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.

Ionotropic receptors mediate nitrogenous waste avoidance in Drosophila melanogaster

Ammonia and its amine-containing derivatives are widely found in natural decomposition byproducts. Here, we conducted biased chemoreceptor screening to investigate the mechanisms by which different concentrations of ammonium salt, urea, and putrescine in rotten fruits affect feeding and oviposition behavior. We identified three ionotropic receptors, including the two broadly required IR25a and IR76b receptors, as well as the narrowly tuned IR51b receptor. These three IRs were fundamental in eliciting avoidance against nitrogenous waste products, which is mediated by bitter-sensing gustatory receptor neurons (GRNs). The aversion of nitrogenous wastes was evaluated by the cellular requirement by expressing Kir2.1 and behavioral recoveries of the mutants in bitter-sensing GRNs. Furthermore, by conducting electrophysiology assays, we confirmed that ammonia compounds are aversive in taste as they directly activated bitter-sensing GRNs. Therefore, our findings provide insights into the ecological roles of IRs as a means to detect and avoid toxic nitrogenous waste products in nature.

Functional Morphology of Gustatory Organs in Caterpillars

The sense of taste plays a pivotal role in the behavior of insects. Caterpillars depend largely on taste cues from plants to detect and locate food sources. Taste stimuli can be either simple or complex as multimolecular mixtures. The insect faces the task of deciphering the nature of these tastants and must then make appropriate feeding choices. Typically, caterpillar larvae possess four types of bilateral gustatory sensilla on their mouthparts. The lateral and medial styloconic sensilla are thought to be the primary organs involved in feeding. These sensilla are in continuous contact with plant sap during feeding and can detect different phytochemicals present in the plant. The gustatory sensory input is encoded as patterns of nerve impulses by gustatory receptor cells housed in these sensilla. Therefore, these gustatory receptor cells form the first layer of a decision-making process that ultimately determines whether food is accepted or rejected by the insect. Caterpillars, such as gypsy moth larvae (Lymantria dispar) (L.) (Lepidoptera: Lymantriidae) are major forest pests in most of the United States. These larvae are highly polyphagous feeders and defoliate a variety of tree species, including forest, shade, fruit, and ornamentals. This chapter discusses morphological, feeding behavioral, and electrophysiological aspects of gustatory sensilla with respect to gypsy moth caterpillars.

Identification of a gustatory receptor tuned to sinigrin in the cabbage butterfly Pieris rapae

Glucosinolates are token stimuli in host selection of many crucifer specialist insects, but the underlying molecular basis for host selection in these insects remains enigmatic. Using a combination of behavioral, electrophysiological, and molecular methods, we investigate glucosinolate receptors in the cabbage butterfly Pieris rapae. Sinigrin, as a potent feeding stimulant, elicited activity in larval maxillary lateral sensilla styloconica, as well as in adult medial tarsal sensilla. Two P. rapae gustatory receptor genes PrapGr28 and PrapGr15 were identified with high expression in female tarsi, and the subsequent functional analyses showed that Xenopus oocytes only expressing PrapGr28 had specific responses to sinigrin; when ectopically expressed in Drosophila sugar sensing neurons, PrapGr28 conferred sinigrin sensitivity to these neurons. RNA interference experiments further showed that knockdown of PrapGr28 reduced the sensitivity of adult medial tarsal sensilla to sinigrin. Taken together, we conclude that PrapGr28 is a gustatory receptor tuned to sinigrin in P. rapae, which paves the way for revealing the molecular basis of the relationships between crucifer plants and their specialist insects.

Genome-Wide Identification of the Gustatory Receptor Gene Family of the Invasive Pest, Red Palm Weevil, Rhynchophorus ferrugineus (Olivier, 1790)

The red palm weevil (Rhynchophorus ferrugineus) is a highly destructive pest of oil palm, date, and coconut in many parts of Asia, Europe, and Africa. The Food and Agriculture Organization of the United Nations has called for international collaboration to develop a multidisciplinary strategy to control this invasive pest. Previous research focused on the molecular basis of chemoreception in this species, particularly olfaction, to develop biosensors for early detection and more effective bait traps for mass trapping. However, the molecular basis of gustation, which plays an essential role in discriminating food and egg-laying sites and chemical communication in this species, is limited because its complete gustatory receptor gene family still has not been characterized. We manually annotated the gene family from the recently available genome and transcriptome data and reported 50 gustatory receptor genes encoding 65 gustatory receptors, including 7 carbon dioxide, 9 sugar, and 49 bitter receptors. This study provides a platform for future functional analysis and comparative chemosensory study. A better understanding of gustation will improve our understanding of this species’ complex chemoreception, which is an important step toward developing more effective control methods.

