Project description:Evaluation of food chemicals is essential to make appropriate feeding decisions. The molecular genetic analysis of Gustatory receptor (Gr) genes and the characterization of the neural circuits that they engage has led to a broad understanding of taste perception in adult Drosophila [1, 2]. For example, eight relatively highly conserved members of the Gr gene family (Gr5a, Gr61a, and Gr64a-f), referred to as sugar Gr genes, are thought to be involved in sugar taste in adult flies [3-8], while the majority of the remaining Gr genes are likely to encode bitter taste receptors [9-11], albeit some function as pheromone [12-14] and carbon dioxide [15, 16] receptors. In contrast to the adult fly, relatively little is known about the cellular and molecular basis of taste perception in larvae. Here, we identify Gr43a, which was recently shown to function as a hemolymph fructose sensor in adult flies [17], as the major larval sugar receptor. We show that it is expressed in taste neurons, proventricular neurons, as well as sensory neurons of the brain. Larvae lacking Gr43a fail to sense sugars, while larvae mutant for all eight sugar Gr genes exhibit no obvious defect. Finally, we show that brain neurons are necessary and sufficient for sensing all main dietary sugars, which probably involves a postingestive mechanism of converting carbohydrates into fructose.
Project description:Single channel custom oligonucleotide array of 147 microRNAs to detect changes in expression of a lgl-hypomorph mutant versus wild-type 0, 3, and 5 day old 3rd instar lgl-hypomorph larvae were compared to 0 day 3rd instar wild-type larvae to test for differences in microRNA expression
Project description:Low temperatures can be fatal to insects, but many species have evolved the ability to cold acclimate, thereby increasing their cold tolerance. It has been previously shown that Drosophila melanogaster larvae perform cold-evoked behaviors under the control of noxious cold-sensing neurons (nociceptors), but it is unknown how the nervous system might participate in cold tolerance. Herein, we describe cold-nociceptive behavior among 11 drosophilid species; we find that the predominant cold-evoked larval response is a head-to-tail contraction behavior, which is likely inherited from a common ancestor, but is unlikely to be protective. We therefore tested the hypothesis that cold nociception functions to protect larvae by triggering cold acclimation. We found that Drosophila melanogaster Class III nociceptors are sensitized by and critical to cold acclimation and that cold acclimation can be optogenetically evoked, sans cold. Collectively, these findings demonstrate that cold nociception constitutes a peripheral neural basis for Drosophila larval cold acclimation.
Project description:Paenibacillus larvae, the causative agent of the devastating honey-bee disease American Foulbrood, produces the cationic polyketide-peptide hybrid paenilamicin that displays antibacterial and antifungal activity. Its biosynthetic gene cluster contains a gene coding for the N-acetyltransferase PamZ. We show that PamZ acts as self-resistance factor in Paenibacillus larvae by deactivation of paenilamicin. Using tandem mass spectrometry, nuclear magnetic resonance spectroscopy and synthetic diastereomers, we identified the N-terminal amino group of the agmatinamic acid as the N-acetylation site. These findings highlight the pharmacophore region of paenilamicin, which we very recently identified as a ribosome inhibitor. Here, we further determined the crystal structure of PamZ:acetyl-CoA complex at 1.34 Å resolution. An unusual tandem-domain architecture provides a well-defined substrate-binding groove decorated with negatively-charged residues to specifically attract the cationic paenilamicin. Our results will help to understand the mode of action of paenilamicin and its role in pathogenicity of Paenibacillus larvae to fight American Foulbrood.
Project description:Cephalic furrow formation (CFF) is a major morphogenetic movement during gastrulation in Drosophila melanogaster embryos that gives rise to a deep, transitory epithelial invagination. Recent studies have identified the individual cell shape changes that drive the initiation and progression phases of CFF; however, the underlying mechanics are not yet well understood. During the progression phase, the furrow deepens as columnar cells from both the anterior and posterior directions fold inwards rotating by 90°. To analyze the mechanics of this process, we have developed an advanced two-dimensional lateral vertex model that includes multinode representation of cellular membranes and allows us to capture the membrane curvature associated with pressure variation. Our investigations reveal some key potential mechanical features of CFF, as follows. When cells begin to roll over the cephalic furrow cleft, they become wedge shaped as their apical cortices and overlying membranes expand, lateral cortices and overlying membranes release tension, internal pressures drop, and basal cortices and membranes contract. Then, cells reverse this process by shortening apical cortices and membranes, increasing lateral tension, and causing internal pressures to rise. Since the basal membranes expand, the cells recover their rotated columnar shape once in the furrow. Interestingly, our findings indicate that the basal membranes may be passively reactive throughout the progression phase. We also find that the smooth rolling of cells over the cephalic furrow cleft necessitates that internalized cells provide a solid base through high levels of membrane tension and internal pressure, which allows the transmission of tensile force that pulls new cells into the furrow. These results lead us to suggest that CFF helps to establish a baseline tension across the apical surface of the embryo to facilitate cellular coordination of other morphogenetic movements via mechanical stress feedback mechanisms.
