Capsaicin responses in Drosophila: Exploring the possibility of establishing a new Non-TRPV1 model

Abstract

Capsaicin, the primary pungent compound in chili peppers, activates the heat-sensitive ion channel transient receptor potential vanilloid 1 (TRPV1) in mammals, eliciting the characteristic burning sensation. On the other hand, Drosophila melanogaster is a powerful invertebrate genetic model for linking gene function to behavior and physiology that lacks an ortholog of TRPV1, providing a unique opportunity to uncover how capsaicin affects organisms that do not possess this canonical receptor. Although Drosophila exhibits measurable responses to capsaicin, it remains unclear whether these effects reflect direct sensory detection or indirect metabolic and stress-related processes, as mechanistic evidence remains inconclusive. Here, we synthesize capsaicin-related studies in Drosophila, with a particular emphasis on reconciling opposing findings. We propose emerging conceptual frameworks based on convergent evidence implicating chemosensory pathways, metabolic processes, and physiological responses, and highlight key directions for future research aimed at clarifying the multifaceted interaction of capsaicin in TRPV1-lacking systems.

Graphical abstract

Highlights

• •We address conflicting findings in capsaicin perception in fruit flies, examining inconsistencies in the literature regarding the presence or absence of capsaicin sensation.
• •The absence of a TRPV1 ortholog in fruit flies suggests that their response to capsaicin may involve alternative chemosensory mechanisms rather than direct TRPV1-like activation.
• •If capsaicin sensation exists in Drosophila, it may occur through stimulation of gustatory chemosensory systems or activation of paralogous TRP channels.
• •If capsaicin sensation is absent, Drosophila may still experience indirect effects from capsaicin exposure through TRPV1-independent mechanisms or capsaicin metabolism.

1. Introduction

Capsaicin, which makes chili peppers hot, activates mammals' transient receptor potential vanilloid 1 (TRPV1) channel [1]. TRPV1 is a nonselective cation channel that belongs to the transient receptor potential (TRP) superfamily [2]. TRPV1 activation in sensory neurons occurs in two phases. First, capsaicin binding triggers channel activation, permitting a rapid influx of cations, predominantly sodium and calcium, which induces membrane depolarization and neuronal excitation. This is then followed by a calcium-dependent inactivation mechanism that modulates channel activity, preventing sustained signaling and ensuring controlled neuronal responses [3,4]. In mammals, TRPV1-mediated activation by capsaicin displays the potential to improve metabolic health, offer neuroprotection, and serve as an analgesic for both acute and chronic pain [[5], [6], [7]].

Drosophila melanogaster is a valuable genetic model for linking genes and their expression to phenotypic outcomes, facilitating the translation of findings to higher organisms due to conserved functional genetic mechanisms [8]. The fruit fly lacks a capsaicin perception mechanism mediated by TRPV1 [9]. Earlier studies exploring neuroactive cues did not report behavioral changes in wild-type flies exposed to capsaicin through the mammalian nociception mechanism [10,11], and Drosophila researchers have taken advantage of this by introducing the TRPV1 channel into the Drosophila system to stimulate neurons and elucidate its functionality, referring to this tool as “chemogenetics” [12,13]. Nevertheless, research focusing on the impact of capsaicin in fruit flies suggests that this phytochemical induces gustatory attraction, repels egg laying, and modulates lifespan under certain concentrations. These studies propose different and sometimes contradictory mechanisms by which flies may sense capsaicin through their chemosensory system, including the activation of gustatory and nociception-associated pathways that ultimately drive sex-specific behavioral and physiological changes [[14], [15], [16]]. Therefore, the existing and limited literature does not yet provide a clear biological mechanism underlying capsaicin's interaction with Drosophila, as the underlying processes remain unresolved and published findings are inconsistent. We propose that this variability may arise from the absence of high-affinity, canonical mammalian TRPV1 activation in flies, together with a predominant focus on TRPV1-dependent mechanisms rather than on systematic exploration of TRPV1-independent pathways.

This mini-review aims to conceptualize the effects of capsaicin in Drosophila and to develop mechanistic frameworks for TRPV1-independent responses. Such mechanisms may be uniquely present in TRPV1-insensitive species or masked by capsaicin's strong affinity for TRPV1 in sensitive species. Ultimately, the goal of this mini-review is to guide future research in Drosophila by applying these frameworks to clarify the dose-dependent effects of capsaicin on taste, sensory processing, metabolism, lifespan, and overall physiology, as well as to understand how these processes shape ecological interactions involving capsaicin.

