The characteristic response of domestic cats to plant iiridoids allows them to gain chemical defense against mosquitoes
Abstract
Domestic cats and other felids rub their faces and heads against catnip (Nepeta cataria) and silver vine (Actinidia polygama) and roll on the ground as a characteristic response. While this response is well known, its biological function and underlying mechanism remain undetermined. Here, we uncover the neurophysiological mechanism and functional outcome of this feline response. We found that the iridoid nepetalactol is the major component of silver vine that elicits this potent response in cats and other felids. Nepetalactol increased plasma β-endorphin levels in cats, while pharmacological inhibition of μ-opioid receptors suppressed the classic rubbing response. Rubbing behavior transfers nepetalactol onto the faces and heads of respondents where it repels the mosquito, Aedes albopictus. Thus, self-anointing behavior helps to protect cats against mosquito bites. The characteristic response of cats to nepetalactol via the μ-opioid system provides an important example of chemical pest defense using plant metabolites in nonhuman mammals.
While learned behaviors allow animals to adapt flexibly to complex and changing environments, some species-specific behaviors are expressed reliably with no requirement for previous exposure or learning (1). This type of fixed behavior in animals is often elicited by chemical signals in the secretions of conspecifics (pheromones) or chemical cues from predators or prey (kairomones), where a reliably evoked behavioral response is important for survival. In addition, some plant odorants can also elicit characteristic responses in animals. A well-known example of plant-induced behavior in mammals is observed in domestic cats (Felis silvestris catus) and other felids such as lions (Panthera leo) and bobcats (Lynx rufus) (2–4). When felids sniff specific plants such as catnip (Nepeta cataria) and silver vine (Actinidia polygama), they exhibit a typical behavioral response that comprises licking and chewing the plants, face and head rubbing against the plants, and rolling over on the ground (2, 4, 5). This catnip and silver vine response usually lasts 5 to 15 min, followed by a period of one or more hours when they are nonresponsive (6). Because cats demonstrate an intoxicated response that does not have any pathophysiological effects (7), dried leaves of these plants are used commercially in toys for domestic cats worldwide.
The first reports of the feline behavioral response to silver vine and catnip were described by a Japanese botanist in 1704 (8) and by a British botanist in 1759 (9), respectively. The behavioral response to silver vine has been captured in Japanese culture: An Ukiyo-e (a type of traditional painting) drawn in 1859 depicts a folk story concerning a battle between cats and mice, wherein mice use silver vine as a weapon to intoxicate cats (10). While silver vine is endemic to Japan and China, its potent effects on cat behavior came to global recognition following its import from China to the United States (11). Bioactive iridoid compounds in catnip (nepetalactone) and silver vine (isoiridomyrmecin, iridomyrmecin, isodihydronepetalactone, and dihydronepetalactone) induce the same characteristic response (12–15). Cats perceive these secondary plant metabolites through the main olfactory system (6), while oral administration of nepetalactone induces no response (16). The strength of behavioral response increases with cat maturity (17), but there is no sexual dimorphism in response among adults. However, despite widespread recognition of this characteristic behavioral response to specific plants by feline carnivores, its functional outcome is not yet understood.
This study aimed to uncover the neurophysiological mechanism and biological function of the silver vine response in domestic cats. To establish a reliable and reproducible behavioral assay for precise control of stimulus presentation, we first purified potent bioactive compounds from silver vine leaves and identified nepetalactol, which had been missed in previous studies (13–15, 18). Using chemically synthesized nepetalactol, we demonstrated that the silver vine response is regulated via μ-opioid receptors that are involved in rewarding and euphoric effects in humans. The rubbing and rolling response transfers nepetalactol from the plant leaves onto the cat’s face and head where it acts as a mosquito repellent, finally revealing the likely biological significance of this enigmatic feline behavior, first observed more than 300 years ago.
