Odorant-Binding Proteins and Chemosensory Proteins in Spodoptera frugiperda: From Genome-Wide Identification and Developmental Stage-Related Expression Analysis to the Perception of Host Plant Odors, Sex Pheromones, and Insecticides.

Spodoptera frugiperda is a worldwide generalist pest with remarkable adaptations to environments and stresses, including developmental stage-related behavioral and physiological adaptations, such as diverse feeding preferences, mate seeking, and pesticide resistance. Insects' odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) are essential for the chemical recognition during behavioral responses or other physiological processes. The genome-wide identification and the gene expression patterns of all these identified OBPs and CSPs across developmental stage-related S. frugiperda have not been reported. Here, we screened for genome-wide SfruOBPs and SfruCSPs, and analyzed the gene expression patterns of SfruOBPs and SfruCSPs repertoires across all developmental stages and sexes. We found 33 OBPs and 22 CSPs in the S. frugiperda genome. The majority of the SfruOBP genes were most highly expressed in the adult male or female stages, while more SfruCSP genes were highly expressed in the larval or egg stages, indicating their function complementation. The gene expression patterns of SfruOBPs and SfruCSPs revealed strong correlations with their respective phylogenic trees, indicating a correlation between function and evolution. In addition, we analyzed the chemical-competitive binding of a widely expressed protein, SfruOBP31, to host plant odorants, sex pheromones, and insecticides. Further ligands binding assay revealed a broad functional related binding spectrum of SfruOBP31 to host plant odorants, sex pheromones, and insecticides, suggesting its potential function in food, mate seeking, and pesticide resistance. These results provide guidance for future research on the development of behavioral regulators of S. frugiperda or other environmentally friendly pest-control strategies.

TRPA5, an Ankyrin Subfamily Insect TRP Channel, is Expressed in Antennae of Cydia pomonella (Lepidoptera: Tortricidae) in Multiple Splice Variants.

Transient receptor potential (TRP) channels are an ancient family of cation channels, working as metabotropic triggers, which respond to physical and chemical environmental cues. Perception of chemical signals mediate reproductive behaviors and is therefore an important target for sustainable management tactics against the codling moth Cydia pomonella L. (Lepidoptera: Tortricidae). However, olfactory behavior strongly depends on diel periodicity and correlation of chemical with physical cues, like temperature, and physical cues thus essentially contribute to the generation of behavioral response. From an antennal transcriptome generated by next generation sequencing, we characterized five candidate TRPs in the codling moth. The coding DNA sequence of one of these was extended to full length, and phylogenetic investigation revealed it to be orthologous of the TRPA5 genes, reported in several insect genomes as members of the insect TRPA group with unknown function but closely related to the thermal sensor pyrexia Reverse transcription PCR revealed the existence of five alternate splice forms of CpTRPA5. Identification of a novel TRPA and its splice forms in codling moth antennae open for investigation of their possible sensory roles and implications in behavioral responses related to olfaction.

Heterologous investigation of metabotropic and ionotropic odorant receptors in ab3A neurons of Drosophila melanogaster.

In insects, antennal ionotropic receptors (IRs) and odorant receptors (ORs) are among the main sensors of olfactory cues. To functionally characterize the subunits from these receptors, the use of ab3A neurons from transgenic Drosophila melanogaster represented one of the most powerful tools, allowing the identification of ligands (deorphanization) and decrypting their pharmacological properties. However, further investigation is needed to shed light on possible metabotropic functionalities behind insect olfactory receptors and test potentials from the up-to-now-used empty neuronal systems to express subunits belonging to variegate receptor classes. In this project, we adopted the most updated system of Drosophila ab3A empty neurons to test various olfactory receptors, ranging from human ORs working as metabotropic G-protein coupled receptors to insect ionotropic IRs and ORs. Testing transgenic Drosophila expressing human ORs into ab3A neurons by single sensillum recording did not result in an OR response to ligands, but it rather re-established neuronal spiking from the empty neurons. When transgenic D. melanogaster expressed ionotropic IRs and ORs, both heterologous and cis-expressed IRs were non-functional, but the Drosophila suzukii OR19A1 subunit responded to a wide asset of ligands, distinguishing phasic or tonic compound-dependent effects. Despite the use of Drosophila ab3A neurons to test the activation of some metabotropic and ionotropic receptor subunits resulted non-functional, this study deorphanized a key OR of D. suzukii demonstrating its binding to alcohols, ketones, terpenes, and esters.

Reviewing findings on the polypeptide sequence of the SARS-CoV-2 S-protein to discuss the origins of the virus.

Several investigations suggested origins of SARS-CoV-2 from the recombination of coronaviruses of various animals, including the bat Rhinolophus affinis and the pangolin Manis javanica, despite the processes describing the adaptation from a reservoir of animals to human are still debated. In this perspective, I will remark two main inconsistencies on the origins of SARS-CoV-2: polypeptide sequence alignment of the S-proteins does not return the expected identity of the receptor-binding motif among most of pangolin-CoVs and SARS-CoV-2; accurate referencing for samplings and sequencing deposition of the ancestral bat coronavirus named RaTG13 was missing since the first reports on the SARS-CoV-2 coronavirus. This contribution aims to stimulate discussion about the origins of SARS-CoV-2 and considers other intermediate hosts as a reservoir for coronavirus.

