SUMMARY Contact chemosensation is required for several behaviors that promote the survival of fruit flies and other insects. These include evasive behaviors such as the suppression of feeding on dangerous repellent compounds, referred to as antifeedants. Contact chemosensation also contributes to the suppression of male-male courtship. However, the identities of the gustatory receptors (GRs) required for the responses to non-volatile avoidance chemicals are largely unknown. Exceptions include GR66a and GR93a, which are required specifically to prevent ingestion of caffeine [1, 2], and GR32a, which is necessary for inhibiting male-male courtship [3]. However, GR32a is dispensable for normal taste. Thus, distinct GRs may function in the sensing of avoidance pheromones and in detecting different subsets of antifeedants. Here, we describe the requirements for GR33a, which is expressed widely in gustatory receptor neurons (GRNs) that respond to aversive chemicals. Gr33a mutant flies were impaired in the avoidance responses to all non-volatile repellents tested, ranging from quinine to denatorium, lobeline and caffeine. Gr33a mutant males also displayed increased male-to-male courtship implying that it functioned in the detection of a repulsive male pheromone. In contrast to the broadly required olfactory receptor (OR) OR83b, which is essential for trafficking of other ORs [4], GR66a and GR93a were localized normally in Gr33a mutant GRNs. Thus, rather than regulating trafficking of GRs, GR33a may function as co-receptor required for sensing all non-volatile repulsive chemicals, including tastants and pheromones.

Caffeine is a methylxanthine present in the coffee tree, tea plant, and other naturally occurring sources and is among the most commonly consumed drugs worldwide. Whereas the pharmacological action of caffeine has been studied extensively, relatively little is known concerning the molecular mechanism through which this substance is detected as a bitter compound. Unlike most tastants, which are detected through cell-surface G protein-coupled receptors, it has been proposed that caffeine and related methylxanthines activate taste-receptor cells through inhibition of a cyclic nucleotide phosphodiesterase (PDE) . Here, we show that the gustatory receptor Gr66a is expressed in the dendrites of Drosophila gustatory receptor neurons and is essential for the caffeine response. In a behavioral assay, the aversion to caffeine was specifically disrupted in flies missing Gr66a. Caffeine-induced action potentials were also eliminated, as was the response to theophylline, the methylxanthine in tea. The Gr66a mutant exhibited normal tastant-induced action potentials upon presentation of theobromine, a methylxanthine in cocoa. Given that theobromine and caffeine inhibit PDEs with equal potencies , these data further support the role of Gr66a rather than a PDE in mediating the caffeine response. Gr66a is the first gustatory receptor shown to be essential for caffeine-induced behavior and activity of gustatory receptor cells in vivo.

Summary The mechanisms by which the fruitfly, Drosophila melanogaster, detect sweet compounds are poorly understood; however, a subset of the family of 68 gustatory receptors (Grs) has emerged as the key receptors. These seven transmembrane receptors include Gr5a and at least one member of the six genes in the Gr64 cluster (Gr64a), which are expressed in sugar-responsive neurons. Disruption of Gr5a prevents the detection of trehalose [1–3], while mutation of Gr64a impairs the responses to sucrose, maltose and glucose [4, 5]. Recent studies suggest that these sugar receptors may require a co-receptor for function in vivo [4–6]; however, the identity of the putative co-receptor is not known. In the current work, we demonstrate that Gr64f is required in combination with Gr5a for the behavioral response to trehalose and for production of action potentials due to application of trehalose. Gr64f was also required in concert with Gr64a to rescue the defects in the sensitivities to sucrose, maltose and glucose, resulting from deletion of the entire Gr64 cluster. These data suggest that Drosophila sugar receptors function as multimers and that Gr64f is required broadly as a co-receptor for the detection of sugars.

The ability of insects to detect and avoid ingesting naturally occurring repellents and insecticides is essential for their survival. Nevertheless, the gustatory receptors enabling them to sense toxic botanical compounds are largely unknown. The only insect gustatory receptor shown to be required for avoiding noxious compounds is the Drosophila caffeine receptor, Gr66a. However, this receptor is not sufficient for the caffeine response, suggesting that Gr66a may be a subunit of a larger receptor. Here, we report that mutations in the gene encoding the gustatory receptor, Gr93a, result in a phenotype identical to that caused by mutations in Gr66a. This includes an inability to avoid caffeine or the related methylxanthine present in tea, theophylline. Caffeine-induced action potentials were also eliminated in Gr93a-mutant animals, while the flies displayed normal responses to other aversive compounds or to sugars. The Gr93a protein was coexpressed with Gr66a in avoidance-gustatory receptor neurons (GRNs), and functioned in the same GRNs as Gr66a. However, misexpression of both receptors in GRNs that normally do not express either Gr93a or Gr66a does not confer caffeine sensitivity to these GRNs. Because Gr93a-and Gr66a-mutant animals exhibit the identical phenotypes and function in the same cells, we propose that they may be caffeine coreceptors. In contrast to mammalian and Drosophila olfactory receptors and mammalian taste receptors, which are monomeric or dimeric receptors, we propose that Drosophila taste receptors that function in avoidance of bitter compounds are more complex and require additional subunits that remain to be identified.Gr93a ͉ gustatory receptor neuron ͉ taste ͉ repellent ͉ chemosensation