RESULTS
Nepetalactol is a potent stimulant for silver vine response
Previous attempts to isolate bioactive compounds from dried leaves of silver vine used steam distillation, alkaline heat treatment, and acid treatment (13–15), all of which have the potential to decompose bioactive components. To avoid this problem, an organic solvent extract from silver vine leaves was resolved into six fractions using silica gel normal-phase column chromatography (Fig. 1A, step 1). The bioactivity of each of these fractions, corresponding to 1.2 g of leaves, was tested using four cats that responded positively to the unfractionated leaf extract (Fig. 1B). Only fraction 3 eluted by n-hexane/ethyl acetate (80:20, v:v) and fraction 4 eluted by n-hexane/ethyl acetate (70:30) induced face rubbing and rolling over in four and three subject cats, respectively. Although fraction 3 stimulated a more prolonged response than fraction 4 in all subjects (Fig. 1C), gas chromatography/mass spectrometry (GC/MS) analysis revealed that compounds with known bioactivity (isoiridomyrmecin, dihydronepetalactone, and isodihydronepetalactone) were at markedly lower levels in fraction 3 compared to fraction 4 (Fig. 1D). This suggests an important contribution of one or more unidentified compounds in fraction 3, which induce the behavioral response. To identify these unknown compounds, bioactive components in fraction 3 were further purified by normal-phase and reversed-phase high-performance liquid chromatography (HPLC) and finally enriched into a bimodal peak by HPLC (Fig. 1A, steps 2 to 5, and movie S1).
GC/MS of the final bioactive fraction (fraction 3-3-2-3) detected five major peaks (Fig. 1E); one, at 43.7 min, had a unique mass spectrum that matched (87%) a standard spectrum of nepetalactol (fig. S1A) in the Wiley MS library. Nepetalactol (Fig. 1F) is a common important biosynthetic precursor of iridoid monoterpenes (19) and shares a very similar structure with cis-trans nepetalactone (Fig. 1F) except for lactol and lactone moieties. A recent study also identified nepetalactol from silver vine leaves but did not examine its bioactivity in cats (20). We therefore synthesized cis-trans nepetalactol for further investigation. The mass spectrum of the 43.7-min peak had 98% similarity with authentic nepetalactol. Further, fraction 3-3-2-3 spiked with authentic nepetalactol yielded a single coeluting GC/MS peak with the same retention index (RI = 2078) as authentic nepetalactol alone (RI = 2080; fig. S1B). These results provide clear evidence that the peak at 43.7 min was cis-trans nepetalactol. We conjectured that the other two peaks (at 39.5 and 39.7 min) were stereoisomers of iridodial (Fig. 1F); the mass spectra (fig. S1C) were in good agreement with published spectra (21, 22). As iridodial has a readily epimerizing dialdehyde structure and is easily oxidized under atmospheric conditions (23), we thought that iridodial is an unsuitable stimulant for a reliable behavioral assay. Thus, further behavioral assays evaluated the bioactivity of chemically synthesized nepetalactol.
Bioactivity of nepetalactol in felid and nonfelid species
In this study, we used 25 laboratory cats that consisted of 18 positive and 7 negative responders to silver vine leaf extract (table S1). In behavioral assays using 15 of the 18 positive responder cats, all subjects exhibited face rubbing and rolling over in response to 50 μg of nepetalactol-impregnated filter paper (nepetalactol-paper); most of them then lost interest in the paper within 10 min after presentation (Fig. 2, A and B, and movie S2), very similar to the behavioral response toward unextracted plant materials (6). No cats exhibited a flehmen-like response, which is a functional behavior that transfers compounds such as pheromones from the oral cavity to the sensory vomeronasal organs (24). The duration of the behavioral response to nepetalactol-paper was more prolonged than that to control solvent filter paper (control-paper) presented simultaneously on the floor (Wilcoxon matched-pair test, one-tailed P = 0.0003; Fig. 2C). To test the generality of the bioactivity of nepetalactol, we also tested this similarly in 30 free-ranging feral cats using nepetalactol-paper versus control-paper. Seventeen of the 30 cats (57%) rubbed their faces against at least one paper. Almost all face rubbing and rolling among these 17 feral cats was directed toward the nepetalactol-paper such that the overall response toward nepetalactol-paper was substantially more prolonged than toward control-paper (P = 0.0001; Fig. 2, D and E, and movie S2), similar to laboratory cats. The other 13 cats did not respond to either papers, suggesting that they either were inherent negative responders (5) or just were not responsive in an unfamiliar test situation.
Next, we compared the behavioral responses of 12 of the 18 positive responder cats to each of the known bioactive iridoids. Nepetalactol (silver vine) and nepetalactone (catnip) induced more prolonged face rubbing and rolling than other iridoids (Fig. 2F; χ2 = 24.0, df = 5, P = 0.0002). At least one cat did not exhibit the characteristic response toward each iridoid except for nepetalactol. Further, the nepetalactol content of silver vine leaves (20.71 μg/g of wet weight) was much higher than isoiridomyrmecin (1.42 μg/g), iridomyrmecin (below the GC/MS detection limit), dihydronepetalactone (<0.18 μg/g), or isodihydronepetalactone content (<0.18 μg/g). Considering also that fraction 3 had stronger bioactivity than fraction 4, as shown in Fig. 1C, and nepetalactol had bioactivity in all of the 18 positive responder cats tested (Fig. 2, C and F), nepetalactol is the most potent and major bioactive iridoid in silver vine leaves. We concluded that nepetalactol is the most suitable stimulant for a reliable and reproducible behavioral assay.
