Characterization and functional analysis of an α-adrenergic-like octopamine receptor in the small brown planthopper Laodelphax striatellus.

As an important biogenic amine in invertebrates and corresponding to the neurotransmitter norepinephrine in vertebrates, octopamine (OA) regulates diverse physiological and behavioral processes by binding to specific octopamine receptors (OARs) in invertebrates. At present, OARs have been identified and characterized in several insects. However, less is known about the OARs of Laodelphax striatellus, one of the most destructive pests in East Asian rice fields. In the present study, an α1-adrenergic-like OAR (LsOA1) from L. striatellus was cloned. LsOA1 has the typical characteristics of G-protein coupled receptors and is clustered with other insect homologs. The transcript level of LsOA1 varied in various stages and tissues, and was highly expressed at the egg stage and in the brain. Silencing of LsOA1 causes a reduction in vitellogenin (LsVg) and vitellogenin receptor (LsVgR) expression. Although LsOA1 interference did not affect the fecundity and survival of L. striatellus, the hatching rate of L. striatellus was significantly reduced, and the hatching period was prolonged. The decrease in the amount of honeydew excreted after silencing LsOA1 indicates that LsOA1 may be involved in regulating the feeding behavior of L. striatellus. In addition, the interference of LsOA1 significantly reduced the expression of capsid protein (CP) and viral RNA3 segment (RNA3) in rice stripe virus (RSV)-viruliferous L. striatellus, but did not affect the vertical transmission rate of RSV. The present study demonstrated that LsOA1 played a crucial role in the physiological and behavioral processes of L. striatellus, which will provide the basis for developing a new target gene for pest control.

ADRA2B and HTR1A: An Updated Study of the Biogenic Amine Receptors Reveals Novel Conserved Motifs Which Play Key Role in Mental Disorders.

Mental disorders are strongly connected with several psychiatric conditions including depression, bipolar disorder, schizophrenia, eating disorder, and suicides. There are many biological conditions and pathways that define these complicated illnesses. For example, eating disorders are complex mental health conditions that require the intervention of geneticists, psychiatrists, and medical experts in order to alleviate their symptoms. A patient with suicidal ideation should first be identified and consequently monitored by a similar team of specialists. Both genetics and epigenetics can shed light on eating disorders and suicides as they are found in the main core of such investigations. In the present study, an analysis has been performed on two specific members of the GPCR family toward drawing conclusions regarding their functionality and implementation in mental disorders. Specifically, evolutionary and structural studies on the adrenoceptor alpha 2b (ADRA2B) and the 5-hydroxytryptamine receptor 1A (HTR1A) have been carried out. Both receptors are classified in the biogenic amine receptors sub-cluster of the GPCRs and have been connected in many studies with mental diseases and malnutrition conditions. The major goal of this study is the investigation of conserved motifs among biogenic amine receptors that play an important role in this family signaling pathway, through an updated evolutionary analysis and the correlation of this information with the structural features of the HTR1A and ADRA2B. Furthermore, the structural comparison of ADRA2B, HTR1A, and other members of GPCRs related to mental disorders is performed.

Structural basis of amine odorant perception by a mammal olfactory receptor.

Odorants are detected as smell in the nasal epithelium of mammals by two G-protein-coupled receptor families, the odorant receptors and the trace amine-associated receptors1,2 (TAARs). TAARs emerged following the divergence of jawed and jawless fish, and comprise a large monophyletic family of receptors that recognize volatile amine odorants to elicit both intraspecific and interspecific innate behaviours such as attraction and aversion3-5. Here we report cryo-electron microscopy structures of mouse TAAR9 (mTAAR9) and mTAAR9-Gs or mTAAR9-Golf trimers in complex with ß-phenylethylamine, N,N-dimethylcyclohexylamine or spermidine. The mTAAR9 structures contain a deep and tight ligand-binding pocket decorated with a conserved D3.32W6.48Y7.43 motif, which is essential for amine odorant recognition. In the mTAAR9 structure, a unique disulfide bond connecting the N terminus to ECL2 is required for agonist-induced receptor activation. We identify key structural motifs of TAAR family members for detecting monoamines and polyamines and the shared sequence of different TAAR members that are responsible for recognition of the same odour chemical. We elucidate the molecular basis of mTAAR9 coupling to Gs and Golf by structural characterization and mutational analysis. Collectively, our results provide a structural basis for odorant detection, receptor activation and Golf coupling of an amine olfactory receptor.

