Monoterpenes alter TAR1-driven physiology in Drosophila species

Drosophila suzukii (Matsumura) (Diptera: Drosophilidae), commonly known as the spotted wing Drosophila, is one of the few Drosophilidae that can lay its eggs on healthy fruits before they become fully ripe (Walsh et al., 2011; Lee et al., 2011). Drosophila suzukii is able to infest most fruit and vine species worldwide, with a particular preference for small fruits (Rota-Stabelli et al., 2013). This species causes serious damage to the horticultural economy especially in South-East Asia and its presence has recently also been reported in North America and Europe (Asplen et al., 2015). Moreover, D. suzukii can spread rapidly (7–15 generations per year) and has a remarkable ability to adapt to different climatic conditions and host plants (Cini et al., 2012). Chemical pesticides are the main D. suzukii control agents, but they need frequent reapplication because of the numerous generations that occur during one crop season. However, repetitive treatments may increase resistance development and have a negative impact on beneficial insects (Desneux et al., 2007; Haviland and Beers, 2012). Alternative and more sustainable control strategies are constantly under investigation (Schetelig et al., 2018). Currently, research on the biology, genetics and physiology of D. suzukii has gained interest in order to develop new tools for more effective and environmentally sensitive pest management. Essential oils (EOs) as botanical pesticides are among the most promising pest control methods for future applications. In fact, studies performed in the last decade showed that pesticides based on plant EOs and their constituents (terpenes) are effective against a large number of insects (Bakkali et al., 2008; Isman, 2020). Members of the Drosophilidae family, D. suzukii included, are particularly sensitive to EO-based pesticides (Park et al., 2016; Kim et al., 2016; Zhang et al., 2016; Dam et al., 2019). Most EOs are complex mixtures of two predominant classes of molecules, terpenes and phenylpropanoids (Regnault-Roger et al., 2012). Although it is clear that EOs have toxic effects against pest insects, their mechanism of action is still unclear (Blenau et al., 2011; Jankowska et al., 2018). Typically, they are able to reduce or disrupt insect growth at several life stages (Konstantopoulou et al., 1992). It has been shown that terpenes can interact with P450 cytochromes, which are involved in insecticide detoxification processes (Jensen et al., 2006; Liao et al., 2016). Some monoterpenes, for example thymol, may induce neuronal degeneration through a direct interaction with GABA receptors (Priestley et al., 2003) or via acetylcholinesterase inhibition (Houghton et al., 2006; Park et al., 2016). Moreover, monoterpenes might interact with the octopamine/tyramine system, analogous to the adrenergic system present in the vertebrates (Enan, 2001; Kostyukovsky et al., 2002; Enan, 2005a,b; Price and Berry, 2006; Gross et al., 2017; Finetti et al., 2020).

In insects, the main biogenic amines are dopamine (DA), serotonin (5-HT), octopamine (OA) and tyramine (TA). Together, they control and modulate a broad range of biological functions essential for the insect's life (Roeder et al., 2003). The insect nervous system contains high levels of OA and TA, suggesting a role as neurotransmitters (Ohta and Ozoe, 2014), but also as neuromodulators and neurohormones in a wide variety of physiological processes (Pauls et al., 2018).

