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.

Knockout of tyramine receptor 1 results in a decrease of oviposition, mating, and sex pheromone biosynthesis in female Plutella xylostella.

BACKGROUND: Mating and oviposition are essential and closely coordinated events in the reproduction of moths. Although tyramine, a biogenic amine, can affect insect reproduction by binding its receptors, the specific regulatory mechanism has not yet been fully elucidated. RESULTS: Plutella xylostella mutant with tyramine receptor 1 (TAR1) knockout (homozygous mutant with 7-bp deletion, Mut7) was developed by the CRISPR/Cas9 system to investigate the effect of TAR1 knockout on the reproduction of the moth. Compared with wild-type (WT), the egg yield of Mut7 female (Mut7F ) was significantly lower, no significant difference was observed in the egg size and hatching ratio between the groups. Further analysis showed that TAR1 knockout adversely affected ovary development, characterized by shorter ovarioles and fewer mature oocyte. Additionally, TAR1 knockout significantly reduced the occurrence of mating, resulting in a decrease in egg yield in Mut7F . The amounts of sex pheromones were quantified using gas chromatography-mass spectrometry. Results showed that the amounts of sex pheromone released by Mut7F were significantly lower before mating. Correspondingly, the messenger RNA (mRNA) levels of sex pheromone biosynthesis enzymes, including acetyl-CoA carboxylase (ACC) and desaturase (DES), were significantly lower in the Mut7F pheromone gland. The decreased sex pheromone biosynthesis in Mut7F , especially before re-mating, may be related to the underexpression of pheromone biosynthesis-activated neuropeptide (PBAN). CONCLUSION: Overall, this study investigated the effect of PxTAR1 on oviposition and mating of P. xylostella. We report for the first time that TAR1 knockout could reduce the sex pheromone biosynthesis. These findings provide insights for developing a novel integrated pest control strategy based on mating interference. © 2023 Society of Chemical Industry.

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.

Molecular and Pharmacological Characterization of ß-Adrenergic-like Octopamine Receptors in the Endoparasitoid Cotesia chilonis (Hymenoptera: Braconidae).

Octopamine (OA) is structurally and functionally similar to adrenaline/noradrenaline in vertebrates, and OA modulates diverse physiological and behavioral processes in invertebrates. OA exerts its actions by binding to specific octopamine receptors (OARs). Functional and pharmacological characterization of OARs have been investigated in several insects. However, the literature on OARs is scarce for parasitoids. Here we cloned three ß-adrenergic-like OARs (CcOctßRs) from Cotesia chilonis. CcOctßRs share high similarity with their own orthologous receptors. The transcript levels of CcOctßRs were varied in different tissues. When heterologously expressed in CHO-K1 cells, CcOctßRs induced cAMP production, and were dose-dependently activated by OA, TA and putative octopaminergic agonists. Their activities were inhibited by potential antagonists and were most efficiently blocked by epinastine. Our study offers important information about the molecular and pharmacological properties of ß-adrenergic-like OARs from C. chilonis that will provide the basis to reveal the contribution of individual receptors to the physiological processes and behaviors in parasitoids.

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.

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.

The octopamine receptor, OA2B2, modulates stress resistance and reproduction in Nilaparvata lugens Stål (Hemiptera: Delphacidae).

The brown planthopper (BPH), Nilaparvata lugens (Stål) is a resurgent pest of rice crops throughout Asia. We recently discovered that octopamine (OA) and OA2B2 operate in the BPH mating system, where it mediates a wide range of molecular, physiological and behavioural changes. Here, we report on outcomes of experiments designed to test the hypothesis that OA/OA2B2 signalling mediates responses to three abiotic stressors, starvation, high temperature (37 °C), and induced oxidative stress. We found per os RNAi-mediated OA2B2 silencing led to significantly decreased survival, measured in days, following exposure to each of these stressors. We selected a biologically costly process, reproductive biology, as a biotic stressor. Silencing of OA2B2 led to decreased total protein content in ovaries and fat bodies, downregulated expression of vitellogenin (Vg) and Vg receptor (VgR), inhibited fat body Vg protein synthesis, shortened the oviposition period, prolonged the preoviposition period, reduced the number of laid eggs, body weight and female longevity. In addition, the silencing treatments also led to inhibited ovarian development, and ovarian Vg uptake, reduced numbers of egg masses and offspring and lower hatching rates and population growth index. These data support our hypothesis that OA2B2 acts in mediating BPH resistance to biotic and abiotic stressors.

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.

Tyramine 1 Receptor Distribution in the Brain of Corbiculate Bees Points to a Conserved Function.