Octopamine receptor gene influences social grouping in the masked birch caterpillar.

OBJECTIVE: Group-living plays a key role in the success of many insects, but the mechanisms underlying group formation and maintenance are poorly understood. Here we use the masked birch caterpillar, Drepana arcuata, to explore genetic influences on social grouping. These larvae predictably transition from living in social groups to living solitarily during the 3rd instar of development. Our previous study showed a notable shift in the D. arcuata transcriptome that correlates with the transition from grouping to solitary behavior. We noted that one differentially regulated gene, octopamine receptor gene (DaOAR), is a prominent 'social' gene in other insect species, prompting us to test the hypothesis that DaOAR influences grouping behavior in D. arcuata. This was done using RNA interference (RNAi) methods by feeding second instar larvae synthetic dsRNAs. RESULTS: RT-qPCR analysis confirmed a significant reduction in DaOAR transcript abundance in dsRNA-fed larvae compared to controls. Behavioral trials showed that caterpillars with reduced transcript abundance of DaOAR remained solitary throughout the observation period compared to controls. These results provide evidence that regulation of the octopamine receptor gene influences social grouping in D. arcuata, and that specifically, a decrease in octopamine receptor expression triggers the larval transition from social to solitary.

PaOctß2R: Identification and Functional Characterization of an Octopamine Receptor Activating Adenylyl Cyclase Activity in the American Cockroach Periplaneta americana.

Biogenic amines constitute an important group of neuroactive substances that control and modulate various neural circuits. These small organic compounds engage members of the guanine nucleotide-binding protein coupled receptor (GPCR) superfamily to evoke specific cellular responses. In addition to dopamine- and 5-hydroxytryptamine (serotonin) receptors, arthropods express receptors that are activated exclusively by tyramine and octopamine. These phenolamines functionally substitute the noradrenergic system of vertebrates Octopamine receptors that are the focus of this study are classified as either α- or ß-adrenergic-like. Knowledge on these receptors is scarce for the American cockroach (Periplaneta americana). So far, only an α-adrenergic-like octopamine receptor that primarily causes Ca2+ release from intracellular stores has been studied from the cockroach (PaOctα1R). Here we succeeded in cloning a gene from cockroach brain tissue that encodes a ß-adrenergic-like receptor and leads to cAMP production upon activation. Notably, the receptor is 100-fold more selective for octopamine than for tyramine. A series of synthetic antagonists selectively block receptor activity with epinastine being the most potent. Bioinformatics allowed us to identify a total of 19 receptor sequences that build the framework of the biogenic amine receptor clade in the American cockroach. Phylogenetic analyses using these sequences and receptor sequences from model organisms showed that the newly cloned gene is an ß2-adrenergic-like octopamine receptor. The functional characterization of PaOctß2R and the bioinformatics data uncovered that the monoaminergic receptor family in the hemimetabolic P. americana is similarly complex as in holometabolic model insects like Drosophila melanogaster and the honeybee, Apis mellifera. Thus, investigating these receptors in detail may contribute to a better understanding of monoaminergic signaling in insect behavior and physiology.

Molecular characterization and functional analysis of Bxy-octr-1 in Bursaphelenchus xylophilus.