Originally, TA was considered only as an intermediate product necessary for the synthesis of OA. Nevertheless, today it is known that TA and OA perform important functions independently of each other (Roeder, 2005; Lange, 2009; Roeder, 2020). TA triggers its physiological effects by interacting with and activating the corresponding receptors, belonging to the G protein-coupled receptor (GPCR) family (Evans and Maqueira, 2005). Tyramine receptors (TARs) play important roles in modulating the biology, physiology and behaviour of invertebrates (Ohta and Ozoe, 2014). In fact, either the inhibition or the over-stimulation of TARs can lead to the death of the insect as well as interfere with physical fitness and reproductive capacity (Audsley and Down, 2015). These receptors are classified into two main groups based on their structure and activity: tyramine receptor type 1 (TA/OA or TAR1) and tyramine receptor types 2 and 3 on the other (TAR2 and TAR3) (Wu et al., 2014). TAR1 transcript localization provides clues to its physiological roles. In D. melanogaster, the receptor is highly expressed in the central nervous system (CNS; Saudou et al., 1990; El-Kholy et al., 2015). A similar expression pattern has also been observed in D. suzukii, Rhodnius prolixus, Chilo suppressalis, Plutella xylostella, Mamestra brassicae and Agrotis ipsilon, suggesting a crucial role for TA as neuromodulator and neurotransmitter (Wu et al., 2013; Hana and Lange, 2017; Ma et al., 2019; Brigaud et al., 2009; Duportets et al., 2010; Finetti et al., 2020). Several studies have reported the importance of TA, through its interaction with TARs, in a variety of processes including olfaction, reproduction, flight, locomotion and metabolic traits (Lange, 2009; Neckameyer and Leal, 2017; Roeder, 2020). In particular, TA appears to play a role in locomotor modulation (Saraswati et al., 2004; Hardie et al., 2007; Rillich et al., 2013; Schützler et al., 2019), egg-laying behaviour (Donini and Lange, 2004; Fuchs et al., 2014), sex pheromone production (Hirashima et al., 2007), metabolic traits including the regulation of energy expenditure (Brembs et al., 2007) and hormone release (Roeder, 2020). Despite the physiological importance of TA in invertebrates, little is known about TARs. In 2000, Kutsukake and co-workers characterized D. melanogasterhonoka, a mutant line with an impaired TAR1, exhibiting a different behaviour towards repellent odours. Furthermore, Li et al. (2017) have shown that TAR1-deficient flies exhibit significant changes in metabolic control such as higher body fat, lower starvation resistance and movement activity. Similar TAR1-mediated metabolic alterations were observed by Ishida and Ozaki (2011) in starved flies. Nevertheless, the existence of crosstalk between the tyraminergic system and other systems, such as the octopaminergic and dopaminergic systems, makes it difficult to precisely dissect the physiological processes controlled by TA (Li et al., 2016).

In the last few years, several studies have suggested that TAR1 might be an interesting target for insecticides, specifically for bioinsecticides. For example, monoterpenes appear to be able to interact with TAR1 directly. In particular, Enan (2005b) was the first to describe an agonistic effect of several monoterpenes (thymol, carvacrol, α-terpineol and eugenol) on D. melanogaster TAR1. However, the same monoterpenes did not show this pharmacological profile with D. suzukii and Rhipicephalus microplus TAR1. They acted instead as positive allosteric modulators, increasing the potency of TA activity (Gross et al., 2017; Finetti et al., 2020). Furthermore, a recent study from our lab has described a possible molecular mechanism underlying the toxicity of these molecules towards insects (Finetti et al., 2020). In particular, the observed downregulation of D. suzukii TAR1 (DsTAR1) after monoterpene exposure might represent a compensatory mechanism in response to enhanced receptor signalling due to the positive allosteric modulatory effect of monoterpenes on the receptor.

The current study presents a detailed investigation on D. suzukii behaviour upon monoterpene treatment, in order to understand whether DsTAR1 downregulation could affect fitness and physiology. Furthermore, a TAR1-defective D. melanogaster line was used as a control to compare the effects of chronic TAR1 impairment on D. melanogaster physiology with monoterpene treatment of D. suzukii flies.

Drosophila suzukii were kindly provided by the Entomological Laboratory of the Agricultural Sciences Department of the University of Padua (Italy) and maintained on an artificial diet with a 16 h:8 h photoperiod, at a temperature of 22±1°C. Drosophila melanogaster mutant lines were as follows: TAR1^PL00408 was generated by the Gene Disruption Project (Bloomington Stock Center, Bloomington, IN, USA, no. 19486; Bellen et al., 2004) and TAR1-Gal4 was previously created in the Molecular Physiology group from the University of Kiel (El-Kholy et al., 2015). The D. melanogaster TAR1^PL00408 defective line, which showed 50% downregulation of the target gene as confirmed by RT-qPCR (Fig. S1), had already been backcrossed several times with y¹w¹¹¹⁸, the control line for all corresponding experiments, as previously described (Li et al., 2016). All D. melanogaster flies were raised on standard food at 25±1°C (12 h:12 h light:dark photoperiod).

