Capsaicin Functions as Drosophila Ovipositional Repellent and Causes Intestinal Dysplasia

Plants generate a plethora of secondary compounds (toxins) that potently influence the breadth of the breeding niches of animals, including Drosophila. Capsaicin is an alkaloid irritant from hot chili peppers, and can act as a deterrent to affect animal behaviors, such as egg laying choice. However, the mechanism underlying this ovipositional avoidance remains unknown. Here, we report that Drosophila females exhibit a robust ovipositional aversion to capsaicin. First, we found that females were robustly repelled from laying eggs on capsaicin-containing sites. Second, genetic manipulations show that the ovipositional aversion to capsaicin is mediated by activation of nociceptive neurons expressing the painless gene. Finally, we found that capsaicin compromised the health and lifespan of flies through intestinal dysplasia and oxidative innate immunity. Overall, our study suggests that egg-laying sensation converts capsaicin into an aversive behavior for female Drosophila, mirroring an adaptation to facilitate the survival and fitness of both parents and offspring.

cells, and they generate two differentiated intestinal cell types: enteroendocrine cells and enterocytes 18 . This regeneration makes the Drosophila intestine a promising model for deciphering stress-related alterations in innate immune signaling and regenerative capacity. Studies have found that dual oxidase (Duox)-mediated production of reactive oxygen species (ROS) is a primary immune mechanism underlying intestinal homeostasis 19,20 . However, the mechanism through which immune activation leads to capsaicin-related pathologies and lifespan impairment is not fully understood.
In the current study, we expected that capsaicin would induce an ovipositional avoidance of Drosophila melanogaster. To pursue this hypothesis, we used this model organism to explore the roles of capsaicin in oviposition decisions and examined how capsaicin affects the lifespan of Drosophila, providing insight into an adaptation to facilitate the survival and propagation of flies.

Results
Egg-laying avoidance to capsaicin. To investigate how gravid females respond to capsaicin, we evaluated their egg-laying preferences by using a two-choice apparatus 10,21 (Fig. 1a). Interestingly, we found that Oregon R (OR) females used as wild-type flies significantly displayed ovipositional avoidance of capsaicin in a dose-dependent manner (Fig. 1b). For instance, OR females laid approximately 35% of their eggs on food halves with capsaicin, and the oviposition index (OI) was −0.46 (Fig. 1b). This result suggested that females were prone to avoiding laying eggs in response to capsaicin. In addition, another wild-type Canton S fly similarly showed oviposition repellence to 80 mM capsaicin, with an OI of −0.63 (Fig. 1b), thus suggesting that genetic variation did not account for this oviposition preference. Since two strains of Drosophila similarly responded to capsaicin, a representative Oregon R was used in our later experiments. Moreover, another closely related Drosophila species, D. yakuba, exhibited an aversion to laying eggs on capsaicin-containing substances (Fig. 1c), implicating that Drosophila has evolved a conserved capability of discriminate capsaicin prior to selecting oviposition sites. Interestingly, we also observed that Thailand chili repelled oviposition in OR females (Fig. 1d). Notably, capsaicin did not inhibit egg laying in general, because OR flies laid comparable numbers of eggs in the absence or presence of 40 mM capsaicin (Fig. 1e). This result suggests that the robustly negative oviposition preference for capsaicin is not simply attributable to a capsaicin-induced decrease in egg laying but instead represents an active choice made by female flies to avoid a repulsive substrate 21 . Together, our results demonstrated that capsaicin triggered ovipositional avoidance in Drosophila. The quantification of ovipositional aversion to chili juice. (e) Capsaicin did not hinder the number of egg-laying in the whole-forced substrate. The average number of eggs laid by female was calculated. The one-sample ttest was used to assess the mean deviation of each column from 0 mM. n = 8-15, 3 replicates. Mean ± S.E.M.; symbols: NS p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001. oviposition after one egg laying event, their oviposition preference might be influenced by the positioning and feeding propensity for capsaicin. In tracking the positions of OR flies during oviposition, we found that females were robustly repelled from capsaicin, with a positional index of −0.18 (Fig. 2a). Additionally, the larvae of OR females were repelled from capsaicin on the surfaces of agar plates (Fig. 2b). The consistency of ovipositional and positional repellence suggests that capsaicin acts as a deterrent that naturally repels Drosophila. We further sought to explore the feeding preferences for capsaicin by using proboscis extension assays. The results showed that OR females showed aversive behavior to capsaicin (Fig. 2c). Concomitantly, feeding aversion was observed for quinine and ethanol treatment compared with H 2 O mock treatment (Fig. 2c). Together, these results demonstrated that capsaicin is a stimulant that simultaneously triggers ovipositional, positional, and feeding repellence.