Bursaphelenchus xylophilus is an invasive plant-parasitic nematode causing the notorious pine wilt disease (PWD) worldwide, which results in huge economic losses. G protein-coupled receptors (GPCRs) play an essential role in mating and reproduction behavior of animals. As a unique biogenic amine in invertebrates, octopamine (OA) can regulate a variety of physiological and behavioral responses by binding specific GPCRs. These specific GPCRs are also called octopamine receptors (OARs), and octr-1 is one of them. However, Bxy-octr-1 is unknown in B. xylophilus. Therefore, we investigated the expression pattern and biological function of Bxy-octr-1. Bioinformatics analysis indicated that Bxy-octr-1 was evolutionarily conserved. The real-time quantitative PCR data revealed that Bxy-octr-1 expression was required throughout the entire life of B. xylophilus. mRNA in situ hybridization showed that Bxy-octr-1 was mainly located in the cephalopharynx, body wall muscle, intestine, and gonadal organs of B. xylophilus. RNA interference (RNAi) showed that embryo hatching rates and locomotion speeds were both dramatically decreased. Obvious abnormal phenotypes were observed in the second-stage of juveniles after RNAi treated. Furthermore, its ontogenesis was stunting. Lack of Bxy-octr-1 reduced fecundity of females, of which 31.25% of them could not successfully ovulate. In addition, the error positioning ratio of the nematode was significantly increased. Our study suggests that Bxy-octr-1 is indispensable for locomotion, early ontogenesis and mating behavior in B. xylophilus.

Evolution of Brain-Expressed Biogenic Amine Receptors into Olfactory Trace Amine-Associated Receptors.

The family of trace amine-associated receptors (TAARs) is distantly related to G protein-coupled biogenic aminergic receptors. TAARs are found in the brain as well as in the olfactory epithelium where they detect biogenic amines. However, the functional relationship of receptors from distinct TAAR subfamilies and in different species is still uncertain. Here, we perform a thorough phylogenetic analysis of 702 TAAR-like (TARL) and TAAR sequences from 48 species. We show that a clade of Tarl genes has greatly expanded in lampreys, whereas the other Tarl clade consists of only one or two orthologs in jawed vertebrates and is lost in amniotes. We also identify two small clades of Taar genes in sharks related to the remaining Taar genes in bony vertebrates, which are divided into four major clades. We further identify ligands for 61 orphan TARLs and TAARs from sea lamprey, shark, ray-finned fishes, and mammals, as well as novel ligands for two 5-hydroxytryptamine receptor 4 orthologs, a serotonin receptor subtype closely related to TAARs. Our results reveal a pattern of functional convergence and segregation: TARLs from sea lamprey and bony vertebrate olfactory TAARs underwent independent expansions to function as chemosensory receptors, whereas TARLs from jawed vertebrates retain ancestral response profiles and may have similar functions to TAAR1 in the brain. Overall, our data provide a comprehensive understanding of the evolution and ligand recognition profiles of TAARs and TARLs.

Fine-tuning spermidine binding modes in the putrescine binding protein PotF.

A profound understanding of the molecular interactions between receptors and ligands is important throughout diverse research, such as protein design, drug discovery, or neuroscience. What determines specificity and how do proteins discriminate against similar ligands? In this study, we analyzed factors that determine binding in two homologs belonging to the well-known superfamily of periplasmic binding proteins, PotF and PotD. Building on a previously designed construct, modes of polyamine binding were swapped. This change of specificity was approached by analyzing local differences in the binding pocket as well as overall conformational changes in the protein. Throughout the study, protein variants were generated and characterized structurally and thermodynamically, leading to a specificity swap and improvement in affinity. This dataset not only enriches our knowledge applicable to rational protein design but also our results can further lay groundwork for engineering of specific biosensors as well as help to explain the adaptability of pathogenic bacteria.

Knockdown of a ß-Adrenergic-Like Octopamine Receptor Affects Locomotion and Reproduction of Tribolium castaneum.

The neurohormone octopamine regulates many crucial physiological processes in insects and exerts its activity via typical G-protein coupled receptors. The roles of octopamine receptors in regulating behavior and physiology in Coleoptera (beetles) need better understanding. We used the red flour beetle, Tribolium castaneum, as a model species to study the contribution of the octopamine receptor to behavior and physiology. We cloned the cDNA of a ß-adrenergic-like octopamine receptor (TcOctß2R). This was heterologously expressed in human embryonic kidney (HEK) 293 cells and was demonstrated to be functional using an in vitro cyclic AMP assay. In an RNAi assay, injection of dsRNA demonstrated that TcOctß2R modulates beetle locomotion, mating duration, and fertility. These data present some roles of the octopaminergic signaling system in T. castaneum. Our findings will also help to elucidate the potential functions of individual octopamine receptors in other insects.