A glass cylinder (10 cm in height, 4.5 cm inner diameter; 150 ml volume) was employed to calculate the monoterpene median (LC₅₀) and maximum lethal concentration (LC₉₀) values for D. suzukii and D. melanogaster y¹w¹¹¹⁸ control and to perform monoterpene exposure. Monoterpenes including thymol, carvacrol and α-terpineol were dissolved in acetone and applied to a piece of filter paper (2 cm×2 cm). The filter paper was placed on the bottom lid of the cylinder, inside a small cage to prevent direct contact of the flies with the monoterpenes. The concentrations ranged between 0.067 and 67 µl l⁻¹ and acetone alone was used as a negative control. After CO₂ anaesthetization, 30 flies (15 males and 15 females) were placed inside the cylinder with 1 ml of solid diet. The top and the bottom of the cylinder were sealed with Parafilm and the assay was maintained at 22±1°C for D. suzukii or 25±1°C for D. melanogaster flies. After 24 h, the flies were collected. For LC₅₀ and LC₉₀ calculation, at least 100 flies were tested, in four replicates.

Total RNA was extracted from D. suzukii or D. melanogaster y¹w¹¹¹⁸ control line adult flies subjected to monoterpene exposure, using an Aurum Total RNA Mini Kit (Bio-Rad, Hercules, CA, USA). A 1 µg sample of RNA was treated with DNase I (Thermo Fisher, Waltham, MA, USA) and used for cDNA synthesis, using a OneScript^® cDNA Synthesis Kit (Abm, Vancouver, BC, Canada), according to the manufacturer's instructions. Quantitative PCR (qPCR) was performed using a CFX Connect Real-Time PCR Detection System (Bio-Rad) in a 12 µl reaction volume containing 1.6 µl cDNA (diluted 1:2), 6 µl SYBR PCR Master Mix (Vazyme, Jiangsu, China), 0.4 µl forward primer (10 µmol l⁻¹), 0.4 µl reverse primer (10 µmol l⁻¹) and 3.6 µl nuclease-free water. Thermal cycling conditions were: 95°C for 2 min, 40 cycles at 95°C for 15 s and 60°C for 20 s. After the cycling protocol, a melting-curve analysis from 55 to 95°C was applied. In D. suzukii, expression of TAR1 was normalized using arginine kinase (AK) and TATA box protein (TBP) genes, which served as reference genes (Zhai et al., 2014). In D. melanogaster y¹w¹¹¹⁸, expression of TAR1 was normalized using actin and tubulin genes that served as reference genes (Ponton et al., 2011). Gene-specific primers (Table 1) were used and four independent biological replicates, made in triplicate, were performed for each sample.

A TAR1-Gal4 Drosophila line was crossed with a UAS-GFP line in order to visualize the complete brain expression pattern of the receptor. The brains were dissected from F1 flies in cold Schneider's Drosophila medium and fixed in 4% (w/v) paraformaldehyde in PBS for 90 min at room temperature. The samples were then washed 3 times in PBST and blocked for 30 min in blocking buffer (1× PBS, 2% NP-40, 10% goat serum) at room temperature. The samples were incubated with primary antibodies in blocking buffer (anti-GFP rabbit 1:300, AB3080, Sigma-Aldrich, St Louis, MO, USA; and anti-Nc82 mouse 1:20, Developmental Studies Hybridoma Bank, University of Iowa) overnight at 4°C and washed 3 times for 5 min in PBST. Subsequently, the samples were incubated with secondary antibodies in blocking buffer (donkey anti-rabbit IgG Alexa Fluor-488 1:300, 711-545-152, Jackson ImmunoResearch, West Grove, PA, USA; and goat anti-mouse IgG Alexa Fluor 555 1:300, 115-165-003, Jackson ImmunoResearch) for 3 h at room temperature and washed twice for 5 min in PBST. Brains were mounted directly on slides and analysed by a Zeiss Axio Imager Z1 microscope equipped with an apotome (Zeiss, Oberkochen, Germany).

Total body triglyceride (TG) content was estimated using the TG colorimetric assay kit GPO-PAP method (Elabscience, Wuhan, China). Three flies were accurately weighed and homogenization medium (9 times the volume, 0.1 mol l⁻¹ phosphate buffered saline, pH 7.4) was added. The sample was mechanically homogenized on ice with a motorized pestle and centrifuged (at 500 g for 10 min); 7 µl of the supernatant was added to 700 µl of working solution, thoroughly mixed and incubated for 10 min at 37°C in the dark. Absorbance was read at 510 nm and distilled water, added to 700 µl of working solution, was used as a blank. TG content was estimated using a glycerol solution (2.26 mmol l⁻¹) as standard. Five independent biological replicates were performed for each sex and genotype.