The nociceptive system mediates ovipositional avoidance in response to capsaicin. Drosophila ranks egg-laying sites via sensory modalities 22 , including vision 23 , gustation 24 , olfaction 14 , and nociception 25 . Next, we attempted to identify potential sensory modalities responsible for the ovipositional aversion to capsaicin. First, we tested the role of vision by allowing flies to lay eggs in darkness. The OR flies were still repelled from oviposition in food halves with capsaicin, and the OI in darkness did not significantly differ from that in light (Fig. 3a). Hence, it is unlikely that vision makes a critical contribution to the ovipositional avoidance to capsaicin. Given that capsaicin has volatile properties, we next examined whether olfaction might be required for this ovipositional avoidance by using the Y-maze assay 14 (Fig. 3b, L). However, the OR flies displayed no bias to capsaicin, with a response index of 0.05 (Fig. 3b, R), indicating that the olfactory system was not required for the ovipositional repellence from capsaicin. To verify these results, we impaired olfaction by surgically dissecting the primary olfactory organs, the third antennal segments. Indeed, antennaectomized OR females still displayed egg-laying aversion to capsaicin (Fig. 3c). Thus, olfaction is not essential for the ovipositional aversion to capsaicin. Capsaicin elicits a burning pain by activating specific receptors (nociceptor) on sensory nerve endings. Because a subset of sensory neurons are embedded within the fly forelegs, we surgically removed the foreleg tips of OR flies. In those flies, the avoidance to capsaicin was markedly lower than that in intact OR flies (Fig. 3d), indicating that pain-sensing neurons were necessary for this avoidance. The forelegs also contain gustatory bristles that guide flies to select hospitable zones to deposit eggs. To rule out the potential roles of gustation, we next sought to identify the nociceptive receptors that regulate egg-laying aversion to capsaicin. We found that painless mutants showed lower avoidance to capsaicin at concentrations below 40 mM than wild-type flies (Fig. 3e). Moreover, the positional and feeding avoidance of painless mutants was completely impaired (Fig. 2a-c). Collectively, our results suggest that nociception is required for ovipositional aversion to capsaicin.
To verify that capsaicin triggered the transcription of pain-associated genes, we further quantified the transcriptional level of painless and dTRPA1 26 (a nonselective cation channel) in capsaicin-treated guts of OR flies through quantitative PCR. As anticipated, our data showed that levels of both painless and dTRPA1 post capsaicin treatment were higher than those in the control group (Fig. 3f). Therefore, we concluded that capsaicin activates the nociceptive modality that mediates the ovipositional avoidance in response to capsaicin in Drosophila.
Capsaicin hinders the growth of Drosophila progeny. In nature, Drosophila populations deposit eggs in suitable sites to enhance the survival of their offspring 10 . To investigate how capsaicin affects the development of progeny, we treated eggs with different doses of capsaicin. The survival rates of pupae and adults were lower after treatment with capsaicin ( Fig. 4a), whereas the animals survived normally in control food. For instance, the survival percentages of pupa and adult formation from eggs declined to 22% and 12% in food with 20 mM capsaicin, whereas the corresponding percentages were 52% and 51% in control food. Furthermore, the pupation and eclosion durations of flies were prolonged with capsaicin treatment compared with the control treatment www.nature.com/scientificreports www.nature.com/scientificreports/ ( Fig. 4b). For example, the duration of formation of pupa and adults from eggs was 6.3 and 9.7 days in control food, respectively. Nevertheless, the corresponding durations were prolonged to 8.1 and 11.8 days in food containing 10 mM capsaicin. Overall, our data support that capsaicin diminishes the fitness of progeny, thus reflecting a strategy of ovipositional avoidance in wild flies.