Sucrose represents an important carbohydrate source for most bee species. In the Western honeybee (Apis mellifera) it was shown that individual sucrose responsiveness correlates with the task performed in the colony, supporting the response threshold theory which states that individuals with the lowest threshold for a task-associated stimuli will perform the associated task. Tyramine was shown to modulate sucrose responsiveness, most likely via the tyramine 1 receptor. This receptor is located in brain areas important for the processing of gustatory stimuli. We asked whether the spatial expression pattern of the tyramine 1 receptor is a unique adaptation of honeybees or if its expression represents a conserved trait. Using a specific tyramine receptor 1 antibody, we compared the spatial expression of this receptor in the brain of different corbiculate bee species, including eusocial honeybees, bumblebees, stingless bees, and the solitary bee Osmia bicornis as an outgroup. We found a similar labeling pattern in the mushroom bodies, the central complex, the dorsal lobe, and the gnathal ganglia, indicating a conserved receptor expression. With respect to sucrose responsiveness this result is of special importance. We assume that the tyramine 1 receptor expression in these neuropiles provides the basis for modulation of sucrose responsiveness. Furthermore, the tyramine 1 receptor expression seems to be independent of size, as labeling is similar in bee species that differ greatly in their body size. However, the situation in the optic lobes appears to be different. Here, the lobula of stingless bees is clearly labeled by the tyramine receptor 1 antibody, whereas this labeling is absent in other species. This indicates that the regulation of this receptor is different in the optic lobes, while its function in this neuropile remains unclear.

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.

Response of tyramine and glutamate related signals to nanoplastic exposure in Caenorhabditis elegans.

Neurotransmission related signals are involved in the control of response to toxicants. We here focused on the tyramine and the glutamate related signals to determine their roles in regulating nanoplastic toxicity in Caenorhabditis elegans. In the range of µg/L, exposure to nanopolystyrene (100 nm) increased the expression of tdc-1 encoding a tyrosine decarboxylase required for synthesis of tyramine, and decreased the expression of eat-4 encoding a glutamate transporter. Both TDC-1 and EAT-4 could act in the neurons to regulate the nanopolystyrene toxicity. Meanwhile, neuronal RNAi knockdown of tdc-1 induced a susceptibility to nanopolystyrene toxicity, and neuronal RNAi knockdown of eat-4 induced a resistance to nanopolystyrene toxicity. In the neurons, TYRA-2 functioned as the corresponding receptor of tyramine and acted upstream of MPK-1 signaling to regulate the nanopolystyrene toxicity. Moreover, during the control of nanopolystyrene toxicity, GLR-4 and GLR-8 were identified as the corresponding glutamate receptors, and acted upstream of JNK-1 signaling and DBL-1 signaling, respectively. Our results demonstrated the crucial roles of tyramine and glutamate related signals in regulating the toxicity of nanoplastics in organisms.

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.

A comprehensive binding study illustrates ligand recognition in the periplasmic binding protein PotF.

Periplasmic binding proteins (PBPs) are ubiquitous receptors in gram-negative bacteria. They sense solutes and play key roles in nutrient uptake. Escherichia coli's putrescine receptor PotF has been reported to bind putrescine and spermidine. We reveal that several similar biogenic polyamines are recognized by PotF. Using isothermal titration calorimetry paired with X-ray crystallography of the different complexes, we unveil PotF's binding modes in detail. The binding site for PBPs is located between two lobes that undergo a large conformational change upon ligand recognition. Hence, analyzing the influence of ligands on complex formation is crucial. Therefore, we solved crystal structures of an open and closed apo state and used them as a basis for molecular dynamics simulations. In addition, we accessed structural behavior in solution for all complexes by 1H-15N HSQC NMR spectroscopy. This combined analysis provides a robust framework for understanding ligand binding for future developments in drug design and protein engineering.

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.

Correlation between octopaminergic signalling and foraging task specialisation in honeybees.

Regulation of pollen and nectar foraging in honeybees is linked to differences in the sensitivity to the reward. Octopamine (OA) participates in the processing of reward-related information in the bee brain, being a candidate to mediate and modulate the division of labour among pollen and nectar foragers. Here we tested the hypothesis that OA affects the resource preferences of foragers. We first investigated whether oral administration of OA is involved in the transition from nectar to pollen foraging. We quantified the percentage of OA-treated bees that switched from a sucrose solution to a pollen feeder when the sugar concentration was decreased experimentally. We also evaluated if feeding the colonies sucrose solution containing OA increases the rate of bees collecting pollen. Finally, we quantified OA and tyramine (TYR) receptor genes expression of pollen and nectar foragers in different parts of the brain, as a putative mechanism that affects the decision-making process regarding the resource type collected. Adding OA in the food modified the probability that foragers switch from nectar to pollen collection. The proportion of pollen foragers also increased after feeding colonies with OA-containing food. Furthermore, the expression level of the AmoctαR1 was upregulated in foragers arriving at pollen sources compared with those arriving at sugar-water feeders. Using age-matched pollen and nectar foragers that returned to the hive, we detected an upregulated expression of a TYR receptor gene in the suboesophageal ganglia. These findings support our prediction that OA signalling affects the decision in honeybee foragers to collect pollen or nectar.

