Comparative transcriptomic analysis of the Drosophila melanogaster antennal olfactory subsystems.
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ABSTRACT: We identified genes whose expression is enriched in the D. melanogaster antennal olfactory subsystems that express Odorant receptors (Ors) or Ionotropic receptors (Irs) by microarray analysis of RNA extracted from antennae of wild-type (Oregon R P2) animals, ato mutants (which lack the Ir subsystem) and amos mutants (which lack the Or subsystem).
Project description:Monarch butterflies (Danaus plexippus) rely on milkweeds as larval host plants. Host plant seeking and verification by female butterflies may be mediated by gustatory (GRs) and olfactory receptors (ORs). Here we employed RNA-Seq, bioinformatics and RT-qPCR techniques to identify sex- and tissue-specific gene expression. We focused on chemosensation related genes and pathways, including putative ORs, GRs, ionotropic receptors (IRs), odorant-binding proteins, chemosensory proteins, and steroid hormone mediated signaling in specific chemosensory tissues (i.e., antennae, legs and proboscis). Twelve butterflies evenly split between males and females were caught and used for RNA extraction. Tissue-specific sequencing libraries were prepared and sequenced using Illumina NovaSeq 6000 or HiSeq 3000, generating 2 billion 150-bp, paired-end reads. Many more genes and gene sets were differentially expressed between tissue types than between sexes. A total of 148 chemosensation-related genes exhibited sex- and/or tissue-biased expression. RT-qPCR of a small set of genes confirmed their differential expression between tissue types or between males and females. These findings laid a solid foundation for further investigations into the biological roles of these identified genes underlying chemosensation-mediated behaviors such as foraging and reproduction.
Project description:Ecdysone-induced protein 93F (E93) regulates local chromatin structure and/or gene transcription, raising the question of whether expression of Ir75c, Ir75b and Ir75a receptors in antenna is affected at the transcriptional level in the absence of this protein. We performed bulk RNA-sequencing (RNA-seq) of control and E93RNAi antennae. In control RNA-seq samples, we predicted the existence of seven distinct transcripts encoded by these three receptor genes. Notably, many transcripts initiating from Ir75c do not terminate at the 3’ end of this gene but rather run through the Ir75b and Ir75a exons. Similarly, a majority of transcripts initiating from Ir75b incorporate exons 2-7 of Ir75a. The pattern of transcription is consistent with the absence of canonical transcription termination/polyadenylation sequences downstream of the Ir75c and Ir75b coding regions; such a sequence is only present 3’ of Ir75a. All transcripts initiating from Ir75c are highly up-regulated in the absence of E93, consistent with the ectopic expression of receptor protein. Transcription initiating from Ir75b is strongly diminished, concordant with the loss of Ir75b protein expression. By contrast, Ir75a transcripts levels were not significantly lower in E93RNAi antennae, despite the loss of detectable Ir75a protein.
Project description:We assessed the differential expression of genes in the adult antennae of pnt RNAi and control flies to understand how the expressions of olfactory receptors could change in response to cell fate alteration in the antennae.
Project description:To better understand the olfactory mechanisms in G. mellonella, we established six transcriptomes from the antennae of male and female adults. A total of 226 chemosensory gene transcripts were identified, which included 36 chemosensory proteins, 52 odorant-binding proteins, 2 sensory neuron membrane proteins, 80 olfactory receptors, and 56 ionotropic receptors. The expression patterns of these genes were calculated from the estimated fragments per kilobase of transcript sequence per million fragments mapped, and the differentially expressed genes (DEGs) related to olfaction were validated by qRT-PCR. Among the 114 DEGs identified, 66 were exclusively or primarily expressed in the female antenna, whereas the rest were specifically expressed in the male antenna. These antenna-specific genes may provide useful subjects for advanced studies of their biological functions; this may provide a theoretical basis for the control of G. mellonella and the prevention of damage caused by this species using the male pheromone as a bait.