Capsaicin decreases the lifespan and climbing behavior of adult flies. Capsaicin widely repels insects 8 and therefore may directly exert adverse effects on adult OR flies. Indeed, the survival of these flies was dramatically less than that of control flies (Fig. 4c,d), indicating that capsaicin was toxic to flies. Notably, male flies were more susceptible to capsaicin than females, partially explained by the fact that bodyweight of males is lighter than that of females. Capsaicin also serves as a contact insecticide that has adverse effects on insect survival. Indeed, abdomen-contacted capsaicin caused greater lethality in females (Fig. 4e,f). Coordinated locomotion is required for fundamental activities of life. As expected, the results showed that capsaicin impaired running and climbing behaviors (Fig. 4g,h). Collectively, our results demonstrated that capsaicin decreased the fitness of both Drosophila parents and progenies.  www.nature.com/scientificreports www.nature.com/scientificreports/

Capsaicin disrupts intestinal integrity in Drosophila.
Wild-type flies may ingest capsaicin-containing food that adversely affects their digestive tracts. Using an unabsorbable blue dye, we assayed for loss of intestinal barrier function, as previously described 18,27 . We found that the percentage of loss of the intestinal barrier was higher in capsaicin-treated flies than in control flies (Fig. 5a,b), suggesting that capsaicin caused dye leakage into the hemolymph and consequently all tissue. Intestinal stem cells generate progeny to replenish cell loss induced by acute injury; therefore, we further assessed the number of dividing cells with an anti-phosphorylated histone 3 (PH3) antibody to determine mitotic activity 28 . The number of PH3-positive cells was significantly greater in flies treated with capsaicin than in control flies (Fig. 5c,d). Overall, our data suggested that capsaicin disrupts intestinal integrity of OR flies.
Capsaicin triggers oxidative innate immunity defense. The expression of the antimicrobial peptides attacin and diptericin was elevated in the guts of OR flies after capsaicin treatment (Fig. 6a). Dual oxidase (Duox) is a member of the ROS-producing nicotinamide adenine dinucleotide phosphate (NADPH) oxidases 29 . As expected, the expression of Duox was significantly higher in the midgut in capsaicin-treated flies than control flies (Fig. 6a). Moreover, the ROS levels were higher in capsaicin-treated OR flies than control OR flies (Fig. 6b,c). To validate the role of ROS in mediating the response to capsaicin, we knocked down Duox in flies with the GAL4/ www.nature.com/scientificreports www.nature.com/scientificreports/ UAS system 20 . The Duox silencing markedly decreased capsaicin's toxicity to flies (Fig. 6d,e). Taken together, these results suggest that capsaicin triggers oxidative innate immunity in the midgut.

Discussion
Egg laying selection is used to detect aversion toward compounds that are toxic to both larvae and adults. In this work, we showed that Drosophila females exhibit a vigorous ovipositional aversion to capsaicin through the nociceptive neurons expressing the painless gene. In addition, capsaicin decreases the fitness of both offspring and parents through intestinal dysplasia and hyperactivation of oxidative innate immunity. Overall, our findings regarding capsaicin avoidance suggest an adaptation promoting the survival and fitness of both parents and offspring 30,31 , providing insight into the resource requirements and ecological behaviors of Drosophila species.