Dye-labelling food intake quantification was performed as described by Deshpande and co-workers (2014), with minor modifications. In brief, five flies of each sex and genotype were placed into a vial with 2 ml of 1× dyed medium (2.5% yeast, 2.5% sucrose, 1% agar and 1% Brilliant Blue FCF; Sigma Aldrich). After 2 h of feeding, the flies were collected and frozen at −80°C. Frozen flies were transferred to 1.5 ml Eppendorf tubes, homogenized with a manual pestle in 50 µl of 1% PBST and centrifuged for 1 min at 12,000 g to clear the debris. The supernatant absorbance was measured at 630 nm on a label-free EnSight Multimode Plate Reader (Perkin Elmer, Waltham, MA, USA). The values obtained from flies fed with non-labelled food were used as a control and subtracted from experimental readings. To determine the dye concentration of each fly homogenate, a standard curve was generated with serial dilutions of an initial 10 µl aliquot of the non-solidified dye-labelled food added to 990 µl of 1% PBST. At least five independent biological replicates were performed for each sex and genotype.

Metabolic rate was assessed by respirometry as described previously (Yatsenko et al., 2014). In brief, for each sex and genotype, three adult flies were placed in each vial and metabolic rate was measured for 2 h using respirometry. The CO₂ yield during the test was calculated based on the volume (µl) of CO₂ produced per hour per fly. Data were obtained from five independent biological replicates.

The negative geotaxis assay was performed based on a published protocol (Gargano et al., 2005). In brief, five flies of each sex and genotype were placed into a 20 cm-tall glass tube without CO₂ anaesthesia. The tube was tapped twice to move flies to the bottom and the climbing height of flies was photographed after 2 s. The average distance climbed (in cm) for each fly was measured using ImageJ software. Five independent biological replicates per sex and genotype were performed.

The starvation resistance assay was performed by placing 25 flies of each sex and genotype into vials containing 1% of agar. The vials were maintained at 22±1°C for D. suzukii or 25±1°C for D. melanogaster. Dead flies were counted every 2 h until all flies were dead. For each genotype and sex, four independent biological replicates were performed (at least 100 flies).

LC₅₀ and LC₉₀ values were evaluated using POLO-plus software. All statistical analyses were performed using GraphPad Prism software (version 6). All data represent means±s.e.m., evaluated using one-way ANOVA followed by Dunnett's test for multiple comparisons.

The results of the LC₅₀ and LC₉₀ estimation as obtained by POLO-plus analysis for each monoterpene, performed on both D. suzukii and D. melanogaster y¹w¹¹¹⁸ flies, are summarized in Table 2. The table reports the LC₅₀ and LC₉₀ values, the 95% confidence limits (Robertson et al., 2017), the slopes (angular coefficients) of lines and the values of χ² for each monoterpene.

To evaluate the effect of exposure to monoterpenes on the expression levels of TAR1 gene in both D. suzukii and D. melanogaster y¹w¹¹¹⁸, flies were exposed to the respective LC₅₀ concentration of thymol, carvacrol and α-terpineol, and mRNA levels were analysed by qPCR. The exposure induced an interesting downregulation of TAR1 gene expression in both genotypes. In D. suzukii, significant differences were observed for thymol and carvacrol (Fig. 1A) but not for α-terpineol. In contrast, in D. melanogaster y¹w¹¹¹⁸, all three monoterpenes induced a significant downregulation of TAR1 although this was less marked than that for D. suzukii (Fig. 1B).

In order to determine the physiological functions controlled by TAR1, the receptor localization in D. melanogaster brain was investigated by immunohistochemistry. The Gal4-UAS system was used to follow TAR1 promotor activity with a GFP reporter, then recognized by the anti-GFP antibody.

The receptor showed specific expression in the pars intercerebralis as well as the lateral horn, suboesophageal ganglion, olfactory and optic lobes (Fig. 2A–C), suggesting that TAR1 might be implicated in important physiological traits in Drosophila.