2. Capsaicin and its recognized mammalian sensation mechanism

The widely recognized mechanism of capsaicin sensation in mammals involves the activation of the TRPV1 channel in nociceptive neurons [1,17]. TRPV1 belongs to the TRPV clade (Fig. 1A), which is part of the TRP superfamily. Members of this superfamily were first identified and named in Drosophila [20,21]. Fruit flies possess 13 TRP channel members, whereas humans have 28. These members are divided into six clades based on sequence homology [2,22]. In contrast to the six TRPV channels in mammals, Drosophila has only two: the inactive (iav) and nanchung (nan) channels. These Drosophila channels are involved in responses to startle, heat, and sound [23,24], and are orthologs of TRPV5 and TRPV6, whose activation is regulated by phospholipid signaling and hormones [25]. In mammals, they share only 30% protein identity with the other TRPV subfamily members [26]. On the other hand, the mammalian TRPV1 is highly sensitive to activation by capsaicin, heat, and acidic pH, and upon activation, the channel becomes permeable to calcium and sodium ions [1]. Specifically, capsaicin interacts with residues Y511, S512, T550, and E570 within the calcium channel domain of Human TRPV1 (Fig. 1B), adopting a tail-up, head-down configuration [17]. Point mutations at these sites have been shown to result in a loss of perception due to the absence of ligand-gated TRPV1 activation [27,28]. In mammalian nociceptive neurons, capsaicin-induced TRPV1 activation triggers ionic influx and neuropeptide release, producing physiological and behavioral responses [4].

Fig. 1.

Drosophila lacks a TRPV1 ortholog among its TRPV members. (A) The evolutionary relationship between TRPV members in vertebrates (grey clade) and invertebrates (green clade). TRPV members in Drosophila are true orthologs of human TRPV5/6. Proteins of TRPV1-studied organisms were collected from the Alliance of Genome Resources (https://www.alliancegenome.org/) and Ensembl (https://useast.ensembl.org/index.html) databases. TRPV phylogeny was analyzed using the Maximum Likelihood method in MEGA11 [18] and visualized with the ape 5.0 package [19]. (B) Conservation of key capsaicin-interacting amino acids among model species in TRPV1 homologs. Drosophila iav is the most homologous to the mammalian TRPV1 channel, with 40% similarity. However, there is no conservation of key amino acids at positions Y511, S512, T550, and E570. The Clustal algorithm performed the multiple sequence alignment with default parameters in AlignmentViewer (https://alignmentviewer.org/). Key capsaicin-related residues are highlighted in red. The height of the residue letter above each position indicates the relative conservation frequency among evaluated organisms.

Conversely, other organisms that cannot digest chili pepper seeds, such as birds, are insensitive to capsaicin, enabling them to function as effective seed-dispersal vectors [29]. Experiments with the chicken TRPV1 ortholog revealed that it is not stimulated by capsaicin, as the key sites for TRPV1 activation by capsaicin are located in transmembrane domain 3 (TM3). Interestingly, mutating alanine at position 578 (corresponding to position 570 in mammals) to glutamic acid, lysine, glutamine, or proline is sufficient to render chicken TRPV1 capsaicin-sensitive, thereby conferring the ability to detect spiciness [30]. Although vertebrates, including fish (e.g., Danio rerio), amphibians (e.g., Xenopus spp.), and birds (e.g., Gallus gallus), possess TRPV1 orthologs, invertebrates such as Drosophila and Caenorhabditis elegans do not. Thus, capsaicin perception, which relies on the highly specific activation of TRPV1, likely evolved more recently and appears to be largely restricted to mammals [31].

3. TRPV1-dependent effects of capsaicin

Capsaicin-mediated activation of TRPV1 has been employed to relieve pain, promote fat oxidation, and confer neuroprotection in mammals, with effects varying depending on the specific cell types that interact with capsaicin [5,7].

The analgesic effect of capsaicin is mediated by desensitization of the TRPV1 channel, in which prolonged or continuous exposure inhibits further activation. A practical example is the FDA-approved use of topical capsaicin creams and the 8% capsaicin patch, both of which reduce sensitivity and induce persistent desensitization [32]. They are used for neuropathic pain conditions such as postherpetic neuralgia [33]. At the molecular level, this process involves calmodulin (CaM), a calcium-binding protein. When intracellular calcium levels rise, CaM binds to TRPV1 in a manner that competes with ATP for its binding pocket. Because ATP is normally required to activate TRPV1, this competition prevents ATP from sustaining channel activation, ultimately leading to TRPV1 deactivation or desensitization [34,35].