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.

Astroglial Calcium Signaling Encodes Sleep Need in Drosophila.

Sleep is under homeostatic control, whereby increasing wakefulness generates sleep need and triggers sleep drive. However, the molecular and cellular pathways by which sleep need is encoded are poorly understood. In addition, the mechanisms underlying both how and when sleep need is transformed to sleep drive are unknown. Here, using ex vivo and in vivo imaging, we show in Drosophila that astroglial Ca2+ signaling increases with sleep need. We demonstrate that this signaling is dependent on a specific L-type Ca2+ channel and is necessary for homeostatic sleep rebound. Thermogenetically increasing Ca2+ in astrocytes induces persistent sleep behavior, and we exploit this phenotype to conduct a genetic screen for genes required for the homeostatic regulation of sleep. From this large-scale screen, we identify TyrRII, a monoaminergic receptor required in astrocytes for sleep homeostasis. TyrRII levels rise following sleep deprivation in a Ca2+-dependent manner, promoting further increases in astrocytic Ca2+ and resulting in a positive-feedback loop. Moreover, our findings suggest that astrocytes then transmit this sleep need to a sleep drive circuit by upregulating and releasing the interleukin-1 analog Spätzle, which then acts on Toll receptors on R5 neurons. These findings define astroglial Ca2+ signaling mechanisms encoding sleep need and reveal dynamic properties of the sleep homeostatic control system.

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.

TrpML-mediated astrocyte microdomain Ca2+ transients regulate astrocyte-tracheal interactions.

Astrocytes exhibit spatially-restricted near-membrane microdomain Ca2+transients in their fine processes. How these transients are generated and regulate brain function in vivo remains unclear. Here we show that Drosophila astrocytes exhibit spontaneous, activity-independent microdomain Ca2+ transients in their fine processes. Astrocyte microdomain Ca2+ transients are mediated by the TRP channel TrpML, stimulated by reactive oxygen species (ROS), and can be enhanced in frequency by the neurotransmitter tyramine via the TyrRII receptor. Interestingly, many astrocyte microdomain Ca2+ transients are closely associated with tracheal elements, which dynamically extend filopodia throughout the central nervous system (CNS) to deliver O2 and regulate gas exchange. Many astrocyte microdomain Ca2+ transients are spatio-temporally correlated with the initiation of tracheal filopodial retraction. Loss of TrpML leads to increased tracheal filopodial numbers, growth, and increased CNS ROS. We propose that local ROS production can activate astrocyte microdomain Ca2+ transients through TrpML, and that a subset of these microdomain transients promotes tracheal filopodial retraction and in turn modulate CNS gas exchange.

The effect of temperature on candidate gene expression in the brain of honey bee Apis mellifera (Hymenoptera: Apidae) workers exposed to neonicotinoid imidacloprid.

Neonicotinoid insecticides are potent agonists of nicotinic acetylcholine receptors and are a major factor in the decline of pollinators worldwide. Several studies show that low doses of this neurotoxin influence honey bee physiology, however, little is known about how insecticides interact with other environmental variables. We studied the effects of two neonicotinoid Imidacloprid doses (IMD, 0, 2.5, and 10 ppb), and three temperatures (20, 28, and 36°C) on gene expression in the brains of worker honey bees (Apis mellifera). Using qRT-PCR we quantified the expression of eight key genes related to the nervous system, stress response, and motor and olfactory capacities. Gene expression tended to increase with the low IMD dose, which was further intensified in individuals maintained in the cold treatment (20°C). At 20°C the octopamine receptor gene (oa1) was underexpressed in bees that were not exposed to IMD, but overexpressed in individuals exposed to 2.5 ppb IMD. Also, heat shock proteins (hsp70 and hsp90) increased their expression at high temperatures (36°C), but not with IMD doses. These results suggest that despite the low insecticide concentrations used in this study (a field-realistic dose), changes in gene expression associated with honey bee physiological responses could be induced. This study contributes to the understanding of how neonicotinoid residual doses may alter honey bee physiology.