To elucidate the role of TAR1 in metabolic traits as well as locomotor control and physiological aspects in Drosophila, the D. melanogaster TAR1^PL00408 strain was enrolled in several behavioural assays. Despite mutant D. melanogaster TAR1^PL00408 and control D. melanogaster y¹w¹¹¹⁸ flies showing no statistical difference in overall body mass (data not shown), the reduced expression of TAR1 translated into a higher propensity for TG accumulation in male flies (Fig. 3A) and a greater food intake in both sexes (Fig. 3B). Therefore, TAR1^PL00408 flies showed higher resistance to starvation than control flies (Fig. 3E,F). These changes were furthermore associated with a slower metabolism in TAR1-impaired insects (Fig. 3C). The increased TG accumulation and the slower metabolism could also be related to the lower propensity to move of the TAR1^PL00408 flies (Fig. 3D). To test whether monoterpenes, besides downregulating TAR1, might also alter the physiology of D. suzukii and D. melanogaster (wild-type or TAR1^PL00408 strain), 24 h after the continuous monoterpene LC₅₀ exposure, flies were challenged with several behavioural tests, as detailed below.

Exposure to monoterpenes for 24 h caused a higher TG content in males of both D. suzukii and D. melanogaster y¹w¹¹¹⁸ flies as compared with females (Fig. 4A–D). In particular, the TG content was significantly higher upon thymol and carvacrol exposure in D. suzukii males only (Fig. 4B), while both D. melanogaster y¹w¹¹¹⁸ females and males showed a significantly higher TG content after carvacrol exposure (Fig. 4C,D). When the same treatments were applied to D. melanogaster TAR1^PL00408 insects, no changes were observed in TG content, which was indistinguishable from that of the untreated control sample (Fig. 4E,F). This evidence would suggest that monoterpenes can induce an increase in total fat deposition that requires TAR1 receptors be functional.

Food consumption was quantified after 2 h of feeding on a dye-labelled diet. A significantly higher food intake was observed only after α-terpineol exposure in both D. suzukii and D. melanogaster y¹w¹¹¹⁸ of both sexes (Fig. 5A–D). The increased food intake might explain the high TG levels observed in D. suzukii and D. melanogaster y¹w¹¹¹⁸ of both sexes after monoterpene exposure. However, monoterpene treatment did not cause any change in food consumption in D. melanogaster TAR1^PL00408 mutant flies (Fig. 5E,F), further suggesting the requirement for an active TAR1.

In order to determine whether monoterpenes and TAR1 downregulation affect metabolism, metabolic rate was analysed in all D. suzukii and D. melanogaster genotypes after treatment with the different monoterpenes. In D. suzukii, males, but not females, treated with the three monoterpenes showed a significantly lower metabolic rate than control flies (Fig. 6A,B). Carvacrol and α-terpineol were able to reduce the metabolic rate in D. melanogaster y¹w¹¹¹⁸ males and females (Fig. 6C,D). Conversely, D. melanogaster TAR1^PL00408 metabolic rate appeared to be unaffected by the treatments and was therefore undistinguishable from that of the untreated controls (Fig. 6E,F).

The observed metabolic changes in terms of energy expenditure and TG content might also affect the physical activity of flies. Therefore, the ability of flies exposed to monoterpenes to walk upwards on a vertical surface in negative geotaxis was used as a motility behavioural assay. In comparison to untreated controls, D. suzukii and D. melanogaster y¹w¹¹¹⁸ males showed a statistically significant reduction in climbing ability after α-terpineol treatment only (Fig. 7B,D). Drosophila melanogaster y¹w¹¹¹⁸ female motility was negatively affected only by thymol (Fig. 7C), while D. suzukii females did not respond to the RING assay at all, in both control and treated samples (Fig. 7A). The climbing ability in both D. melanogaster TAR1^PL00408 sexes was unaffected by exposure to monoterpenes (Fig. 7E,F), confirming the hypothesis of TAR1 involvement in this behavioural trait.