The proposed mechanism of visceral fat reduction through lipolysis and fatty-acid oxidation induction is the increment of calcium signaling in adipocyte cells due to TRPV1 ionic influx during capsaicin consumption [36,37]. Capsaicin also inhibits fatty acid and triglyceride synthesis via a TRPV1-dependent pathway that involves NR1D1, a transcription factor regulating circadian rhythm and metabolism. This interaction helps prevent excessive lipid accumulation, suggesting a link between TRPV1 activation, metabolic regulation, and sleep-related processes [[38], [39], [40], [41]].

Capsaicin has been proposed as a neuroprotective agent in Alzheimer's and Parkinson's diseases, although the pathophysiology of these conditions is not fully understood [42,43]. Specifically, capsaicin is believed to enhance the clearance of β-amyloid plaques by promoting microglial phagocytosis and to reduce tau hyperphosphorylation through the activation of TRPV1-expressing neurons, together suggesting a neuroprotective role in neurodegenerative conditions.

4. TRPV1-independent effects of capsaicin

Capsaicin is likely to interact with alternative membrane structures and neuronal excitation targets because of its chemical properties [44]. For example, capsaicin has been shown to impair mitochondrial function by inhibiting electron transport chain complexes I and III, resulting in reduced ATP production, increased generation of reactive oxygen species (ROS), and subsequent oxidative stress [45]. It also affects mitochondrial membrane proteins known as prohibitins, causing their relocalization within the cell and triggering apoptosis through downstream interaction with the key regulator p53 [46]. Moreover, capsaicin has been suggested to affect voltage-gated Na⁺, K⁺, and Ca²⁺ channels through direct binding and alterations in lipid membranes [47,48]. For instance, capsaicin may induce nociception by inhibiting voltage-gated potassium channels via protein kinase-dependent pathways, leading to increased neuronal excitability [49].

The metabolic processing of capsaicin does not involve TRPV1. Instead, it undergoes biotransformations, such as hydroxylation and dehydrogenation, catalyzed by cytochrome P450s (CYP450s), glycosyltransferases, and decarboxylases. However, its complete metabolic pathway and tissue specificity remain unclear. These metabolic modifications are thought to be similar to those observed for other phytochemicals, including piperine and curcumin, which also undergo enzymatic processing that can affect their bioavailability and biological activity [[50], [51], [52], [53]]. In line with these biotransformations, glutathione and glycine conjugates have also been proposed to contribute to the excretion of capsaicin-derived metabolites [52]. A proposed capsaicin-associated, TRPV1-independent mechanism of CYP450 activation in mammalian cell lines is mediated through the human Pregnane X Receptor (hPXR) [54] and the Aryl hydrocarbon Receptor (AhR) [55]. Specifically, capsaicin has been shown to induce the transcription of CYP3A4 and CYP1A1 through PXR- and AhR-dependent mechanisms. Although these effects have not been demonstrated in other model organisms or explored in depth, evidence suggests that the Drosophila nuclear receptor ortholog HR96 is involved in the degradation of xenobiotics and pesticides [56,57].

Lastly, behavioral evidence suggests that capsaicin influences taste-related dietary decisions. TRPV1 knockout mice show increased preference for capsaicin-laced diets [58], and capsaicin enhances sweet consumption through the sweet transduction pathway [[59], [60], [61], [62]]. Additionally, mammals such as mice can shift their dietary preference toward spicy food after chronic exposure and the subsequent desensitization of TRPV1-mediated pungency [63]. These observations imply that preference for spicy food may reflect a masked mechanism of capsaicin taste that is evident once TRPV1 is inactivated through habituated exposure. It is also possible that this behavior occurs in populations carrying less-sensitive TRPV1 alleles.

5. Capsaicin and insects

Capsaicin has been suggested as an insect deterrent pesticide [64]. Caterpillars of Spodoptera latifascia (Lepidoptera: Noctuidae) and Euplectrus platyhypenae (Hymenoptera: Eulophidae) fed a diet with high capsaicin supplementation exhibited reduced survival to adulthood and decreased larval weight [65]. However, evidence suggests that some insects can metabolize relatively high capsaicin doses. For example, Helicoverpa assulta, a specialist herbivore of capsaicin-rich plants, shows greater tolerance to capsaicin than other non-spicy-feeding specialists (e.g., H. subflexa, S. frugiperda, H. virescens, H. armigera, H. zea), as demonstrated by enhanced larval survival and growth under chronic capsaicin exposure [66]. A possible mechanistic explanation is their enhanced capacity for capsaicin detoxification via CYP450-mediated pathways, specifically through hydroxylation and dehydrogenation [[67], [68], [69]]. In particular, members of the CYP6B and CYP9A subfamilies (e.g., CYP6B6, CYP9A12, CYP9A14, CYP9A17) have been shown to biotransform capsaicin through hydroxylation and dehydrogenation, converting it into more readily excretable metabolites and demonstrating a CYP-selective transformation in which the specific CYP isoform determines the site of modification on the capsaicin molecule.