Nondomesticated captive felids tested at zoos in Japan (an Amur leopard, P. pardus orientalis; two jaguars, P. onca; two Eurasian lynx, L. lynx) also exhibited more prolonged face rubbing and rolling on nepetalactol-paper than on control-paper (exact P = 0.031; Fig. 2, G and H, and movie S3), a bias that did not differ significantly from the laboratory cats (Mann-Whitney U test of bias; z = 1.27, P = 0.23).
We also tested the response of domestic dogs (Canis lupus familiaris, n = 8) and laboratory mice (C57BL/6 or BALB/cAJcl strain males, n = 10) to nepetalactol. All animals tested were uninterested in nepetalactol, and none exhibited a silver vine response (movie S3). The lack of response to nepetalactol in dogs and mice differed substantially from the positive response found among 72% of the 25 laboratory cats tested in this study (Fisher’s exact tests, domestic dogs: P < 0.0005; mice: P < 0.0005).
Activation of the μ-opioid system during silver vine response in cats
Subjective observations of the behavioral response of cats to catnip suggest that they may experience a positive reaction that has often been interpreted as extreme pleasure (17, 25). Thus, we hypothesized that olfactory reception of nepetalactol stimulates the μ-opioid system, which controls rewarding and euphoric effects in humans (26). First, we examined temporal changes in plasma levels of β-endorphin (a peptide hormone and an endogenous opiate) in five cats 5 min before and after exposure to 200 μg of nepetalactol (corresponding to the contents in approximately 10 leaves) on day 1 (inducing the behavioral response) and then to a blank stimulus control 4 days later (Fig. 3A). Plasma β-endorphin concentration was markedly elevated after exposure to nepetalactol but not after exposure to a control stimulus [repeated-measures analysis of variance (ANOVA), interaction between stimulus and time point: F1,4 = 9.97, P = 0.034; Fig. 3B].
To test whether the μ-opioid system is directly involved in regulation of the behavioral response to nepetalactol, we examined the behavioral response to nepetalactol in six cats that had been administered saline (day 1) or naloxone (day 2), an antagonist of μ-opioid receptors (Fig. 3C). While all cats exhibited a typical response to nepetalactol-paper after saline administration, the duration of their characteristic rubbing and rolling response was reduced significantly after naloxone administration on the following day (Wilcoxon matched-pair test, two-tailed exact P = 0.031; Fig. 3D and movie S4). By contrast, six cats administered saline on both days as a control showed no reduction in response to nepetalactol (P = 1.00; Fig. 3E), differing significantly from the reduced response caused by naloxone (Mann-Whitney test of change between day 1 and day 2; P = 0.04). Naloxone did not affect the duration of other activities compared to vehicle alone (walking: P = 0.31; grooming: P = 0.31; fig. S2, A and B), confirming that the naloxone dose administered did not disturb locomotor activities or motor functions during the observations. Inhibition of the μ-opioid system specifically suppressed the rubbing and rolling response in the cats. These results demonstrate that the μ-opioid system is involved in the induction of the feline behavioral response.
Mosquito-repellent activity of nepetalactol
The consistent expression of such a characteristic response to nepetalactol suggests that the response has an important adaptive function for cats. On the basis of reports that nepetalactone from catnip has mosquito-repellent activity when applied to humans (27–29), we hypothesized that the characteristic rubbing and rolling against plants allows cats to transfer nepetalactol or nepetalactone onto the fur for chemical defense against mosquitoes and possibly also against other biting arthropods. In this study, we tested whether nepetalactol is repellent to Aedes albopictus, a mosquito common in Japan and China (30). A. albopictus avoided both silver vine leaves (five leaves, containing approximately 100 μg of nepetalactol) and nepetalactol alone (50 μg, 200 μg, and 2 mg) compared to a solvent control, when each was placed separately into test cages that had shelters into which A. albopictus could move (Fig. 4A; ANOVA, effect of stimulus F4,15 = 79.93, P < 0.0001; planned contrasts confirmed that avoidance of each test stimulus was significantly greater than the control, P < 0.003; Fig. 4B). This indicates that nepetalactol acts as a repellent against A. albopictus, consistent with the previously reported repellent activity of nepetalactone (29).