Project description:G protein-coupled receptors in intracellular organelles can be activated in response to membrane permeant ligands, which contributes to the diversity and specificity of agonist action. The opioid receptors (ORs) provide a striking example, where small molecule opioid drugs activate ORs in the Golgi apparatus within seconds of drug addition. To date, our knowledge on the signaling of intracellular GPCRs remains incomplete and it is unknown if the downstream events triggered by ORs in plasma membrane and Golgi apparatus differ. To address this gap, we analyzed OR-mediated phosphorylation changes in cells treated with SNC80 (signaling control) or ICI-SNC80 (Golgi-restricted signaling) for 5 min or 25 min. We show that OR activation in the plasma membrane or Golgi apparatus have strikingly different downstream effects on protein phosphorylation. The study delineates OR signal transduction with unprecedented resolution and reveals that the subcellular location defines the signaling effect promoted by opioid drugs.
Project description:Queen discrimination behavior in the red imported fire ant Solenopsis invicta maintains its two types of societies: colonies with one (monogyne) or many (polygyne) queens, yet the underlying genetic mechanism is poorly understood. This behavior is controlled by two supergene alleles, SB and Sb, with ~600 genes. Polygyne workers, having either the SB/SB or SB/Sb genotype, accept additional SB/Sb queens into their colonies but kill SB/SB queens. While monogyne workers, all SB/SB, reject all additional queens regardless of genotype. Because the SB and Sb alleles do not recombine, it is difficult to determine which genes within the supergene mediate this differential worker behavior. We hypothesized that the alternate worker genotypes sense queens differently because of different patterns of gene expression in their main sensory organ, the antennae. To identify such differentially expressed genes, we sequenced RNA from four biological replicates of pooled antennae from three groups of workers: monogyne SB/SB, polygyne SB/SB, and polygyne SB/Sb. We identified 81 differentially expressed protein coding genes with 14 encoding potential odor metabolism and perception proteins. We focused on the two differentially expressed odorant perception genes: an odorant binding protein SiOBP12 and an odorant receptor SiOR463. We found that the SiOR463 was lost in the Sb-genome. In contrast, the SiOBP12 has an Sb-specific duplication SiOBP12b’, which was expressed in the SB/Sb worker antennae, while both paralogs SiOBP12 and SiOBP12b’ were expressed in the body. This result indicates that SiOBP12b’ has gained an antennal promoter or enhancer and suggests neofunctionalization, perhaps for queen discrimination behavior.
Project description:Queen discrimination behavior in the red imported fire ant Solenopsis invicta maintains its two types of societies: colonies with one (monogyne) or many (polygyne) queens, yet the underlying genetic mechanism is poorly understood. This behavior is controlled by two supergene alleles, SB and Sb, with ~600 genes. Polygyne workers, having either the SB/SB or SB/Sb genotype, accept additional SB/Sb queens into their colonies but kill SB/SB queens. While monogyne workers, all SB/SB, reject all additional queens regardless of genotype. Because the SB and Sb alleles do not recombine, it is difficult to determine which genes within the supergene mediate this differential worker behavior. We hypothesized that the alternate worker genotypes sense queens differently because of different patterns of gene expression in their main sensory organ, the antennae. To identify such differentially expressed genes, we sequenced RNA from four biological replicates of pooled antennae from three groups of workers: monogyne SB/SB, polygyne SB/SB, and polygyne SB/Sb. We identified 81 differentially expressed protein coding genes with 14 encoding potential odor metabolism and perception proteins. We focused on the two differentially expressed odorant perception genes: an odorant binding protein SiOBP12 and an odorant receptor SiOR463. We found that the SiOR463 was lost in the Sb-genome. In contrast, the SiOBP12 has an Sb-specific duplication SiOBP12b’, which was expressed in the SB/Sb worker antennae, while both paralogs SiOBP12 and SiOBP12b’ were expressed in the body. This result indicates that SiOBP12b’ has gained an antennal promoter or enhancer and suggests neofunctionalization, perhaps for queen discrimination behavior.