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In insects, the biogenic amines octopamine (OA) and tyramine (TA) are involved in controlling several physiological and behavioural processes. OA and TA act as neurotransmitters, neuromodulators or neurohormones, performing their functions by binding to specific receptors belonging to the G protein-coupled receptor (GPCR) superfamily. OA and TA along with their receptors are involved in reproduction, smell perception, metabolism, and homeostasis. Moreover, OA and TA receptors are targets for insecticides and antiparasitic agents, such as the formamidine Amitraz.In the dengue and yellow fever vector,Aedes aegypti, limited research has been previously reported on their OA or TA receptors. Here, we identify and characterize the OA and TA receptors inA. aegypti. Bioinformatic tools have been used to identify four OA and three TA receptors in the genome ofA. aegypti. The seven receptors are expressed in all developmental stages ofA. aegypti; however, their highest transcript abundance is observed in the adult compared to the larval stages. Among several adultA. aegyptitissues examined, including the central nervous system, antennae and rostrum, midgut, Malpighian tubules, ovaries, and testes, the type 2 TA receptor (TAR2) transcript is most abundant in the ovaries and the type 3 TA receptor (TAR3) is enriched in the Malpighian tubules, leading us to hypothesize putative roles for these receptors in reproduction and diuresis, respectively. Furthermore, a blood meal influenced OA and TA receptor transcript expression patterns in adult female tissues at several time points post blood meal, suggesting these receptors may play key physiological roles associated with feeding. To better understand OA and TA signaling inA. aegypti, the transcript expression profiles of key enzymes in their biosynthetic pathway, namely tyrosine decarboxylase (Tdc) and tyramine β-hydroxylase (Tβh), were examined in developmental stages, adult tissues, and brains from blood-fed females.These findings provide information for better understanding the physiological roles of OA, TA, and their receptors inA. aegypti, and additionally, may help in the development of novel strategies for the control of these human disease vectors.Author summaryAedes aegyptiis the primary vector for dengue, chikungunya, and yellow fever – debilitating diseases that together are responsible for hundreds of millions of infections and thousands of deaths annually worldwide. Understanding theA. aegyptiphysiology may be critical for the development of new control strategies. In insects, the biogenic amines dopamine, serotonin, tyramine and octopamine play important roles in controlling various physiological processes. InA. aegypti, both serotonin and dopamine are implicated in blood feeding behavior and development. Conversely, the role of octopamine (OA) and tyramine (TA) inA. aegyptiphysiology is still poorly characterized. Both OA and TA exert their physiological actions by interacting with and activating different receptors, the tyramine (TAR) and the octopamine (OAR) receptors. Here, we show the characterization of the OA and TA receptors inA. aegypti. In theA. aegyptigenome we identify a total of four OA receptors and three TA receptors, suggesting for each receptor a particular role in the development and physiology of this insect.This work contributes to better understanding the roles of OA, TA, and their receptors, inA. aegyptidevelopment and physiology. Furthermore, it may be crucial in identifying novel strategies for the mosquitoes control.

Double-stranded (ds) RNA-based technologies could provide novel and potential tool for pest management with efficiency and specificity of action. However, before applying this technique in the field, it is necessary to identify effective delivery methods and evaluate the non-target effects that may occur. In this article, we evaluated the effectiveness of dsRNA by topical delivery on a species of great agricultural interest, Halyomorpha halys. The specificity of action of the dsRNA was also investigated in Rhodnius prolixus, an insect phylogenetically close to H. halys. Of the three investigated genes (putative ATPase N2B, ATPase, serine/threonine-protein phosphatase PP1-β catalytic subunit, PP1, and IAP repeat-containing protein 7-B-like, IAP), IAP and ATPase were able to induce higher mortality in H. halys nymphs compared to the control, with specific concentrations for each gene targeted. However, when the same RNAs were topically delivered to both R. prolixus 2nd and 3rd instar nymphs, no gene silencing and mortality were observed. For this reason, to assess dsRNA application-mediated non-target effects, we injected both H. halys and R. prolixus specific dsRNA in R. prolixus 5th instar nymphs. When the dsRNA targeting H. halys IAP was microinjected into R. prolixus 5th instar nymphs, no mortality was observed, suggesting a strong RNAi specificity. Together, these data suggest that the topical delivery could be suitable for the dsRNA to control H. halys population. Furthermore, its specificity of action would allow treatments towards single harmful species with limited non-target effects.