The function of rubbing behavior in nepetalactol-stimulated cats
Next, we examined whether the characteristic rubbing and rolling response functions to transfer nepetalactol to the cat’s face, head, and body. To establish the importance of contact with the source (to rub nepetalactol onto the fur), seven laboratory cats were tested with 200 μg of nepetalactol versus control on papers placed on the test cage walls or ceiling. In this arrangement, cats could rub the papers with their faces, but rolling would not allow rubbing contact with the stimulus. As expected, all subjects rubbed their faces and heads on nepetalactol-paper placed on the cage walls more frequently than on control-paper (Wilcoxon matched-pair test, exact P = 0.008; Fig. 5A and movie S5). When papers were more difficult to contact on the cage ceiling, five of seven subject cats stood on their hind legs, held on to the ceiling mesh with their fore paws, and rubbed their faces and heads on nepetalactol-paper more frequently than on control-paper (exact P = 0.031; Fig. 5B and movie S5). However, no subject cat rolled on the ground when test papers were on the cage walls or ceiling, in stark contrast to typical rolling observed in all subjects when filter papers were placed on the floor (χ2 = 29.0, df = 1, P < 0.0001). Strong motivation to contact nepetalactol was further evidenced in high-ceiling cages, when two of seven subjects climbed the 116-cm walls to reach nepetalactol-paper on the ceiling and then proceeded to rub their faces and heads on nepetalactol-paper as before (Fig. 5C and movie S6). Thus, rubbing is specifically targeted at the nepetalactol source.
Rubbing against the source (200 μg of nepetalactol) should transfer the material to the fur, but the quantity of nepetalactol in ethanol-soaked cotton used for wiping the face and head fur was below the limit of detection (2.2 μg) by our experimental procedure using GC/MS as a detector, indicating that no more than 1% of the material was recovered on the cotton wipe. To provide a more sensitive test, subject cats were tested with face and head wipes from donors that had rubbed nepetalactol-papers with or without direct physical contact versus wipes from unstimulated donors (Fig. 5D). We predicted that subjects would detect nepetalactol on papers used to wipe donors that had contact-rubbed nepetalactol-paper, but they would not respond to wipes from unstimulated donors or from donors that rubbed in response to nepetalactol when they could not physically contact the source. In agreement with our prediction, subjects rubbed and rolled only in response to wipes from donors that had physically contact-rubbed nepetalactol-papers (Fig. 5E; interaction between donor nepetalactol stimulation and direct contact, F1,4 = 9.97, P = 0.034; behavioral response to donor with nepetalactol physical contact, F1,4 = 12.35, P = 0.025; behavioral response to donor without nepetalactol physical contact, F1,4 = 0.32, P = 0.60). Thus, face rubbing transfers nepetalactol onto the cat’s fur.
Silver vine response provides cats with mosquito repellency
To examine whether nepetalactol on the fur protects cats from mosquito bites, the heads of six pairs of anesthetized cats were placed into opposite sides of a test cage. One cat’s head had been treated with nepetalactol (500 μg) and the other with the appropriate solvent control. The number of A. albopictus landing on the nepetalactol-treated head was half the number landing on the control head on average (repeated-measures ANOVA, F1,5 = 8.56, P = 0.033; Fig. 5F), showing significant repellence. Lastly, to investigate a more natural situation, we assessed whether cats responding to silver vine leaves transfer sufficient active compound(s) to repel A. albopictus, compared to unstimulated cat controls in the same two-head test. Cats that had rubbed against silver vine leaves were significantly avoided by A. albopictus compared to control cats that had not been stimulated by the leaves (repeated-measures ANOVA, F1,5 = 11.78, P = 0.019; Fig. 5G). By contrast, there was no significant difference in the number of A. albopictus landing on the head of control cats versus cats that had not rubbed when presented with silver vine leaves (F1,5 = 0.029, P = 0.87; Fig. 5H; difference in bias between tests, F1,10 = 9.21, P = 0.013). These results show that nepetalactol, transferred to face and head fur by rubbing against silver vine leaves, functions as a repellent against A. albopictus in cats. This is convincing evidence that the characteristic rubbing and rolling response functions to transfer plant chemicals that provide mosquito repellency to cats.