Altered functional properties of the codling moth Orco mutagenized in the intracellular loop-3.

Amino acid substitutions within the conserved polypeptide sequence of the insect olfactory receptor co-receptor (Orco) have been demonstrated to influence its pharmacological properties. By sequence analysis and phylogenetic investigation, in the Lepidopteran subgroup Ditrysia we identified a fixed substitution in the intracellular loop-3 (ICL-3) of a conserved histidine to glutamine. By means of HEK293 cells as a heterologous system, we functionally expressed Orco from the Ditrysian model Cydia pomonella (CpomOrco) and compared its functional properties with a site-directed mutagenized version where this ICL-3-glutamine was reverted to histidine (CpomOrcoQ417H). The mutagenized CpomOrcoQ417H displayed decreased responsiveness to VUAA1 and reduced response efficacy to an odorant agonist was observed, when co-transfected with the respective OR subunit. Evidence of reduced responsiveness and sensitivity to ligands for the mutagenized Orco suggest the fixed glutamine substitution to be optimized for functionality of the cation channel within Ditrysia. In addition, contrary to the wild type, the mutagenized CpomOrcoQ417H preserved characteristics of VUAA-binding when physiologic conditions turned to acidic. Taken together, our findings provide further evidence of the importance of ICL-3 in forming basic functional properties of insect Orco- and Orco/OR-channels, and suggest involvement of ICL-3 in the potential functional adaptation of Ditrysian Orcos to acidified extra-/intracellular environment.

Current Status on the Functional Characterization of Chemosensory Receptors of Cydia pomonella (Lepidoptera: Tortricidae).

Cydia pomonella (Lepidoptera: Tortricidae) is a major pest of apple, pear and walnuts. For its control, alternative strategies targeting the olfactory system, like mating disruption, have been combined with insecticide applications. The efficacy of these strategies headed the direction of efforts for the functional characterization of codling moth chemosensory receptors to implement further control methods based on chemical sensing. With the advent of transcriptomic analysis, partial and full-length coding sequences of chemosensory receptors have been identified in antennal transcriptomes of C. pomonella. Extension of partial coding sequences to full-length by polymerase chain reaction (PCR)-based techniques and heterologous expression in empty neurons of Drosophila melanogaster and in Human Embryonic Kidney cells allowed functional studies to investigate receptor activation and ligand binding modalities (deorphanization). Among different classes of antennal receptors, several odorant receptors of C. pomonella (CpomORs) have been characterized as binding kairomones (CpomOR3), pheromones (CpomOR6a) and compounds emitted by non-host plants (CpomOR19). Physiological and pharmacological studies of these receptors demonstrated their ionotropic properties, by forming functional channels with the co-receptor subunit of CpomOrco. Further investigations reported a novel insect transient receptor potential (TRPA5) expressed in antennae and other body parts of C. pomonella as a complex pattern of ribonucleic acid (RNA) splice-forms, with a possible involvement in sensing chemical stimuli and temperature. Investigation on chemosensory mechanisms in the codling moth has practical outcomes for the development of control strategies and it inspired novel trends to control this pest by integrating alternative methods to interfere with insect chemosensory communication.

Candidate pheromone receptors of codling moth Cydia pomonella respond to pheromones and kairomones.

Olfaction plays a dominant role in the mate-finding and host selection behaviours of the codling moth (Cydia pomonella), an important pest of apple, pear and walnut orchards worldwide. Antennal transcriptome analysis revealed a number of abundantly expressed genes related to the moth olfactory system, including those encoding the olfactory receptors (ORs) CpomOR1, CpomOR3 and CpomOR6a, which belong to the pheromone receptor (PR) lineage, and the co-receptor (CpomOrco). Using heterologous expression, in both Drosophila olfactory sensory neurones and in human embryonic kidney cells, together with electrophysiological recordings and calcium imaging, we characterize the basic physiological and pharmacological properties of these receptors and demonstrate that they form functional ionotropic receptor channels. Both the homomeric CpomOrco and heteromeric CpomOrco + OR complexes can be activated by the common Orco agonists VUAA1 and VUAA3, as well as inhibited by the common Orco antagonists amiloride derivatives. CpomOR3 responds to the plant volatile compound pear ester ethyl-(E,Z)-2,4-decadienoate, while CpomOR6a responds to the strong pheromone antagonist codlemone acetate (E,E)-8,10-dodecadien-1-yl acetate. These findings represent important breakthroughs in the deorphanization of codling moth pheromone receptors, as well as more broadly into insect ecology and evolution and, consequently, for the development of sustainable pest control strategies based on manipulating chemosensory communication.