An indication of a capsaicin-perception mechanism in some insects comes from thermoregulatory behavioral changes observed in the American cockroach (Periplaneta americana), which prefers lower temperatures after capsaicin treatment [70,71]. Capsaicin also interacts with slow-inactivated insect voltage-gated sodium channels (VGSCs), inhibiting sodium currents and blocking neuronal excitation by altering steady-state activation at an optimal concentration of 0.130 mM in this species. Members of the lophotrochozoan clade, such as the medicinal leech, have likewise been reported to sense capsaicin through a putative TRPV1 homolog channel [72]. Although these organisms provide insights into the evolutionary development of capsaicin perception in mammals, they appear to have a lower affinity for capsaicin, responding only to higher doses than mammalian receptors and exciting their polymodal nociceptive neurons. This contrasts with the widely accepted view that invertebrates such as Drosophila and C. elegans do not exhibit nociceptive responses to capsaicin exposure [11,73,74], and that only the introduction of mammalian TRPV1 into nociceptive neurons, such as Painless-expressing or multidendritic neurons, can confer capsaicin sensation and aversion [9,75]. Still, the literature on capsaicin and insects remains limited, with relatively few studies investigating its effects across diverse insect taxa and providing mechanistic explanations of capsaicin perception and metabolism. Consequently, our understanding of how insects detect, tolerate, or metabolize capsaicin at the molecular level is still at an early stage, highlighting the need for more targeted and comprehensive investigations. We propose that, due to its genetic tractability and well-characterized nervous system, Drosophila represents an ideal model for studying the molecular and cellular mechanisms underlying TRPV1-independent responses to capsaicin in insects.

6. Capsaicin effects in Drosophila

In fruit flies, dietary supplementation with chili pepper has been found to affect physiology, resulting in reduced body weight, glucose levels, and triglyceride levels. Additionally, it influences lifespan in a sex-specific and dose-dependent manner, with lower doses proving beneficial for lifespan extension [14,76,77]. The molecular mechanisms underlying these effects involve modulation of the glycerolipid pathway, implicated in fat metabolism, and insect hormone biosynthesis, which affects metamorphosis and developmental processes; however, the hypothesized improvements in antioxidant responses were not observed. Nevertheless, chili peppers are known to contain a variety of phytochemicals, including carotenoids, which are recognized for their ability to influence oxidative processes [78]. These effects are distinct from those attributed to capsaicin itself or other capsaicinoids. For example, changes in the abundance of bacterial families Lactobacillaceae and Acetobacteraceae in the gut microbiome of flies reared on pepper-containing diets show a strong correlation with phenolic and carotenoid content [79].

Studies using pure capsaicin in fruit flies revealed a lack of a nociceptive response. For example, efforts to understand the cellular substrates of taste neurons and their behavioral impact with several compounds revealed that wild-type flies do not exhibit any differential behavioral preference when subjected to capsaicin [11]. A subsequent study found that a sugary diet supplemented with capsaicin resulted in increased preference, proposing that the mechanisms underlying capsaicin attraction might involve chemosensory receptors, including the olfactory and gustatory systems, rather than TRP channels in Drosophila [10]. Although this hypothesis has not been experimentally demonstrated, it is not inconsistent with the fact that flies are insensitive to this compound, which is normally considered pungent [68,[80], [81], [82], [83]]. Conversely, Li et al. [15] showed that capsaicin deterred egg laying, indicating an aversion. The authors proposed that this ovipositional behavior likely arises from the recognition of capsaicin via Painless, a TRP channel associated with nociception and known to be activated by temperature and isothiocyanates in fruit flies. However, this interpretation appears flawed, as previous work [10] demonstrated the opposite: knockout lines of Painless neither abolished capsaicin attraction nor produced repulsion. Moreover, that study did not include experiments to directly investigate or characterize this mechanism. Similarly, the aforementioned study also reported a decrease in fly fitness, but this contrasts with the finding from Shen et al. [16] that female flies exposed to a low concentration of capsaicin (1 × 10⁻⁷ mM) exhibited an increased lifespan. To explain this effect, the authors proposed that capsaicin may activate iav, the most homologous, though not orthologous, TRPV channel in Drosophila, within a specific set of neurons that subsequently release Diuretic hormone 31 (Dh31), a regulator of circadian activity and locomotor rhythms, thereby extending lifespan. However, this proposed mechanism remains unverified, as no additional experiments were performed to test it. Moreover, previous studies have reported the opposite effect, showing that higher concentrations of capsaicin (1.3 × 10⁻³ to 1.15 × 10⁻² mM) actually shorten the lifespan of flies [14].