Because of their limited mobility, larvae are vulnerable to predators and toxicants. Female flies must select preferred oviposition sites to increase the survival of their offspring 11 . As such, selecting an appropriate site for oviposition has become an innate behavior in Drosophila females. For herbivorous insects, Drosophila feed on fruits and engage in a persistent battle with host plants 32 . However, many plants use a chemical defense system to generate many secondary metabolites, including capsaicin 4 . For example, capsaicin has been shown to hinder feeding by many invertebrates 32 . Recent studies, including our work, suggest a general theme in which Drosophila is robustly repelled from many toxicants, including harmful molds 12 . Ovipositional deterrents have not previously been tested for the onion fly with host models 6 . Flies at all times must balance the benefits and threats of progeny fitness and their survival, because survival and reproduction strategies are used in the context of systemic ecology 10 . Interestingly, a previous study has found a feeding preference for capsaicin when wild-type flies are given a choice between capsaicin-laced sucrose and sucrose alone 4 . The difference of these results may be explained by the differences in capsaicin concentrations. Because capsaicin affects thermoregulation of insects through TRPA receptors, we speculate that low concentration of capsaicin could provoke excitement, but high concentration would activate the defense reaction. Indeed, capsaicin in high concentrations has known insecticidal properties 6,8 , potentially resulting in fly feeding avoidance of capsaicin. Further exploration of the subtle interactions involved will be of great interest. Of note, it is unlikely that the ovipositional avoidance of capsaicin stems simply from the feeding and positioning avoidance of capsaicin (Fig. 2). The oviposition preference does not, in fact, constantly correspond to a positional preference, because Drosophila balances multiple environment cues, even competing behavioral drives before behavioral output 22 . For example, acetic acid is an ovipositional stimulant that flies www.nature.com/scientificreports www.nature.com/scientificreports/ otherwise avoid 21 . Our results showed that up to 200 mM capsaicin efficiently decreased the survival of flies in a contact toxicity bioassay (Fig. 4e,f). Therefore, it is postulated that female adults can endure capsaicin in sites with concentrations of capsaicin (20-80 mM) when they are laying eggs. In addition, female flies laid comparable numbers of eggs in the absence or presence of 40 mM capsaicin (Fig. 1e), suggesting that low concentrations of capsaicin do little harm to adult females. Overall, our findings suggest that female flies make an independent decision to avoid ovipositing in response to capsaicin.
Nociception is caused by physical (heat, cold, and pressure) or chemical (acid, irritants, and inflammatory mediators) stimuli 33 . Nociceptive sensory neurons detect these stimuli and activate neural circuits that elicit stereotyped escape responses 34 . Our findings showed that the ovipositional aversion toward capsaicin is mediated by the activation of the nociceptive neurons expressing the painless gene (Fig. 3). In Drosophila, the painless genean evolutionary homolog of the mammalian isothiocyanate receptor TRPA1/ANKTM1 4 -is required for the foraging avoidance of isothiocyanates, because isothiocyanates from wasabi and capsaicin can give rise to a burning sensation 25 . Painful or threatening experiences trigger escape responses that are guided by nociceptive neuronal circuitry 3 . Efficient and rapid escape behavior in reaction to threatening sensory stimuli is vital for defense and ultimately survival. Drosophila larvae must forage for food while avoiding noxious stimuli and predators, such as parasitoid wasps 14 . Capsaicin is derived from the genus Capsicum, its receptor, transient receptor potential vanilloid subfamily member 1 (TRPV1), is localized to many human organs 34 , including the brain, intestine, liver, pancreas, lung, and kidney. Nociception is the encoding of a noxious stimulus and its transduction into electric signals. Noxious stimuli are detected by nerve endings found throughout the body and originating from the PSNs. The activation of TRPV1 induces cation (Ca 2+ ) influx, which further triggers voltage-gated sodium channels and generates an action potential 26 .
Animals across multiple species must navigate a complicated and ever-changing environment for survival and propagation. For instance, oxidative stress induces immune activation in both Drosophila and humans 35 . Capsaicin ingested from the environment transiently interacts with intestine epithelia by passing through the alimentary flowing stream. Consequently, capsaicin results in intestinal epithelium impairment and intestinal barrier dysfunction in adult flies (Fig. 5). Intestinal barrier dysfunction is a good predictor of stress-induced mortality. To adapt to acute stress in the gut, animals have evolved to modulate their innate immunity to achieve intestinal homeostasis 35 . Although immunity is critical for host fitness in response to stress, the specific signaling pathways through which capsaicin triggers immunity are not yet entirely clear. This study was based on the well-characterized DUOX-dependent intestine immunity in flies. We found that capsaicin resulted in DUOX-dependent intestinal immune activation (Fig. 6). DUOX, a member of the NADPH oxidase family, functions as the first line of defense against intestinal stress by producing ROS. DUOX-dependent ROS are considered to be involved directly or indirectly in epithelial turnover through activation of intestinal stem cells in the presence of stress 36 . In lines with previous findings 37 , capsaicin triggers the immune pathway via de novo production of AMPs. Thus, capsaicin-induced DUOX-activating signaling exerts a pronounced effect on the lifespan and health of Drosophila.
