Spodoptera littoralis genome mining brings insights on the dynamic of expansion of gustatory receptors in polyphagous noctuidae

The bitter taste, triggered via gustatory receptors, serves as an important natural defense against the ingestion of poisonous foods in animals, and the diversity of food diet is usually linked to an increase in the number of gustatory receptor genes. This has been especially observed in polyphagous insect species, such as noctuid species from the Spodoptera genus. However, the dynamic and physical mechanisms leading to these gene expansions and the evolutionary pressures behind them remain elusive. Among major drivers of genome dynamics are the transposable elements but, surprisingly, their potential role in insect gustatory receptors expansion has not been considered yet. In this work, we hypothesized that transposable elements and possibly positive selection would be involved in the active dynamic of gustatory receptor evolution in Spodoptera spp. We first sequenced de novo the full 465Mb genome of S. littoralis, and manually annotated all chemosensory genes, including a large repertoire of 373 gustatory receptor genes (including 19 pseudogenes). We also improved the completeness of S. frugiperda and S. litura gustatory receptor repertoires. Then, we annotated transposable elements and revealed that a particular category of class I retrotransposons, the SINE transposons, was significantly enriched in the vicinity of gustatory receptor gene clusters, suggesting a transposon-mediated mechanism for the formation of these clusters. Selection pressure analyses indicated that positive selection within the gustatory receptor gene family is cryptic, only 7 receptors being identified as positively selected. Altogether, our data provide a new good quality Spodoptera genome, pinpoint interesting gustatory receptor candidates for further functional studies and bring valuable genomic information on the mechanisms of gustatory receptor expansions in polyphagous insect species.

Ribosome protein mutant cells rely on the GR64 cluster of gustatory receptors for survival and proteostasis in Drosophila

Mutations in ribosome protein (Rp) genes and ribosome biogenesis factors result in debilitating diseases, known as ribosomopathies. Recent studies in Drosophila have shown that cells heterozygous mutant for Rp genes (Rp/+) exhibit proteotoxic stress and aggregates, which drive stress pathway activation and apoptosis. Understanding how Rp/+ cells fend off proteotoxic stress could suggest mechanisms to ameliorate these and other conditions caused by proteotoxic stress. Here we find that Rp/+ epithelial cells express all six Gustatory Receptor 64 (Gr64) genes, a cluster of sugar receptors involved in taste sensation. We show that Rp/+ cells depend on Gr64 for survival and that loss of Gr64 autonomously exacerbates stress pathway activation and proteotoxic stress by negatively effecting autophagy and proteasome function in Rp/+ cells. This work identifies a non-canonical role in proteostasis maintenance for a family of gustatory receptors known for their function in neuronal sensation.

Drosophila sensory receptors—a set of molecular Swiss Army Knives

Genetic approaches in the fruit fly, Drosophila melanogaster, have led to a major triumph in the field of sensory biology—the discovery of multiple large families of sensory receptors and channels. Some of these families, such as transient receptor potential channels, are conserved from animals ranging from worms to humans, while others, such as “gustatory receptors,” “olfactory receptors,” and “ionotropic receptors,” are restricted to invertebrates. Prior to the identification of sensory receptors in flies, it was widely assumed that these proteins function in just one modality such as vision, smell, taste, hearing, and somatosensation, which includes thermosensation, light, and noxious mechanical touch. By employing a vast combination of genetic, behavioral, electrophysiological, and other approaches in flies, a major concept to emerge is that many sensory receptors are multitaskers. The earliest example of this idea was the discovery that individual transient receptor potential channels function in multiple senses. It is now clear that multitasking is exhibited by other large receptor families including gustatory receptors, ionotropic receptors, epithelial Na+ channels (also referred to as Pickpockets), and even opsins, which were formerly thought to function exclusively as light sensors. Genetic characterizations of these Drosophila receptors and the neurons that express them also reveal the mechanisms through which flies can accurately differentiate between different stimuli even when they activate the same receptor, as well as mechanisms of adaptation, amplification, and sensory integration. The insights gleaned from studies in flies have been highly influential in directing investigations in many other animal models.