In Drosophila, detection of tastants is thought to be mediated by members of a family of 68 gustatory receptors (Grs). However, only one receptor, Gr5a, has been associated with a sugar, and it appears to be activated specifically by trehalose. It is unclear whether other sugar receptors are activated by single or multiple sugars. Currently, no Grs are known to colocalize with Gr5a. Such Grs would be candidate sugar receptors because Gr5a-expressing cells function in the responses to attractive tastants. Here we use an ''mRNA tagging'' approach to identify Gr RNAs that are coexpressed with Gr5a. We found that all seven Grs most related to Gr5a (Gr64a-f and Gr61a) were expressed in Gr5a-expressing cells, whereas none of the other Grs examined were enriched in these Gr neurons (GRNs). We characterized the role of one Gr5a-related receptor, Gr64a, and found that it was required for the behavioral responses to glucose, sucrose, and maltose. Gr64a was required for GRN function because action potentials induced by these sugars were dependent on expression of Gr64a in GRNs. These data demonstrate that multiple Grs are coexpressed with Gr5a and that Drosophila Gr64a is required for the responses to multiple sugars. gustatory receptor neuron ͉ taste ͉ tip recordings ͉ sugars ͉ behavior

Summary Animals are often confronted with the decision as to consume a diet that contains competing attractive and aversive compounds. Here, using the fruit fly, Drosophila melanogaster, we describe a mechanism that influences this decision. Addition of bitter compounds to sucrose suppressed feeding behavior, and this inhibition depended on the odorant binding protein, OBP49a. In wild-type flies, bitter compounds suppressed sucrose-induced action potentials, and the inhibition was impaired in Obp49a mutants. However, loss of OBP49a did not affect action potentials in sugar- or bitter-activated gustatory receptor neurons (GRNs) when the GRNs were presented with just one type of tastant. OBP49a was expressed in accessory cells, and acted non-cell autonomously to attenuate nerve firings in sugar-activated GRNs when bitter compounds were combined with sucrose. These findings demonstrate an unexpected role for an OBP in taste, and identify a molecular player involved in the integration of opposing attractive and aversive gustatory inputs.

The Drosophila lymph gland, the larval hematopoietic organ comprised of prohemocytes and mature hemocytes, has been a valuable model for understanding mechanisms underlying hematopoiesis and immunity. Three types of mature hemocytes have been characterized in the lymph gland: plasmatocytes, lamellocytes, and crystal cells, which are analogous to vertebrate myeloid cells, yet molecular underpinnings of the lymph gland hemocytes have been less investigated. Here, we use single-cell RNA sequencing to comprehensively analyze heterogeneity of developing hemocytes in the lymph gland, and discover previously undescribed hemocyte types including adipohemocytes, stem-like prohemocytes, and intermediate prohemocytes. Additionally, we identify the developmental trajectory of hemocytes during normal development as well as the emergence of the lamellocyte lineage following active cellular immunity caused by wasp infestation. Finally, we establish similarities and differences between embryonically derived- and larval lymph gland hemocytes. Altogether, our study provides detailed insights into the hemocyte development and cellular immune responses at single-cell resolution.

SUMMARYThis paper presents the problem definition for the second generation of benchmark structural control problems for cable-stayed bridges. The goal of this study is to provide a testbed for the development of strategies for the control of cable stayed-bridges. Based on detailed drawings of the Bill Emerson Memorial Bridge, a three-dimensional evaluation model has been developed to represent the complex behavior of the full-scale benchmark bridge. Phase II considers more complex structural behavior than phase I, including multi-support and transverse excitations. Evaluation criteria are presented for the design problem that are consistent with the goals of seismic response control of a cable-stayed bridge. Control constraints are also provided to ensure that the benchmark results are representative of a control implementation on the physical structure. Each participant in this benchmark bridge control study is given the task of defining, evaluating and reporting on their proposed control strategies. Participants should also evaluate the robust stability and performance of their resulting designs through simulation with an evaluation model which includes additional mass due to snow loads. The problem and a sample control design have been made available in the form of a set of MATLAB equations.