In insects, tyramine receptor 1 (TAR1) has been shown to control several physiological functions, including olfaction. We investigated the molecular and functional profile of the Halyomorpha halys type 1 tyramine receptor gene (HhTAR1) and its role in olfactory functions of this pest. Molecular and pharmacological analyses confirmed that the HhTAR1 gene codes for a true TAR1. RT-qPCR analysis revealed that HhTAR1 is expressed mostly in adult brain and antennae as well as in early development stages (eggs, 1st and 2nd instar nymphs). In particular, among the antennomeres that compose a typical H. halys antenna, HhTAR1 was more expressed in flagellomeres. Scanning electron microscopy investigation revealed the type and distribution of sensilla on adult H. halys antennae: both flagellomeres appear rich in trichoid and grooved sensilla, known to be associated with olfactory functions. Through an RNAi approach, topically delivered HhTAR1 dsRNA induced a 50% downregulation in gene expression after 24 h in H. halys 2nd instar nymphs. An innovative behavioural assay revealed that HhTAR1 RNAi-silenced 2nd instar nymphs were less susceptible to the alarm pheromone component (E)-2 decenal as compared with controls. These results provide critical information concerning the role of TAR1 in olfaction regulation, especially alarm pheromone reception, in H. halys. Furthermore, considering the emerging role of TAR1 as target of biopesticides, this work opens the way for further investigation on innovative methods for controlling H. halys.

Background: Mutations in leucine-rich repeat kinase 2 (LRRK2) are associated with familiar and sporadic Parkinson’s disease. LRRK2 modulates the autophagy-lysosome pathway (ALP), a clearance process subserving the quality control of cellular proteins and organelles. Since dysfunctional ALP might lead to α-synuclein accumulation and, hence, Parkinson’s disease, LRRK2 kinase modulation of ALP, its age-dependence and relation with pSer129 a-synuclein inclusions in striatal and nigral neurons were investigated in vivo. Methods: Striatal ALP markers were analyzed by Western blotting in 3, 12 and 20-month-old LRRK2 G2019S knock-in mice (bearing enhanced kinase activity), LRRK2 knock-out mice, LRRK2 D1994S knock-in (kinase-dead) mice and wild-type controls. The lysosomotropic agent chloroquine was used to investigate the autophagic flux in vivo. Quantitative Real-time PCR was used to quantify the transcript levels of key ALP genes. The activity of the lysosomal enzyme glucocerebrosidase was measured using enzymatic assay. Immunohistochemistry was used to co-localize LC3B puncta with pSer129 a-synuclein inclusion in striatal MAP-positive and nigral TH-positive neurons. Results: No genotype differences in macroautophagy and chaperone-mediated autophagy markers were observed at 3 months. Conversely, increase of LC3-I, p62, LAMP2 and GAPDH levels, decrease of p-mTOR levels and downregulation of mTOR and TFEB expression was observed in 12-month-old kinase-dead mice. The LC3-II/LC3-I ratio was reduced following administration of chloroquine, suggesting a defective autophagic flux. G2019S knock-in mice showed LAMP2 accumulation and downregulation of ALP key genes MAP1LC3B, LAMP2, mTOR, TFEB and GBA1. Subacute administration of the LRRK2 kinase inhibitor MLi-2 in wild-type and G2019S knock-in mice did not replicate the pattern of kinase-dead mice. Lysosomal glucocerebrosidase activity was increased in 3 and 12-month-old knock-out and kinase-dead mice, and GBA1 expression reduced in 12-month-old G2019S knock-in mice. Immunofluorescence revealed a dissociation between LC3B puncta accumulation and pSer129 a-synuclein inclusions in striatal neurons of kinase-dead and G2019S knock-in mice. Conclusions: We conclude that constitutive LRRK2 kinase silencing results in early deregulation of GCase activity followed by late impairment of macroautophagy and chaperone-mediated autophagy. In G2019S knock-in mice, pSer129 a-synuclein inclusions observed under basal conditions appear unrelated to autophagy impairment.