Two key factors that may contribute to these contrasting results are the capsaicin concentration and the fly genotype background used in the experiments (Table 1). First, studies reporting neutral or attractive behavior and lifespan extension used capsaicin concentrations comparable to those that activate TRPV1 in mammals and occur naturally in chili peppers. Binary food choice and food intake assays showed that capsaicin is not aversive to flies and has no significant effects on stress responses, body composition, or fecundity. However, the female lifespan was significantly increased. Fernández-Bedmar and Alonso-Moraga [14] reported a decrease in lifespan, but they used non–wild-type genetic backgrounds, specifically mwh/flr3 mutants; therefore, we cannot conclude that this effect is common in wild-type flies. On the other hand, Li et al. [15] evaluated positioning, feeding aversion, sensory modulation, oxidative stress, and the expression of nociception-related genes, such as painless and TRPA1, in flies exposed to capsaicin concentrations ranging from 10 mM to 80 mM. These concentrations greatly exceed the standard 0.1 mM used to activate the heterologously expressed mammalian TRPV1 in Drosophila as a chemogenetic tool for neuronal characterization [13,82], as well as the levels naturally found in chili peppers, revealing significant deterrent and injury effects of capsaicin.

Table 1.

Studies on the behavioral and physiological effects of capsaicin in adult Drosophila.

Genotype	Capsaicin Concentration (mM)	Experiment	Phenotypic effect	Proposed mechanism	Reference
w1118	1 × 10−3, 1 × 10−2, and 0.1	Taste preference	Increased positive preference was observed at all tested concentrations, but it was not statistically significant	Not discussed	[11]
	1 × 10−8, 1 × 10−7, 1 × 10−6, 1 × 10−5, 0.1, and 1, 10	Lifespan, feeding, stress resistance, activity, body composition, and climbing	Lifespan extension was observed only at 1 × 10−7 mM, and daytime activity was decreased, while no differences were observed in other parameters	iav-dependent modulation of Dh31+ neurons	[16]
Canton-S pain1 pain2 pain-GAL4	3 × 10−3, 6 × 10−3, and 1.2 × 10−2	Food preference	A positive preference for capsaicin was observed in all strains across all tested concentrations	Activation of gustatory nociceptive pathways (painless-dependent)	[10]
Canton-S Oregon-R Painless	10, 20, 40, and 80	Oviposition, proboscis extension reflex (PER), survival, and intestinal integrity	Ovipositional aversion starting at 10 mM in wild types. Positive increase in oviposition and PER in Painless flies at 20 mM. Lifespan reduction and compromised intestinal integrity at 80 mM in wild types	Activation of Painless + nociceptive neurons	[15]
mwh/mwh, flr3/TM3, BdS	1.3 × 10−3 and 1.15 × 10−2	Oxidative stress and lifespan	The highest inhibition of oxidative damage occurred at 1.15 × 10−2 mM, but supplementation decreased lifespan	Antioxidant activity	[14]

Therefore, the varied capsaicin concentrations and methodologies used to assess its effects in Drosophila have been inconsistent and limited. Indeed, both the detrimental and beneficial effects of dietary compounds like capsaicin, such as lethal dosage versus lifespan or healthspan extension, are likely determined by effective dosage [84]. For example, a neuroprotective effect can be biphasic, with high doses being harmful while low doses are ineffective. Although lifespan has been evaluated in flies, no study demonstrating an impact on neuroprotection or lifespan in Drosophila has yet provided an experimentally validated mechanistic explanation. Thus, modes of action may vary depending on the dosage tested. One possibility is that capsaicin modulates behavior and physiology in Drosophila through the activation of gustatory sensory neurons, similar to other tastants like l-alanine [85], thereby influencing activity, sleep, and feeding, and triggering downstream neural signaling that reshapes circuits controlling homeostatic processes.