Structure-dependent receptor subtype selectivity and G protein subtype preference of heterocyclic agonists in heterologously expressed silkworm octopamine receptors.

(R)-Octopamine (OA), a major invertebrate biogenic amine, plays an important role in a wide variety of physiological processes as a neurohormone, neuromodulator, and neurotransmitter in insects. OA receptors (OARs) are class A G protein-coupled receptors that specifically bind OA to activate downstream signaling cascades by coupling to G proteins and presumably other regulatory proteins. These receptors are broadly classified as α- and ß-adrenergic-like OARs (α- and ß-ALOARs). OARs are considered important targets of insecticides and acaricides. In the present study, we examined the actions of an array of 13 heterocyclic OAR agonists with the moieties that correspond to the phenyl group and the basic nitrogen atom of OA on α- and ß-ALOARs from the silkworm (Bombyx mori) and the signaling pathways activated through these actions. The results indicated that these compounds display structure-dependent receptor subtype selectivity and G protein subtype preference, underscoring the need to determine which subtype and signaling pathway mediates toxicologically relevant effects for the efficient discovery of novel pest control chemicals. The results of insecticidal assays using B. mori larvae suggested that the activation of signal transduction pathways via α-ALOARs might be mainly responsible for the toxicological effects of the heterocycles.

Genomic Characterization of the Barnacle Balanus improvisus Reveals Extreme Nucleotide Diversity in Coding Regions.

Barnacles are key marine crustaceans in several habitats, and they constitute a common practical problem by causing biofouling on man-made marine constructions and ships. Despite causing considerable ecological and economic impacts, there is a surprising void of basic genomic knowledge, and a barnacle reference genome is lacking. We here set out to characterize the genome of the bay barnacle Balanus improvisus (= Amphibalanus improvisus) based on short-read whole-genome sequencing and experimental genome size estimation. We show both experimentally (DNA staining and flow cytometry) and computationally (k-mer analysis) that B. improvisus has a haploid genome size of ~ 740 Mbp. A pilot genome assembly rendered a total assembly size of ~ 600 Mbp and was highly fragmented with an N50 of only 2.2 kbp. Further assembly-based and assembly-free analyses revealed that the very limited assembly contiguity is due to the B. improvisus genome having an extremely high nucleotide diversity (π) in coding regions (average π ≈ 5% and average π in fourfold degenerate sites ≈ 20%), and an overall high repeat content (at least 40%). We also report on high variation in the α-octopamine receptor OctA (average π = 3.6%), which might increase the risk that barnacle populations evolve resistance toward antifouling agents. The genomic features described here can help in planning for a future high-quality reference genome, which is urgently needed to properly explore and understand proteins of interest in barnacle biology and marine biotechnology and for developing better antifouling strategies.

Molecular and pharmacological characterization of biogenic amine receptors from the diamondback moth, Plutella xylostella.

BACKGROUD: Insect biogenic amines play important roles in mediating behavioral and physiological processes. They exert their effects by binding to biogenic amine receptors (BARs), which are specific receptor proteins in the G-protein-coupled receptor superfamily. BAR genes have been cloned and characterized from multiple model insects, including Drosophila melanogaster, Anopheles gambiae, Bombyx mori, Apis mellifera and Tribolium castaneum. However, relatively little work has addressed the molecular properties, expression profiles, and pharmacological characterization of BARs from other insects, including important pests. RESULTS: In this study, we cloned 17 genes encoding putative biogenic amine receptor proteins from Plutella xylostella, a global pest of Brassica crops. These PxBAR genes were five octopamine receptors (PxOA1, PxOA2B1, PxOA2B2, PxOA2B3, and PxOA3), three tyramine receptors (PxTAR1A, PxTAR1B, and PxTAR2), four dopamine receptors (PxDOP1, PxDOP2, PxDOP3, and PxDopEcR), and five serotonin receptors (Px5-HT1A , Px5-HT1B , Px5-HT2A , Px5-HT2B , and Px5-HT7 ). All PxBARs showed considerable sequence identity with orthologous BARs, and phylogenetic analysis clustered the receptors within their respective groups while preserving organismal evolutionary relationships. We investigated their molecular properties and expression profiles, and pharmacologically characterized the dopamine receptor, PxDOP2. CONCLUSIONS: Our study provides important information and resources on biogenic amine receptors from P. xylostella, which suggests potential target sites for controlling this pest species. © 2021 Society of Chemical Industry.