Description of Chemosensory Genes in Unexplored Tissues of the Moth Spodoptera littoralis

Illumina-based transcriptome sequencing of chemosensory organs has become a standard in deciphering the molecular bases of chemical senses in insects, especially in non-model species. A plethora of antennal transcriptomes is now available in the literature, describing large sets of chemosensory receptors and binding proteins in a diversity of species. However, little is still known on other organs such as mouthparts, legs and ovipositors, which are also known to carry chemosensory sensilla. This is the case of the noctuid Spodoptera littoralis, which has been established as a model insect species in molecular chemical ecology thanks to the description of many—but not all—chemosensory genes. To fulfill this gap, we present here an unprecedented transcriptomic survey of chemosensory tissues in this species. RNAseq from male and female proboscis, labial palps, legs and female ovipositors allowed us to annotate 115 putative chemosensory gene transcripts, including 30 novel genes in this species. Especially, we doubled the number of candidate gustatory receptor transcripts described in this species. We also evidenced ectopic expression of many chemosensory genes. Remarkably, one third of the odorant receptors were found to be expressed in the proboscis. With a total of 196 non-overlapping chemosensory genes annotated, the S. littoralis repertoire is one of the most complete in Lepidoptera. We further evaluated the expression of transcripts between males and females, pinpointing sex-specific transcripts. We identified five female-specific transcripts, including one odorant receptor, one gustatory receptor, one ionotropic receptor and one odorant-binding protein, and one male-specific gustatory receptor. Such sex-biased expression suggests that these transcripts participate in sex-specific behaviors, such as host choice for oviposition in females and/or mating partner recognition in both sexes.

Requirement for an Otopetrin-Like protein for acid taste in Drosophila

Many of the Drosophila receptors required for bitter, sugar and other tastes have been identified. However, the receptor required for the taste of acid has been elusive. In Drosophila, the major families of taste receptors, such as Gustatory Receptors and Ionotropic Receptors are unrelated to taste receptors in mammals. Previous work indicated that members of these major families do not appear to be broadly required acid sensors. Here, to identify the enigmatic acid taste receptor, we interrogated three genes encoding proteins distantly related the mammalian Otopertrin1 proton channel. We found that RNAi knockdown or mutation of Otopetrin-Like A (OtopLA) by CRISPR/Cas9, severely impairs the behavioral rejection of sugary foods laced with HCl or carboxylic acids. Mutation of OtopLA also greatly reduces acid-induced action potentials. We identified an isoform of OtopLA that was expressed in the proboscis and was sufficient to restore acid sensitivity to OtopLA mutant flies. OtopLA functioned in acid taste in a subset of bitter-activated gustatory receptor neurons that senses protons. This work highlights an unusual functional conservation of a receptor required for a taste modality in flies and mammals.

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.

Salty surfaces deter feeding in a blood-sucking disease vector

Salts are essential nutrients required for many physiological processes, and deficient or excessive salt results in adverse health problems. Taste is the ultimate sensory modality involved in resource quality assessment, resulting in acceptance or rejection. Here, we show that detection of high-salt substrates by a salt-sensitive antennal gustatory receptor neuron, S1-GRN, results in feeding avoidance in the hematophagous bug Rhodnius prolixus. Knock-down of two antennal-expressed amiloride-sensitive pickpocket channel receptors (PPKs; RproPPK014276 and RproPPK28) using RNA interference, prevents avoidance of bugs to high-salt substrates. Tracing antennal GRNs to the central nervous system reveals the antennal lobes as a gustatory processing center. The identification of the gustatory basis of high-salt detection in a blood feeder provides novel targets to prevent biting and feeding, as well as to promote substrate avoidance in a relevant disease vector.Significance StatementDetection of aversive gustatory stimuli induces avoidance responses in animals. Avoidance acquires particular interest if it reduces the biting rates of blood-feeding insects of medical relevance. Here we describe the molecular and physiological basis of high-salt detection in the blood-sucking disease vector Rhodnius prolixus. We show that detection of high-salt substrates through two PPK receptors expressed in an antennal gustatory receptor neuron produces feeding avoidance. Understanding these gustatory-driven aversive responses allows the hitherto overlooked use of gustatory molecules as a complement to known olfactory repellents.