Finally, a starvation resistance assay was performed to investigate whether the monoterpene-mediated metabolic modifications could affect general fitness. Given the higher food intake and TG content caused by monoterpene treatment, an enhanced resistance to starvation was expected. Drosophilasuzukii and D. melanogaster y¹w¹¹¹⁸ showed different results depending on the monoterpene used as compared with the control (Fig. 8A–D). According to log-rank statistical analysis, a significant reduction in starvation resistance was detected in D. suzukii, for both males and females, after carvacrol treatment (Fig. 8A,B), while both D. melanogaster y¹w¹¹¹⁸ sexes were less resistant to starvation after thymol exposure. Moreover, α-terpineol treatment reduced starvation resistance only in D. melanogaster y¹w¹¹¹⁸ females (Fig. 8C,D). Conversely, carvacrol exposure significantly increased starvation resistance in D. melanogaster y¹w¹¹¹⁸ males (Fig. 8C). Drosophilamelanogaster TAR1^PL00408 flies were again unaffected by the treatment, showing starvation resistance comparable to that of controls (Fig. 8E,F).

The biogenic amine TA is a mediator of several physiological functions in invertebrates (Roeder, 2005; Lange, 2009), but its mechanism of action is still far from being fully characterized. TA activates intracellular responses by interacting with specific GPCRs: the tyramine receptors, TARs (Saudou et al., 1990; Roeder et al., 2003). TAR1 is highly expressed in the CNS of numerous insects, thus suggesting its involvement in essential behavioural processes (El-Kholy et al., 2015; Hana and Lange, 2017; Finetti et al., 2020). Furthermore, several studies have suggested that TAR1 is a direct target for biomolecules with insecticidal action, such as monoterpenes. In fact, it has been reported that the D. melanogaster and R. microplus TAR1, when expressed in a heterologous cell system, respond to the administration of monoterpenes with an increased release of cytosolic calcium (Enan, 2005a; Gross et al., 2017). Recently, the same intracellular response has been observed in our laboratory for D. suzukii TAR1, allowing us to hypothesize that the interaction between monoterpene and receptor causes a downregulation of the gene coding for the receptor (Finetti et al., 2020). To further study the effects of the monoterpenes on TAR1 and on the insect physiology, a D. melanogaster TAR1-defective line (TAR1^PL00408) was evaluated together with corresponding controls and D. suzukii. Comparative studies using these two Drosophila species are possible as they are phylogenetically highly related and their TAR1 share a high degree of homology (98%) (Finetti et al., 2020).

Firstly, the identification of the LC₅₀ for the three monoterpenes thymol, carvacrol and α-terpineol, for both D. suzukii and D. melanogaster y¹w¹¹¹⁸ via a fumigant assay (Park et al., 2016), revealed that the most toxic monoterpene was carvacrol with an LC₅₀ of 0.844 µl l⁻¹ for D. suzukii and 0.592 µl l⁻¹ for D. melanogaster. Similarly, Zhang and co-workers (2016) observed that carvacrol was the most toxic monoterpene for D. melanogaster. Interestingly, when TAR1^PL00408 flies were treated with the monoterpenes at the LC₅₀ calculated for the y¹w¹¹¹⁸ strain, a 40% reduction in mortality was observed as compared with the control (data not shown), suggesting a strong correlation between TAR1 and the insecticidal activity of these monoterpenes. A similar observation was made in a D. melanogaster TAR1-deficient strain (specifically TyrR^Neo30), which appeared to be insensitive to thymol and carvacrol when topically applied (Enan, 2005a).

All three monoterpenes tested, thymol, carvacrol and α-terpineol, by 24 h of fumigant treatment, were able to induce TAR1 downregulation not only in D. suzukii (as already established, Finetti et al., 2020) but also in D. melanogaster. As TAR1 is mainly expressed in the CNS, the greatest impact of its downregulation might be expected in this region.