Sensitivity to expression levels underlies differential dominance of a putative null allele of the Drosophila tßh gene in behavioral phenotypes.

The biogenic amine octopamine (OA) and its precursor tyramine (TA) are involved in controlling a plethora of different physiological and behavioral processes. The tyramine-ß-hydroxylase (tßh) gene encodes the enzyme catalyzing the last synthesis step from TA to OA. Here, we report differential dominance (from recessive to overdominant) of the putative null tßhnM18 allele in 2 behavioral measures in Buridan's paradigm (walking speed and stripe deviation) and in proboscis extension (sugar sensitivity) in the fruit fly Drosophila melanogaster. The behavioral analysis of transgenic tßh expression experiments in mutant and wild-type flies as well as of OA and TA receptor mutants revealed a complex interaction of both aminergic systems. Our analysis suggests that the different neuronal networks responsible for the 3 phenotypes show differential sensitivity to tßh gene expression levels. The evidence suggests that this sensitivity is brought about by a TA/OA opponent system modulating the involved neuronal circuits. This conclusion has important implications for standard transgenic techniques commonly used in functional genetics.

Phenyl imidazolidin-2-ones antagonize a ß-adrenergic-like octopamine receptor in diamondback moth (Plutella xylostella).

BACKGROUND: The diamondback moth (Plutella xylostella) is one of the most destructive lepidopteran pests on cruciferous vegetables. However, resistance has emerged to current chemical and biological insecticides used for P. xylostella control, indicating the necessity of screening new targets on P. xylostella, and finding new insecticides against P. xylostella. In particular, octopamine receptors are representative G protein-coupled receptors found only in invertebrates and are potential targets for identifying novel insecticides. RESULTS: A ß-adrenergic-like octopamine receptor gene (PxOA2B1) was cloned, and its pharmacological characteristics in P. xylostella were studied. The results demonstrated that octopamine could activate the PxOA2B1 receptor, with a half-maximal effective concentration (EC50 ) of 49.5 nm. Amitraz, an insecticide and acaricide, and its metabolite (N-2,4-dimethylphenyl-N'-methylformamidine; DPMF) were also found to act as PxOAB1R agonists. We synthesized phenyl imidazolidin-2-one derivatives 3a-h using DPMF as the lead compound, and compounds 3a-h showed similar antagonist activities as phentolamine, mianserin and chlorpromazine. In particular, 3d, with an EC50 of 25.2 nm, showed very similar antagonist activity to mianserin. CONCLUSION: This research found that PxOAB1R might be a potential target for P. xylostella control. Phenyl imidazolidin-2-ones could be novel potential antagonists targeted at octopamine receptors and would be useful tools for the design and development of novel insecticides. © 2021 Society of Chemical Industry.

Characterization and functional analysis of a ß-adrenergic-like octopamine receptor from the oriental armyworm (Mythimna separata Walker).