The whole body transcriptome of Coleophora obducta reveals important olfactory proteins

Background The tiny casebearer moth Coleophora obducta, an important defoliator of Larix spp., is a major threat to ecological security in north China. Studies have shown that C. obducta is strongly specific to host plants; it is unable complete its life cycle without Larix spp. The sex pheromones of C. obducta Z5-10:OH have been elucidated; and eight types of antennae sensilla, have been detected, indicating that an exploration of its olfactory proteins is necessary, due to the general lack of information on this topic. Methods We investigated the whole body transcriptome of C. obducta, performed a phylogenetic analysis of its olfactory proteins and produced expression profiles of three pheromone-binding proteins (CobdPBPs) by qRT–PCR. Results We identified 16 odorant binding proteins, 14 chemosensory proteins, three sensory neuron membrane proteins, six odorant degrading enzymes, five antennal esterases, 13 odorant receptors, seven ionotropic receptors and 10 gustatory receptors, including three PBPs and one odorant co-receptor. Additionally, three putative pheromone receptors, two bitter gustatory receptors and five functional ionotropic receptors were found by phylogenetic analysis. The expression profiles of three PBPs in males and females showed that all of them exhibited male-specific expression and two were expressed at significantly higher levels in males. These data provide a molecular foundation from which to explore the olfactory recognition process and may be useful in the development of a new integrated pest management strategy targeting olfactory recognition of C. obducta.

Identification, characterization and use of novel thermogenetic tools in drosophilia melanogaster

Extrinsic control of neural activity is necessary to decipher the neural mechanisms underlying behavior. Molecular tools that employ light (optogenetics) or temperature (thermogenetics) are primarily used for extrinsic manipulation of neurons. While the available tools offer precise temporal and spatial resolution, their caveats lie in the limited number of tools that can be used simultaneously to alter neuronal activity. The overlapping spectrum of activation of optogenetic tools prevents their simultaneous use in preparations. Similarly, the lack of thermogenetic tools that can function in the physiological range of organisms has restricted their use. The use of thermogenetic tools is limited to two members from the Transient receptor family of proteins, TrpA1 and TrpM8 to activate neurons, and one protein that reduces synaptic output, Shibirets. A major drawback to the Trp channels is their response to both temperature and voltage changes. Hence, the discovery of a new temperature sensitive Gustatory Receptor protein provided an opportunity to mine for other temperature sensitive proteins and develop novel thermogenetic tools. In this thesis we report the identification of several thermosensitive proteins, their characterization, and use in studying the learning and memory of freely moving flies. In the first chapter, we probed several Gustatory receptors for their temperature sensitivity using the heat box. The heat box is a high throughput system that enables us to test and track the behavior of single flies in response to temperature. The top and bottom of heat box chamber has Peltier elements that allow for control of temperature with a resolution of 1[degrees]C. We overexpressed several Gustatory receptors one at a time pan neuronally in Drosophila melanogaster and exposed them to various assays. Our initials results imply that at least 2 Drosophila melanogaster Gustatory receptors other than Gr28bD are temperature sensitive. To increase the repertoire of thermosensitive proteins, we assayed for temperature response properties of Gr28bD orthologs from 5 other Drosophila species that occupy different habitats in the world. We rationalized that flies in different habitats will have Gr28bD orthologs with unique temperature response properties designed to sustain in that habitat. Of the 5 proteins we tested, we found that 4 proteins are temperature sensitive at different temperatures. While pan-neuronal overexpression is a robust method to determine the temperature responsiveness of a protein, it does not recapitulate the natural environment the protein is present in. In D. melanogaster, Gr28bD is present in specialized heat sensing cells in the antenna, called Hot Cells. There are 3 Hot cells on each of the two antennae. There is however no physiological information on the where the orthologs are expressed. Since Gr28bD is used for rapid heat avoidance in flies, we rationalized that its orthologs too sever a similar function in their host species and are expressed in the peripheral regions. Hence, in the second chapter, we tested for the avoidance behavior of flies using two choice assays. We made mutant flies that lacked Gr28b proteins, including Gr28bD in the antennae. We then examined the ability of the orthologs to rescue the heat avoidance behavior in these mutants. We found that all the orthologs respond to temperature differences albeit, at different temperatures. Above a threshold temperature, flies rescued with some orthologs could not differentiate between small temperature differences, suggesting that the activity of the orthologs might saturate beyond certain temperatures. Some homologs responded to temperature only when expressed in Hot Cells, thus leading us to examine the presence of accessory proteins it the hot cells that might be enhancing the thermosensitive properties of these homologs. We found several candidate proteins that can studied further to determine their role in the temperature sensing in the hot cells. When used as thermogenetic tools, thermosensitive proteins are in localized environments in small cluster of cells. In the third chapter, we expressed Gr28bD in small clusters of dopaminergic neurons in the fly brain with an aim to understand the role of activation of dopaminergic neurons in operant place learning and memory paradigm. In addition to examining their learning scores at different temperatures, we investigated other behaviors of the flies during the training. Contrary to previous results from our lab that showed that dopaminergic neurons are not important for place learning and memory, we found that activation of a specific subset of dopaminergic neurons does alter place learning and memory. Our findings new laid the groundwork for more experiments to investigate dopaminergic modulation of place learning and memory.