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: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: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: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: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 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.
Project description:The understanding of the electrophysiological mechanisms that underlie mechanosensitivity of the sinoatrial node (SAN), the primary pacemaker of the heart, has been evolving over the past century. The heart is constantly exposed to a dynamic mechanical environment; as such, the SAN has numerous canonical and emerging mechanosensitive ion channels and signaling pathways that govern its ability to respond to both fast (within second or on beat-to-beat manner) and slow (minutes) timescales. This review summarizes the effects of mechanical loading on the SAN activity and reviews putative candidates, including fast mechanoactivated channels (Piezo, TREK, and BK) and slow mechanoresponsive ion channels [including volume-regulated chloride channels and transient receptor potential (TRP)], as well as the components of mechanochemical signal transduction, which may contribute to SAN mechanosensitivity. Furthermore, we examine the structural foundation for both mechano-electrical and mechanochemical signal transduction and discuss the role of specialized membrane nanodomains, namely, caveolae, in mechanical regulation of both membrane and calcium clock components of the so-called coupled-clock pacemaker system responsible for SAN automaticity. Finally, we emphasize how these mechanically activated changes contribute to the pathophysiology of SAN dysfunction and discuss controversial areas necessitating future investigations. Though the exact mechanisms of SAN mechanosensitivity are currently unknown, identification of such components, their impact into SAN pacemaking, and pathological remodeling may provide new therapeutic targets for the treatment of SAN dysfunction and associated rhythm abnormalities.
Project description:The cryopreservation techniques proposed for embryos of the fruit fly Drosophila melanogaster are not yet ready for practical use. Alternative methods for long-term storage of D. melanogaster strains, although urgently needed, do not exist. Herein, we describe a narrow interval of low temperatures under which the larvae of D. melanogaster can be stored in quiescence for up to two months. The development of larvae was arrested at the pre-wandering stage under fluctuating thermal regime (FTR), which simultaneously resulted in diminishing the accumulation of indirect chill injuries. Our physiological, metabolomic, and transcriptomic analyses revealed that compared to larvae stored at constant low temperatures, the larvae stored under FTR conditions were able to decrease the rates of depletion of energy substrates, exploited brief warm episodes of FTR for homeostatic control of metabolite levels, and more efficiently exerted protection against oxidative damage.