Project description:Behavioral studies have established that Drosophila appetitive taste responses towards fatty acids are mediated by sweet sensing Gustatory Receptor Neurons (GRNs). Here we show that sweet GRN activation requires the function of the Ionotropic Receptor genes IR25a, IR76b and IR56d. The former two IR genes are expressed in several neurons per sensillum, while IR56d expression is restricted to sweet GRNs. Importantly, loss of appetitive behavioral responses to fatty acids in IR25a and IR76b mutant flies can be completely rescued by expression of respective transgenes in sweet GRNs. Interestingly, appetitive behavioral responses of wild type flies to hexanoic acid reach a plateau at ~1%, but decrease with higher concentration, a property mediated through IR25a/IR76b independent activation of bitter GRNs. With our previous report on sour taste, our studies suggest that IR-based receptors mediate different taste qualities through cell-type specific IR subunits.
Project description:CO(2) elicits a response from many insects, including mosquito vectors of diseases such as malaria and yellow fever, but the molecular basis of CO(2) detection is unknown in insects or other higher eukaryotes. Here we show that Gr21a and Gr63a, members of a large family of Drosophila seven-transmembrane-domain chemoreceptor genes, are coexpressed in chemosensory neurons of both the larva and the adult. The two genes confer CO(2) response when coexpressed in an in vivo expression system, the "empty neuron system." The response is highly specific for CO(2) and dependent on CO(2) concentration. The response shows an equivalent dependence on the dose of Gr21a and Gr63a. None of 39 other chemosensory receptors confers a comparable response to CO(2). The identification of these receptors may now allow the identification of agents that block or activate them. Such agents could affect the responses of insect pests to the humans they seek.
Project description:The detection of water and the regulation of water intake are essential for animals to maintain proper osmotic homeostasis. Drosophila and other insects have gustatory sensory neurons that mediate the recognition of external water sources, but little is known about the underlying molecular mechanism for water taste detection. Here we identify a member of the degenerin/epithelial sodium channel family, PPK28, as an osmosensitive ion channel that mediates the cellular and behavioural response to water. We use molecular, cellular, calcium imaging and electrophysiological approaches to show that ppk28 is expressed in water-sensing neurons, and that loss of ppk28 abolishes water sensitivity. Moreover, ectopic expression of ppk28 confers water sensitivity to bitter-sensing gustatory neurons in the fly and sensitivity to hypo-osmotic solutions when expressed in heterologous cells. These studies link an osmosensitive ion channel to water taste detection and drinking behaviour, providing the framework for examining the molecular basis for water detection in other animals.
Project description:Living cells respond to mechanical forces applied to their outer membrane through processes referred to as "mechanosensation". Faced with hypotonic shock, to circumvent cell lysis, bacteria open large solute-passing channels to reduce the osmotic pressure gradient. In the vascular beds of vertebrate animals blood flow is regulated directly through mechanical distention-induced opening of stretch-activated channels in smooth muscle cells. Touch sensation is thought to originate in mechanically sensitive ion channels in nerve endings, and hearing in mechanically sensitive ion channels located in specialized cells of the ear. While the ubiquity of mechanosensation in living cells is evident, the ion channels underlying the transduction events in vertebrate animals have remained elusive. Here we demonstrate through electrophysiological recordings that voltage-dependent K(+) (Kv) channels exhibit exquisite sensitivity to small (physiologically relevant in magnitude) mechanical perturbations of the cell membrane. The demonstrated mechanosensitivity is quantitatively consistent with membrane tension acting on a late-opening transition through stabilization of a dilated pore. This effect causes a shift in the voltage range over which Kv channels open as well as an increase in the maximum open probability. This mechanically induced shift could allow Kv channels and perhaps other voltage-dependent ion channels to play a role in mechanosensation.
Project description:The mosquito Anopheles gambiae is the principal Afrotropical vector for human malaria. A central component of its vectorial capacity is the ability to maintain sufficient populations of adults. During both adult and preadult (larval) stages, the mosquitoes depend on the ability to recognize and respond to chemical cues that mediate feeding and survival. In this study, we used a behavioral assay to identify a range of odorant-specific responses of An. gambiae larvae that are dependent on the integrity of the larval antennae. Parallel molecular analyses have identified a subset of the An. gambiae odorant receptors (AgOrs) that are localized to discrete neurons within the larval antennae and facilitate odor-evoked responses in Xenopus oocytes that are consistent with the larval behavioral spectrum. These studies shed light on chemosensory-driven behaviors and represent molecular and cellular characterization of olfactory processes in mosquito larvae. These advances may ultimately enhance the development of vector control strategies, targeting olfactory pathways in larval-stage mosquitoes to reduce the catastrophic effects of malaria and other diseases.