellular messenger.