Chemosensory Receptors in the Larval Maxilla of Papilio hospiton

Abstract

Among the butterflies of the genus Papilio (Lepidoptera: Papilionidae), Papilio hospiton (Géné) has a geographical distribution limited to the Mediterranean islands of Sardinia (Italy) and Corsica (France). This is mainly due to the host range that includes only a few plant species of Apiaceae and Rutaceae growing on these islands. In a previous electrophysiological investigation conducted on the maxillary gustatory system of larvae of P. hospiton and its closely phylogenetically related species Papilio machaon, a significantly higher spike activity was shown for the gustatory neurons of lateral and medial styloconic sensilla in P. hospiton when bitter compounds were tested. This effect was possibly correlated to the limited host choice range for P. hospiton. To shed light on the molecular aspects of this phenomenon, we investigated the expression pattern of sensory-related sequences by conducting a transcriptomic analysis from total RNA isolates of P. hospiton larval maxillae. We identified several transcripts that may be involved in taste (one gustatory receptor, one divergent ionotropic receptor, and several transient receptor potential channels, TRPs) as well as transcripts supporting an olfactory function for this appendage, including odorant receptors (ORs), antennal ionotropic receptors (A-IRs), sensory neuron membrane proteins (SNMPs), and odorant-binding proteins (OBPs). We used Human Embryonic Kidney (HEK293A) cells to heterologously express two of the identified receptors, PhospOR1 and PhospPain, together with their orthologs from P. machaon, for functional characterization. While our data suggest no activation of these two receptors by the ligands known so far to activate the electrophysiological response in larval maxillary neurons of Papilio species, nor temperature activation of both Papilio TRPA-channel Painless, they represent the first attempt in connecting neuronal activity with their molecular bases to unravel diet specialization between closely related Papilio species.

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

“…In the last decades, most of the investigation of lepidopterans targeted their chemoreceptors [ 49 , 50 , 51 , 52 ]. Among various reports, some provided in-depth analysis of specific OBPs [ 53 , 54 ], CSPs [ 18 , 55 ], or both [ 30 , 56 ]. However, to our knowledge, this is among the few [ 57 ], or may be the sole study in lepidopterans, in which a systematic investigation of soluble proteins merges with a variegate analysis including genomic loci, gene and polypeptide structures, expression patterns across every developmental stage, and functional evidence of semiochemical/pesticide binding.…”

Heterologous expression of insect IRs in transgenicDrosophila melanogaster

“…In this work, we also explored the heterologous expression in HEK cells, which is a single-cell ex vivo system such as Xenopus oocytes. HEK cells have been largely used in the deorphanization of ORs (Cattaneo et al, 2017; Bobkov et al, 2021, Crava et al, 2022, Miazzi et al, 2019, Grosse-Wilde et al, 2007; 2006) whereas, to our knowledge, this method has not been previously utilized for IR subunit deorphanization. We conducted several experiments using this approach but did not achieve successful deorphanization of IRs, despite observing correct expression of IRs in transfected cells through immunohistochemistry experiments (Figure 5).…”

“…The full-length coding sequences of C. pomonella IR41a1, IR25a and IR76b and of D. suzukii IR8a were obtained from previous studies (Walker et al, 2016; Walker et al, 2023; Ramasamy et al, 2016). Sequences were submitted to GeneArt TM gene synthesis service (Thermos Fischer Scientific, Waltham, MA, USA) requesting integration of the 5’-upstram CACC Kozak-sequence, as done previously (Bobkov et al, 2021; Cattaneo et al, 2017; Crava et al, 2022), codon optimization for expression in human cells (Roberts et al, 2021) and integration into pcDNA3.1 plasmids. To test expression of D. melanogaster IR8a+IR84a, the Gibson-assembled combination of these two CDS into pcDNA3.1 was courtesy provided by Dr. Hayden R. Schmidt (International AIDS Vaccine Initiative, USA).…”

“…Activation of HEK293T cells transfected with CpomOrco+IR25a+IR76b+IR41a1 was tested using the same procedures we previously described (Bobkov et al, 2021; Cattaneo et al, 2017; Crava et al, 2022). Mat Tek petri dishes containing HEK293T cells co-transfected with Calcium (GCaMP) or voltage (ArcLightQ239) indicators, underwent to imaging experiments directly upon replacing HEK cell media with 2.0 mL HEK Ca ++ Ringer (mM: 140 NaCl, 5 KCl, 2 CaCl 2 , 10 HEPES, pH 7.4).…”

“…The full-length coding sequences of C. pomonella IR41a1, IR25a and IR76b and of D. suzukii IR8a were obtained from previous studies (Walker et al, 2016;Walker et al, 2023;Ramasamy et al, 2016). Sequences were submitted to GeneArt TM gene synthesis service (Thermos Fischer Scientific, Waltham, MA, USA) requesting integration of the 5'-upstram CACC Kozak-sequence, as done previously (Bobkov et al, 2021;Cattaneo et al, 2017;Crava et al, 2022), codon optimization for expression in human cells (Roberts et al, 2021) media was replaced with 2.0 mL fresh media to incubate cells at 37ºC for up to 6-8 additional hours. Part of the cell culture was split in the middle of Mat Tek P35G-1.5-14-C dishes (Ashland, MA USA) as individual cells or small clusters and rinsed at the sides with 2.0 ml fresh HEK media.…”