In a recent review [86], the authors discussed signaling and molecular pathway targets implicated in capsaicin responses that occur independently of TRPV1, which we could infer are present in flies. Among the mechanisms mentioned, only Pyruvate Kinase Isoenzyme Type M2, Lactate Dehydrogenase A, Secretory Phospholipase A2, Carbonic Anhydrases IX and XII, and HSP90 and HSP70 have annotated fly ortholog proteins, namely Pyk, Ldh, PLA2, CAH7 and CAH9, and Hsp83, respectively (consulted using the curated database at https://www.alliancegenome.org/). However, we cannot assume that these mechanisms are directly translatable to the fly model system, since the supporting evidence originates from in vitro, computational, and mammalian cell culture studies and lacks validation in whole organisms with full bioavailability and tissue-specific dynamics. Nonetheless, these pathways provide valuable starting points for identifying conserved molecular players that could mediate capsaicin responses in model organisms like Drosophila, guiding future in vivo experiments and helping bridge mechanistic gaps across species. These mechanisms can also differ between taxa, especially between invertebrates and vertebrates, potentially giving rise to alternative or less specialized sensory systems, such as those involved in the chemosensory detection of capsaicin. For example, functional divergence of TRPV channels in invertebrates may exist between ecdysozoans and lophotrochozoans, distinguishing organisms like Drosophila and C. elegans from other suggested capsaicin-responsive non-mammalian species, such as leeches and mollusks [72].

7. Drosophila as a model for capsaicin research: diverging perspectives

There is still no clear mechanistic and conclusive evidence to explain the divergent phenotypic outcomes observed in capsaicin–Drosophila interactions, likely due to inconsistencies across studies employing different experimental setups, focuses, concentrations, and genotypes. Many studies lack the methodological rigor to investigate the underlying mechanisms of their observations and instead rely on pathways characterized in other organisms to interpret their findings. Therefore, we propose two conceptual frameworks to address these differing results and guide future research to better understand how fruit flies respond to different capsaicin concentrations, allowing the study of TRPV1-independent mechanisms linked to this compound.

7.1. Perspective 1: the hypothesis that Drosophila does not possess a capsaicin-sensing mechanism

As our first proposed hypothetical framework, the effects of capsaicin may be mediated by metabolic byproducts, such as an increase in ROS, that accumulate as a consequence of substantial metabolic transformation under high-dose conditions, rather than through binding to a specific receptor. Similar effects have been observed for other non-polar phytochemicals [87]. Detoxification enzymes such as CYP450s, glycosyltransferases, and decarboxylases have been implicated in the capsaicin detoxification pathway in insects, and their activity correlates with increased oxidative stress [69,88]. In Drosophila, homologs of the Helicoverpa CYP6B6 and CYP9A families are present, with the closest matches identified as CYP6a2 and CYP9f2 according to BLAST searches of the NCBI RefSeq protein database; these genes are annotated as participating in the metabolism of insecticides and environmental food substrates [89].

This metabolism may lead to indirect effects such as gut damage and the activation of nociception-related genes reported in the study of Li et al. [15]. Consistent with this proposal, previous work has shown that insects exhibit increased tolerance to capsaicin due to enhanced metabolic machinery [50]. Oxidative stress may give rise to phenotypes such as accelerated aging and tissue damage, which can phenocopy the molecular activation of nociception-related genes in response to capsaicin. These effects are driven by ROS acting on TRP channels [90], including TRPA1 and Painless, both of which can exhibit increased ion gating in nociceptive neurons under oxidative conditions [91,92]. This aligns with findings that the Helicoverpa gut undergoes the most substantial changes during capsaicin consumption due to its high metabolic activity [50,69], and that capsaicin can indirectly activate TRPA1 through a metabolic pathway. In this pathway, capsaicin activates phospholipase A2, leading to the release of arachidonic acid, which is subsequently converted by cyclooxygenase-2 (COX2) into prostaglandin E2 and then prostaglandin A2, a known agonist of TRPA1 [93]. Although the exact triggering step remains under investigation [94], available evidence suggests that COX2 may act as a potential capsaicin target mediating this pathway, resulting in diverse downstream phenotypes, including reduced inflammatory responses [95].

From these observations, we can suggest that capsaicin interacts primarily with xenobiotic detoxification pathways in fruit flies, exerting indirect physiological effects through elevated ROS levels (Fig. 2).

Fig. 2.

Perspective 1: The hypothesis that Drosophila does not possess a capsaicin-sensing mechanism. Capsaicin's effects in the fruit fly may be driven primarily by its metabolism. Drosophila may tolerate capsaicin concentrations comparable to those found in natural chili peppers without sensing capsaicin, thereby exhibiting no behavioral or physiological changes. However, higher concentrations, such as those used experimentally, reveal the fly's metabolic capacity through enzymes such as CYP450s, glycosyltransferases, and decarboxylases. These enzymes metabolize capsaicin and generate reactive species that indirectly affect lifespan, behavior, physiology, and molecular processes, ultimately leading to protein disruption and tissue damage. This damage is proposed to underlie the reported activation and expression of nociceptive signatures, such as TRP channels.