The ß-adrenergic-like octopamine receptor (OA2B2), which binds the biogenic amine octopamine, belongs to the class of G-protein coupled receptors and significantly regulates many physiological and behavioral processes in insects. In this study, the putative open reading frame sequence of the MsOA2B2 gene in Mythimna separata was cloned, the full-length complementary DNA was 1191 bp and it encoded a 396-amino acid protein (GenBank accession number MN822800). Orthologous sequence alignment, phylogenetic tree analysis, and protein sequence analysis all showed that the cloned receptor belongs to the OA2B2 protein family. Real-time quantitative polymerase chain reaction of spatial and temporal expression analysis revealed that the MsOAB2 gene was expressed in all developmental stages of M. separata and was most abundant in egg stages and second and fourth instars compared with other developmental stages, while the expression level during the pupal stage was much lower than that at the other stages. Further analysis with sixth instar M. separata larvae showed that the MsOA2B2 gene was expressed 1.81 times higher in the head than in integument and gut tissues. Dietary ingestion of dsMsOA2B2 significantly reduced the messenger RNA level of MsOA2B2 and decreased mortality following amitraz treatment. This study provides both a pharmacological characterization and the gene expression patterns of OA2B2 in M. separata, facilitating further research for insecticides using MsOA2B2 as a target.

Molecular Characterization of the ß2-like Octopamine Receptor of Helicoverpa armigera.

Helicoverpa armigera is a devastating polyphagous and cosmopolitan crop pest. There are reports of this insect being resistant to a variety of pesticides raising concern worldwide. The Octopamine (OA) binding ß2-like receptor (OAR), a GPCR, is widely distributed in the nervous system of the insect and plays essential roles in the physiology and development and thus is an important target for insecticides. Yet, the molecular characterization of the H. armigera OAR (HarmOAR) and rational design of compounds based on this receptor is lacking. As a first step, we performed multiple sequence alignment of all insect OARs, which revealed that the sequences contained all conserved class A GPCR motifs. Phylogenetic studies showed clade-specific variations in the protein sequences primarily arising owing to differences in the ICL3 loop region. Further, a structural model of HarmOAR was built using the inactive human ß2AR as a template. 0.9 µs atomistic simulations revealed conserved inter helical contacts and water molecules of HarmOAR. The detailed binding of octopamine was studied using molecular docking and 0.3 µs atomistic simulations. Twenty-two insecticides active against octopamine receptors of other insects were compiled and docked to HarmOAR followed by rescoring with binding free energies to prioritize them for H. armigera. Our study suggests α-terpineol to be a good candidate as an insecticide or insect repellent for Helicoverpa armigera.

Are insect GPCRs ideal next-generation pesticides: opportunities and challenges.

The increasing human population, combined with low inefficiency and adverse effects of available pesticides, has magnified the urgent need of developing next-generation pesticides. Among the available approaches, strategies targeting invertebrate G protein-coupled receptors (GPCRs) are very promising as these receptors are the targets of endogenous neuropeptides/neuromodulators that upon binding to their receptors induce profound changes in insect physiology. Therefore, exploring GPCR regulators has great potential in the development of targeted next-generation pesticides. Despite the great potential of such alternative pesticides, so far there has been only one approved compound, Amitraz, which conveys its anti-pest activity via the GPCR Octopamine receptor. Here, we review the current status of pesticide development, hazards associated with conventional pesticide compounds, alternative strategies that involve next-generation of pesticides, structural features of GPCRs, and opportunities and challenges of targeting the members of this superfamily in invertebrates to develop anti-pest agents. In conclusion, we emphasize that the potential of GPCRs cannot be utilized in full without more genomic and transcriptomic data to improve our understanding of the complex network of peptidergic signaling pathways. We argue how vital it is to obtain three-dimensional (3D) structural data on physiologically important target GPCRs and encourage the readers to use the state of the art in silico methods such as virtual screening for the discovery of new pesticide compounds.

Genotyping amitraz resistance profiles in Rhipicephalus microplus Canestrini (Acari: Ixodidae) ticks from Punjab, India.