In insects, the tyramine receptor 1 (TAR1) has been shown to control several physiological functions, including olfaction. We investigated the molecular and functional profile of the Halyomorpha halys type 1 tyramine receptor gene (HhTAR1) and its role in olfactory functions of this pest. Molecular and pharmacological analyses confirmed that the HhTAR1 gene codes for a true TAR1. The RT-qPCR analysis revealed that HhTAR1 is expressed mostly in adult brain and antennae as well as in early development stages (eggs, 1st and 2nd instar nymphs). In particular, among the antennomeres that compose a typical H. halys antenna, HhTAR1 was more expressed in flagellomeres. Scanning electron microscopy (SEM) investigation revealed the type and distribution of sensilla on adult H. halys antennae: both flagellomeres appear rich in trichoid and grooved sensilla, known to be associated with olfactory functions. Through a RNAi approach, topically delivered HhTAR1 dsRNA induced a 50 % gene downregulation after 24 h in H. halys 2nd instar nymphs. An innovative behavioral assay revealed that HhTAR1 RNAi-silenced 2nd instar nymphs were less susceptible to the alarm pheromone component (E)-2 decenal as compared to control. These results provide critical information concerning the TAR1 role in olfaction regulation, especially alarm pheromone reception, in H. halys. Furthermore, considering the emerging role of TAR1 as target of biopesticides, this work paves the way for further investigation on innovative methods for controlling H. halys.

Monoterpenes are molecules with insecticide properties whose mechanism of action is, however, not completely elucidated. Furthermore, they seem to be able to modulate the monoaminergic system and several behavioural aspects in insects. In particular, tyramine (TA) and octopamine (OA) and their associated receptors orchestrate physiological processes such as feeding, locomotion and metabolism. Here, we show that monoterpenes not only act as biopesticides in

This study proposes a biochemical and molecular model for the interaction between the Drosophila suzukii type 1 tyramine receptor (DsTAR1) and monoterpenes. A preliminary molecular and functional characterization of DsTAR1 cDNA revealed that a 1.8 kb long ORF codes for a 600 amino acid polypeptide featuring seven transmembrane domains, as expected for a GPCR. A stable HEK 293 cell line expressing DsTAR1 was tested for responsiveness to tyramine (TA) and octopamine (OA). In intracellular calcium mobilization studies, TA led to a concentration-dependent increase in [Ca2+]i (pEC50 ~ 6.40), completely abolished by pre-incubation with the antagonist yohimbine 1 μM. Besides, in dynamic mass redistribution (DMR) studies, TA evoked a positive DMR signal in a concentration-dependent manner (pEC50 ~ 6.80). The recombinant cell line was then used to test three monoterpenes (thymol, carvacrol and α-terpineol) as putative ligands for DsTAR1. The terpenoids showed no agonist effects in both DMR and calcium mobilization assays, but they increased the potency of the endogenous ligand, TA, acting as positive allosteric modulators. Moreover, expression analysis on adults D. suzukii, exposed for 24, 72 or 120 h to a sublethal concentration of the three monoterpenes, showed a downregulation of DsTAR1. This evidence has led to hypothesize that the downregulation of DsTAR1 might be a compensatory mechanism in response to the positive allosteric modulation of the receptor induced by monoterpenes. Therefore, these findings might be useful for the development of a new generation of biopesticides against Drosophila suzukii, targeting TAR1.

Background: The peptide nociceptin/orphanin FQ (N/OFQ) and its receptor (NOP) are implicated in the modulation of emotional states. Previous human and rodent findings support NOP antagonists as antidepressants. However, the role played by the N/OFQ–NOP receptor system in resilience to stress is unclear. Aims: The present study investigated the effects of activation or blockade of NOP receptor signaling before exposure to acute stress. Methods: The behavioral effects of the administration before stress of the NOP agonists Ro 65-6570 (0.01–1 mg/kg) and MCOPPB (0.1–10 mg/kg), and the NOP antagonist SB-612111 (1–10 mg/kg) were assessed in mice exposed to inescapable electric footshock and forced swim as stressors. The behavioral phenotype of mice lacking the NOP receptor (NOP(-/-)) exposed to inescapable electric footshock was also investigated. Results: The activation of NOP receptor signaling with the agonists increased the percentage of mice developing helpless behavior and facilitated immobile posture. In contrast, the blockade of NOP receptor reduced the acquisition of depressive-like phenotypes, and similar resistance to develop helpless behaviors was observed in NOP(-/-) mice. Under the same stressful conditions, the antidepressant nortriptyline (20 mg/kg) did not change the acquisition of helpless behavior and immobile posture. Conclusions: These findings support the view that NOP activation during acute stress facilitates the development of depressive-related behaviors, whereas NOP blockade has a protective outcome. This study showed for first time that NOP antagonists are worthy of investigation as preemptive treatments in patients with severe risk factors for depression.