7.2. Perspective 2: the hypothesis that Drosophila does possess a capsaicin-sensing mechanism

Our second proposed hypothetical framework, based on the existing literature, suggests that a mechanism of capsaicin sensation may exist in Drosophila; however, two non-complementary interpretations emerge. First, flies appear to behave differently from mammals, recognizing capsaicin through their chemosensory system, including gustatory and olfactory pathways, as a chemical cue that promotes food acceptance (Fig. 3). Alternatively, low-affinity interactions with TRP channels may activate nociceptive signaling, eliciting aversive responses only at high concentrations.

Fig. 3.

Perspective 2: The hypothesis that Drosophila does possess a capsaicin-sensing mechanism. At capsaicin concentrations naturally present in chili peppers, Drosophila may show reduced activity due to Dh31 release triggered by gut neuronal excitation through the activation of the capsaicin-responsive TRPV1 homolog iav. This modulation of activity may conserve energy and ultimately increase lifespan. Behaviorally, flies may be attracted to dietary capsaicin through activation of sweet-sensing gustatory neurons. Conversely, at high capsaicin concentrations, low-affinity TRPV1-homolog channels such as painless, iav, and/or TRPA1 may be activated in nociceptive sensory neurons located in the forelegs, driving aversion and altering oviposition behavior as flies perceive the compound as noxious.

In fruit fly research, the widely accepted assumption is that capsaicin is a neutral compound and can therefore be used to excite neurons that are ‘humanized’ with TRPV1 when expressed in specific cell types using GAL4 patterning [9]. Nonetheless, capsaicin has been shown to influence dietary decisions in flies, which demonstrate increased attraction and preference for capsaicin-containing diets. This suggests that capsaicin may be detected through interactions with acceptance neurons expressing gustatory, olfactory, or ionotropic receptors [10]. In this context, capsaicin may function similarly to a pheromone, modulating preference behaviors through taste or smell independently of nutrient sensing, given that (1) capsaicin is a secondary, non-energetic metabolite, and (2) the absence of a principal macronutrient seems essential for observing this trait.

On the other hand, under high-capsaicin conditions, iav, Painless, and TRPA1, channels homologous but not orthologous to TRPV1, have been suggested to be involved with weak affinity in capsaicin recognition at the high concentrations used by Li et al. [15]. Because behavioral output is defined by the activated cell type rather than the receptor itself, it is also plausible that capsaicin-induced neuronal activation produces opposing behaviors due to second-order circuit effects, even when the same first-order neurons are stimulated. Examples already exist in which taste neurons drive opposite food preference and egg-laying behaviors, such as food avoidance accompanied by egg-laying attraction [13]. It has also been proposed that high homolog channels such as iav and Painless might be expressed in food acceptance neurons and could therefore drive capsaicin preference in flies [96]. However, this hypothesis is not supported by the recent single-cell RNA sequencing atlas [97], which reveals no co-expression of these genes within the same cell type (https://scope.aertslab.org/#/FlyCellAtlas). An explanatory bridge to understanding the influence of capsaicin on neuronal activity lies in its ability to inhibit VGSCs, a phenomenon that has been observed in the non-TRPV1 American cockroach (Periplaneta americana) [98] as well as in TRPV1 knockout mice [99]. In these models, reduced sodium conductance blocks neuronal firing and, consequently, overall activity. Nevertheless, a detailed characterization of the neuronal populations influenced by capsaicin, along with their connectomic organization, remains necessary.

Given the ambiguity in the literature, three hypothetical and non-overlapping scenarios may explain capsaicin sensation in flies, each requiring experimental validation. First, capsaicin may be detected by sweet-sensing neurons via the canonical sweet-transduction pathway, potentially driving opposing behavioral outcomes. Second, flies may exhibit an adaptive tolerance to capsaicin, perceiving it only at high concentrations, possibly through low-affinity interactions with TRP channel members. Together, these possibilities support a third scenario: capsaicin may be sensed by sugar-responsive neurons at biologically relevant (“natural”) concentrations, while less-sensitive TRP homologs may contribute only at substantially higher levels.