Acaricide resistance is one of the greatest threats to sustainable and effective control of vector ticks worldwide. The amitraz resistance status in cattle tick, Rhipicephalus microplus populations collected from 18 districts of Punjab in north-western India were characterized using bioassay and molecular assays. The modified larval packet test was used and the resistance factors (RF) against amitraz for the field populations were in the range of 0.36-4.85, indicating level I resistance status in ten populations. Characterization of a partial segment of the octopamine/tyramine (OCT/Tyr) receptor gene of R. microplus field populations from Punjab revealed a total of 18 nucleotide substitutions in the coding region out of which 5 were non-synonymous substitutions. Three of these non-synonymous substitutions (T8P, V15I and A20 T) were earlier reported in American and South African populations of R. microplus. Among the two single nucleotide polymorphisms (A22C-T8P; T65C-L22S) potentially linked to amitraz resistance in American, South African and Zimbabwean resistant populations, only the T8P substitution was recorded from the Barnala population. The PCR-RFLP assay using EciI restriction enzyme was used for genotyping of the larvae as homozygous resistant (RR), homozygous susceptible (SS) and heterozygous (SR). Genotyping of 514 larval DNA samples from 18 field populations revealed 92.8 % larval population as SR and the remaining 7.2 % as RR genotypes. The percentage of resistant alleles in the tick populations was 53.6 (range 50.0-57.2) indicating its moderate distribution in the region. The present study is the pioneer report establishing the hypothesis that amitraz-resistance is recessively inherited and heterozygous individuals show phenotypic susceptibility to the drug in the Indian tick populations.

Forager age and foraging state, but not cumulative foraging activity, affect biogenic amine receptor gene expression in the honeybee mushroom bodies.

Foraging behavior is crucial for the development of a honeybee colony. Biogenic amines are key mediators of learning and the transition from in-hive tasks to foraging. Foragers vary considerably in their behavior, but whether and how this behavioral diversity depends on biogenic amines is not yet well understood. For example, forager age, cumulative foraging activity or foraging state may all be linked to biogenic amine signaling. Furthermore, expression levels may fluctuate depending on daytime. We tested if these intrinsic and extrinsic factors are linked to biogenic amine signaling by quantifying the expression of octopamine, dopamine and tyramine receptor genes in the mushroom bodies, important tissues for learning and memory. We found that older foragers had a significantly higher expression of Amdop1, Amdop2, AmoctαR1, and AmoctßR1 compared to younger foragers, whereas Amtar1 showed the opposite pattern. Surprisingly, our measures of cumulative foraging activity were not related to the expression of the same receptor genes in the mushroom bodies. Furthermore, we trained foragers to collect sucrose solution at a specific time of day and tested if the foraging state of time-trained foragers affected receptor gene expression. Bees engaged in foraging had a higher expression of Amdop1 and AmoctßR3/4 than inactive foragers. Finally, the expression of Amdop1, Amdop3, AmoctαR1, and Amtar1 also varied with daytime. Our results show that receptor gene expression in forager mushroom bodies is complex and depends on both intrinsic and extrinsic factors.

AmOctα2R: Functional Characterization of a Honeybee Octopamine Receptor Inhibiting Adenylyl Cyclase Activity.

The catecholamines norepinephrine and epinephrine are important regulators of vertebrate physiology. Insects such as honeybees do not synthesize these neuroactive substances. Instead, they use the phenolamines tyramine and octopamine for similar physiological functions. These biogenic amines activate specific members of the large protein family of G protein-coupled receptors (GPCRs). Based on molecular and pharmacological data, insect octopamine receptors were classified as either α- or ß-adrenergic-like octopamine receptors. Currently, one α- and four ß-receptors have been molecularly and pharmacologically characterized in the honeybee. Recently, an α2-adrenergic-like octopamine receptor was identified in Drosophila melanogaster (DmOctα2R). This receptor is activated by octopamine and other biogenic amines and causes a decrease in intracellular cAMP ([cAMP]i). Here, we show that the orthologous receptor of the honeybee (AmOctα2R), phylogenetically groups in a clade closely related to human α2-adrenergic receptors. When heterologously expressed in an eukaryotic cell line, AmOctα2R causes a decrease in [cAMP]i. The receptor displays a pronounced preference for octopamine over tyramine. In contrast to DmOctα2R, the honeybee receptor is not activated by serotonin. Its activity can be blocked efficiently by 5-carboxamidotryptamine and phentolamine. The functional characterization of AmOctα2R now adds a sixth member to this subfamily of monoaminergic receptors in the honeybee and is an important step towards understanding the actions of octopamine in honeybee behavior and physiology.