Using Drosophila, we investigated an ecological phenomenon whereby capsaicin repels Drosophila females from oviposition. Consistent with this finding, capsaicin decreases the fitness of Drosophila through intestinal dysplasia. Future research will facilitate more comprehensive understanding of the relationship between the molecular pathology and ecological behaviors of Drosophila.

Materials and Methods
Fly husbandry and stocks. All fly stocks were raised at 25 °C, 60% humidity in a 12/12 h light/dark cycle on standard cornmeal-yeast-sucrose food unless otherwise noted 10 . Oregon R and Canton S strains were used as wild-type flies. NP3084 and DUOX mutants were a gift from Prof. Gao Guanjun (Shanghai Technology University) 20 . Painless mutants and D. yakuba were from the Core Facility of Drosophila Resource and Technology, Shanghai Institute of Biochemistry and Cell Biology, CAS, China.
Oviposition preference assays. The two-choice apparatus was assembled with a transparent 80-mm column with a 60-mm Petri dish at the bottom 10,21 . The two-choice dishes were generated by evenly dividing food into halves with a razor blade. In brief, capsaicin (MACKLIN) was dissolved in absolute alcohol, and diluted capsaicin-containing solutions (10 mM, 20 mM, 40 mM, and 80 mM) were added to the surfaces of the egg-laying substrate. The two-choice dishes were placed in air for 3 h to volatilize the alcohol. For each test, 25 newly eclosing females were collected and mated for 3 days in the presence of yeast paste. Flies were gently transferred into assay cages without CO 2 anesthesia and were allowed to lay eggs for 8 h in darkness. To assess oviposition preference, we counted the number of eggs on each half and determined the OI as follows: [OI = (number of eggs laid on experimental food -number of eggs laid on control food)/total number of eggs laid]. For one-forced assays, 25 mated females were transferred to cages totally with capsaicin-containing substrate, where they were allowed to lay eggs for 12 h. Flies were removed, and laid eggs were counted. Egg laying was calculated by dividing the number of eggs by the total number of live females.
Position preference assays. The two-choice apparatus was the same as that used in oviposition preference assays 10,21  For surgeries, females were anesthetized with CO 2 on a pad, and the antennae and tarsi were removed with fine forceps. Flies were allowed to recover for 48 h before testing.

Scientific RepoRtS |
(2020) 10:9963 | https://doi.org/10.1038/s41598-020-66900-2 www.nature.com/scientificreports www.nature.com/scientificreports/ Feeding preference assays. To assay feeding preferences, each compound was dissolved in 5% sucrose solution as previously described 14 . Briefly, files were collected on the day of eclosion and kept in standard corn meal food for 3-7 days at 25 °C. Before the feeding assays, females were starved for 24 h at 25 °C in vials with a water-saturated Whatman filter paper. Flies were anaesthetized by chilling on ice, mounted by their backs/wings on a microscope slide using double-sided Scotch tape and allowed to recover for 1 h at room temperature. Taste solutions were delivered with a 10 μl pipette to legs for up to 20 s, and the time of proboscis extension was examined. The feeding index (FI) was calculated as follows: FI = the total time of proboscis extension/20 s ×100%.
Contact toxicity bioassay. The contact toxicity bioassay was performed as previously described 38 . Briefly, files were collected on the day of eclosion and kept in standard corn meal food for 5 days at 25 °C. Flies were anaesthetized with CO 2 , mounted by their wings on a microscope slide using double-sided Scotch tape and allowed to recover for 30 min at room temperature. Each compound was dissolved in 75% EtOH solution, and 10 μL solutions with different concentrations of capsaicin were evenly spread on their abdomens (n = 5, 4 replicates). Flies without leg movements were designed as dead flies every 15 minutes, and the survival rate was calculated. Survival ratio assays. In feeding experiments 39 , flies were starved at 25 °C for 4 h before transfer to cages containing a filter paper with 1 ml capsaicin-sucrose. Capsaicin feeding was performed at 25 °C. For viability analysis, we used 20 flies per vial, and the experiments were repeated four times. Flies were transferred to new cages with new capsaicin-sucrose filter paper discs every 24 h and counted every day. The percentage of survival was calculated.