8. Conclusions and further research directions

The main goal of this mini-review was to provide explanations for contrasting observations in the literature, helping readers compare TRPV1-dependent pathways, distinguish opposing phenotypes, and clarify controversial points that make current fly studies difficult to interpret. Despite these complexities and the lack of comprehensive evidence in the literature, we propose two conceptual frameworks based on compatible findings: one in which capsaicin sensation may be absent in Drosophila, and another in which it may be present. Presenting these two perspectives underscores that the study of capsaicin interactions in flies remains an open field, requiring solid experimental evidence rather than assumptions or mechanistic interpretations derived from TRPV1 models. Although flies do not possess a canonical TRPV1 receptor, they nevertheless seem to exhibit physiological and behavioral responses under specific experimental and dietary conditions, suggesting the involvement of TRPV1-independent pathways, metabolic interactions, and context-dependent chemosensory processes. To fully address the effects of capsaicin on behavior and physiology in Drosophila, both short- and long-term investigations across a range of capsaicin dosages are necessary. Such a comprehensive approach will help resolve the conflicting evidence presented in past studies, which spans a broad and often incomparable set of concentrations. In addition, understanding how specific neurons mediate chemosensory responses could uncover conserved pathways that regulate feeding behavior and nutrient detection across animals, including humans who show tolerance or insensitivity to spiciness. A deeper understanding of the relationship between capsaicin and fruit flies also requires evaluating both naturally occurring concentrations and those relevant to therapeutic or experimental applications. This approach will allow researchers to investigate how capsaicin functions in the diet and as a supplementary compound, while accounting for variations across species. If capsaicin is sensed only at high, non-natural concentrations in Drosophila, a thorough mechanistic characterization is needed to distinguish direct sensory effects from secondary outcomes driven by detoxification or metabolism. Natural variation in chemosensory systems, including gustatory and olfactory pathways, has been documented within Drosophila [[100], [101], [102]], across the Drosophilidae family [103,104], and in other higher organisms [105]. Accordingly, a promising future direction involves leveraging the Drosophila Genetic Reference Panel (DGRP) to identify genes underlying evolutionary differences in responses to capsaicin exposure, such as short-term dietary preference and metabolic capacity. The outcomes of this research may provide insights applicable to other closely related arthropods and to higher organisms that lack capsaicin-mediated TRPV1 activation but still encounter capsaicin in their ecological niches [106]. Similarly, this knowledge has the potential to inform the development of novel strategies for modulating dietary choices, designing targeted pest control approaches, or identifying molecular targets for drug discovery in systems where TRPV1-independent capsaicin signaling influences sensory perception, metabolic regulation, and overall physiology.

Thus, Drosophila offers a unique platform to investigate TRPV1-independent mechanisms that influence animal behavior. Moreover, it serves as a valuable model for exploring capsaicin-mediated, TRPV1-dependent mechanisms in humanized flies and human disease models, allowing direct comparison with conventional flies, in which such responses may be absent due to the high-affinity interaction of capsaicin with the TRPV1 channel. Our research group is currently working to fully understand the effects of capsaicin on behavior and physiology in Drosophila. Using a combination of behavioral, physiological, imaging, and genomic approaches, we aim to identify the key molecular players that interact with capsaicin and resolve discrepancies reported in prior studies.

Ethical statement

This literature review is based solely on published data and does not involve studies with human participants or animals; thus, ethical approval and informed consent were not required.

Declaration of generative AI and AI-assisted technologies in the writing process

During the preparation of this work, the authors used AI chatbots to proofread the manuscript for grammar and typos. The authors reviewed and edited the content as needed and take full responsibility for the final publication.

Declaration of funding

This research was supported by the United States Department of Agriculture - National Institute of Food and Agriculture (USDA-NIFA) under grant numbers 2024-38821-42101 and 2022-38821-37343 and National Science Foundation (NSF) under grant numbers 2242771 and 1920920.

CRediT authorship contribution statement

Gerardo Flores-Iga: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Software, Visualization, Writing – original draft. Mohankumar Amirthalingam: Conceptualization, Investigation, Visualization, Writing – review & editing. Carlos Lopez-Ortiz: Conceptualization, Visualization, Writing – review & editing. Padma Nimmakayala: Conceptualization, Funding acquisition, Project administration, Supervision, Visualization, Writing – review & editing. Umesh K. Reddy: Conceptualization, Funding acquisition, Project administration, Supervision, Visualization, Writing – review & editing.

Declaration of competing interest

The authors declare they have no conflicts of interest.

Data availability

No new data were generated for this article. Publicly available databases were used. The FASTA files and R script used to construct and visualize the TRPV phylogeny have been uploaded to GitHub (https://github.com/gerapskx/TRPV-phylogeny).