As shown by El-Kholy et al. (2015), in a study focused on D. melanogaster brain, TAR1 is expressed in the pars intercerebralis, mushroom bodies and ellipsoid body, as also confirmed by Li et al. (2016). Our study revealed that TAR1 is strongly expressed not only in the pars intercerebralis and the mushroom bodies but also in the lateral horn, suboesophageal ganglia and antennae mechanosensory centre. Even if the physiological significance of these specific TAR1 expression patterns in the Drosophila CNS is still unclear, they could be connected to the functions associated with the corresponding brain areas. The pars intercerebralis is an important insect neuroendocrine centre, composed of neurosecretory cells that regulate feeding (olfactory/gustatory perception of food sources; feedback information from the intestinal tract and body cavity regarding the urgency of feeding) and reproductive behaviours (de Velasco et al., 2007). TAR1^PL00408 flies showed a phenotypic profile that correlates with these observations. These flies are in fact characterized by increased body fat, higher food intake and starvation resistance as well as reduced locomotor activity and metabolic rate in comparison to y¹w¹¹¹⁸ controls (Li et al., 2016, 2017). These metabolic alterations were not sex dependent, although the effects in TAR1^PL00408 males appeared to be more pronounced as compared with those seen in females. This could be related to sex-dependent differences in TAR1 expression, the mRNA of which accumulated at higher levels in males than in females (Finetti et al., 2020). Despite all this, little is still known about the precise mechanism by which the tyraminergic system modulates essential metabolic traits such as fat body, food intake, starvation resistance, locomotor activity and metabolic rate.

In insects, fat is mainly stored in the fat body, which is also one of the most important metabolic centres (Arrese and Soulages, 2010). Lipid storage and release are mainly controlled by two hormones, the Drosophila insulin-like peptides (mainly dILP2) and adipokinetic hormone (AKH, analogous to mammalian glucagon) (Roeder, 2020). During an acute stress situation, the mobilization of lipids is essential for survival. This mechanism appears to be also controlled by both OA and TA, presumably through modulation of dILP secretion (Fields and Woodring, 1991; Orchard et al., 1993). In fact, it has recently been observed that in Caenorhabditiselegans, during acute stress, TA accumulates, which in turn modulates the insulin signal (De Rosa et al., 2019). Therefore, the increased TG level observed in TAR1^PL00408, as compared with y¹w¹¹¹⁸ control flies, might be related to a direct tyraminergic action on the release of dILPs. RNAi-mediated TAR1 silencing, targeted to the fat body, triggered a reduction of dILP2 in insulin-producing cells in the D. melanogaster pars intercerebralis and an increase in TG accumulation (Li et al., 2017). The increased TG levels in TAR1^PL00408 flies could also be linked to enhanced food intake as well as to lower movement propensity and metabolic rate. It has recently been proposed, in fact, that TAR1 could be involved in processes related to sugar sensibility and food intake regulation (Ishida and Ozaki, 2011). For example, honoka flies showed a reduced sugar response (Damrau et al., 2018) linked to differences in food intake. It is worth noting that TAR1 is highly expressed in neurons located in the suboesophageal ganglia that are presumably associated with the salivary glands and neck muscle control, and are thus linked with feeding.

After monoterpene treatment, both D. melanogaster y¹w¹¹¹⁸ and D. suzukii showed alterations in all behavioural assays performed. The link between monoterpene treatment and TAR1 downregulation is supported by the higher food intake observed in response to this treatment. When the D. melanogaster TAR1^PL00408 deficient line was considered, no phenotypic changes were observed whatsoever after exposure to monoterpenes, suggesting that the alterations observed in the other genotypes require the correct expression of a functioning receptor. This further confirms the relationship between monoterpene-induced behavioural changes and TAR1. TAR1-mediated physiological alterations due to monoterpenes were also observed in Phormiaregina. In fact, d-limonene treatment decreased TA levels in P. regina brain, causing a direct modification of food intake (Nishimura et al., 2005). This different response to food stimuli was subsequently attributed to a probable alteration of TAR1 expression at the level of the suboesophageal ganglion (Ishida and Ozaki, 2011). Furthermore, thymol and carvacrol appeared to play a crucial role modulating ant behaviour (locomotion and aggression), through aminergic regulation (Mannino et al., 2018).

In conclusion, this study shows that monoterpenes might be instrumental in the manipulation of the insect behaviour via TAR1. In fact, sublethal concentrations of thymol, carvacrol and α-terpineol downregulate TAR1 expression, ultimately affecting important metabolic traits such as starvation resistance and energy storage. Moreover, this work demonstrates that monoterpenes, in addition to their insecticidal properties, can modify the metabolism and fitness of surviving D. suzukii, opening the way to innovative applications of these molecules in pest control.

We would like to thank Dr Morena de Bastiani (University of Ferrara) for excellent technical assistance and Dr Federica Albanese (University of Ferrara, Italy) for linguistic improvement of the manuscript.