Dye penetration test (smurf). The intestinal barrier dysfunction assay was conducted on flies with starved at 25 °C for 5 h. The feeding experiment was performed as described above. Twenty flies were used in every vial and kept at 25 °C. Flies were first exposed to capsaicin-sucrose filter paper for 5 d and then transferred to new cages with filter paper mixed with 2.5% Erioglaucine (FD&C Blue #1) 18 and 5% sucrose. For the dye penetration test, 20 wild-type flies were fed on filter paper soaked with capsaicin-sucrose solution and Erioglaucine. A fly was counted as a Smurf when dye coloration was observed outside the digestive tract. Smurf % = number of smurf flies/(number of non-Smurf flies + number of smurf flies) × 100%.
Development timing assays. The embryos were inoculated into fly food with 0 mM, 6 mM, 10 mM, or 20 mM capsaicin (n = 20-35, 5 replicates). The number of embryos was counted with a stereo microscope. The numbers of pupae and emerging adults were counted everyday following embryo incubation. Developmental timing was calculated with the following formula: T = (T1 × N1 + T2 × N2 + … + Tm × Nm)/(N1 + N2 + … + Nm), where T is the developmental timing; Tm is the number of days to form pupae and adults after egg laying; and Nm is the number of pupae and adults on the m th day 12 .
Climbing and running assays. Polystyrene vials were prepared by placing a Kimwipe moistened with 5% sucrose at the bottom. Capsaicin solution (80 mM) was delivered to the vials everyday. Flies were treated for 5 d and then transferred to a glass graduated cylinder or a serological pipette (5 ml). Locomotion assays were conducted as described with the following modifications 40 . To analyze climbing ability, all flies were synchronized to be tapped down to the bottom of the cylinder. Climbing was recorded using a video camera, and climbing distances at the 6 th second were calculated. To analyze running velocity, 5 flies was gently pounded down to the end of a serological pipette in a dark room. This apparatus was horizontal and perpendicular to the light source 15 cm away. Running was recorded using a video camera, and the time to pass the line at the other end of the serological pipette was calculated.
Real time-PCR analysis. Flies were challenged with 80 mM capsaicin for 48 h, and the guts were dissected and transferred to cold PBS buffer. A total of 40 guts were homogenized with TRIzol reagent (Invitrogen), and RNA was extracted as previously described 12 . RNA was reverse transcribed with an oligo-dT primer. The primer sequences are available upon request. Expression of these genes was assessed with a Bio-Rad CFX instrument. The ΔCt method was used to analyze data with rp49 as the reference gene. The relative expression value was calculated with the following formula: △Ct = Ct (target gene) -Ct (reference gene), and the relative expression was equal to 2 −△△Ct .
Immunofluorescence staining. The immunofluorescence assay was performed as described in previous studies 28 . The flies were fed with 5% sucrose solution with or without 80 mM capsaicin for 5 d. Guts were dissected in PBS and fixed with 4% paraformaldehyde for 30 min at room temperature. Samples were blocked with blocking solution (PBS with 0.3% Triton X-100, 0.2% goat serum and 0.1% fetal calf serum) for 30 min. Guts were incubated in phospho-histone 3 (EMD Millipore, 1:1000 dilution) overnight at 4°C, washed in PBS supplemented with 0.3% Triton X-100, and incubated in PBS with anti-rabbit secondary antibodies (Invitrogen, 1:1000 dilution) and DAPI (Invitrogen, 1:1000 dilution) for 2 h at room temperature. Samples were mounted with Vectshield and observed under a fluorescence microscope (Leica DM4000).
In vivo detection of reactive oxygen species. ROS production in intestinal epithelial cells was measured with the intracellular ROS-sensitive fluorescent dye dihydroethidium 19 . After capsaicin feeding for 48 h, the midguts of flies were dissected in PBS and incubated in a 5 μM concentration of the intracellular ROS-sensitive fluorescent dye dihydroethidium (hydroethidine) (Invitrogen) for 30 min at room temperature in the dark. Then, the midguts were washed three times with PBS, and the tissues were immediately immobilized with 4% paraformaldehyde for 10 min. Next, the tissues were washed three times with PBS. Subsequently, the gut samples were