DISCUSSION
This study has found that the iridoid nepetalactol is the major bioactive compound in the leaves of silver vine that induces characteristic rubbing and rolling in cats (Fig. 6). Further, nepetalactol had similar bioactivity in Amur leopard, jaguar, and Eurasian lynx. As most of the Felidae species so far tested have shown positive responses toward catnip (13 of 21 species tested from a total of 41 living species in this family) (2–4), it is likely that this characteristic response to nepetalactol will also be common across many of the Felidae. Using synthesized nepetalactol, we have demonstrated that the μ-opioid system regulating euphoric and rewarding effects in humans (26) is involved in the expression of the catnip and silver vine response in cats. We have uncovered an adaptive benefit of the behavioral response in cats: Rubbing and rolling on the leaves of silver vine transfers nepetalactol to the heads, faces, and bodies of cats. As a consequence, this reduces the number of A. albopictus mosquitoes that land on the animal’s head, helping to protect from mosquito bites. These findings provide new insight into this well-known and characteristic plant-induced feline response, for which the biological function was first questioned in popular science culture more than 300 years ago.
We have yet to understand fully how the μ-opioid system is activated by nepetalactol in cats. In previous reports, essential oils from N. caesarea, of which nepetalactone is a major component, had an analgesic effect involving μ-opioid receptors in rats, but these compounds were injected intraperitoneally (31). Oral administration of nepetalactone to cats has no marked physiological effects (16). Further, cats do not require naso-oral contact with nepetalactol-paper for this to stimulate the silver vine response. This suggests that nepetalactol and other bioactive iridoids activate the μ-opioid system via chemical sensing through the olfactory system for the response. The vomeronasal system does not appear to be involved in inducing the response in cats (6). Previous studies have reported functional connections between the olfactory and opioidergic processes in mammals such as rats (32–35), suggesting that cats may have neural circuitry that connects the olfactory neurons that detect bioactive iridoids with the μ-opioid system. While our results indicate that taste is not essential for the silver vine response, cats exhibiting the response commonly lick the stimulus when this is possible. As previous studies have reported that tastes and other orosensory stimulation can activate β-endorphin release in mammals (36, 37), the possibility cannot be excluded that the taste system also participates in regulating the silver vine response in cats.
The feline response to specific chemicals in plant materials is nonaddictive (38). This may be because the μ-opioid system is stimulated by an increase in endogenous β-endorphin secretion when olfactory neurons are activated by these iridoids. This contrasts with the development of addiction to exogenous opiates such as morphine in mammals including cats (39), when μ-opioid receptors are directly activated by opiates via the bloodstream (40, 41).
Although we only tested for a repellent effect on A. albopictus in this study, we might also expect nepetalactol to be repellent to other mosquito species including A. aegypti, which is a common vector of yellow fever, dengue, and Zika viruses (42), consistent with the broad repellence of nepetalactone across a range of mosquito and other biting arthropods (27–29). Our findings suggest that nepetalactol may be a new natural candidate repellent to help reduce mosquito problems in human society.
We propose that silver vine and catnip response provides repellency against A. albopictus by transferring nepetalactol or nepetalactone from plants onto a cat’s fur. Face rubbing against plant sources of the repellent will help to protect the face and head of the animal, as the mouth, eyelids, ears, and nose of felines have relatively little fur and are therefore easy targets for mosquitoes. Although the rolling response following face rubbing, which exposes the belly, may look like a defenseless behavior, it enables cats to pick up repellent iridoids on other areas of their bodies. Notably, cats did not roll on the ground when stimuli were placed such that rolling would not bring the cat into contact with the stimulus. Therefore, rolling is a functional behavior rather than an indicator of euphoria or extreme pleasure.
The silver vine and catnip response is an important example of how animals use plant metabolites for protection against insect pests. There are other examples that nonhuman animals may exploit some chemicals emitted from other species for protection against insect pests: boat-tailed grackles (Quiscalus major) and white-nosed coatis (Nasua narica) rub fruits of Citrus spp. against themselves (43), chimpanzees (Pan troglodytes schweinfurthii) use sleeping platforms created from specific trees as a source of repellents (44), house sparrows (Passer domesticus) and house finches (Carpodacus mexicanus) living in urban habitats bring cigarette butts to the nest (45), and capuchin monkeys (Cebus olivaceus) anoint themselves with millipedes (Orthoporus dorsovittatus) (46). Each species may select plants and other materials as insect repellents during evolution. The examples so far uncovered of animals self-anointing or using prophylactic self-medication (47) with secondary plant metabolites to protect against pests and diseases typically occur in individual species, although the same pests and diseases may affect many species. However, self-anointing with plant iridoids is common in Felidae, although not in the Carnivora more generally, suggesting that the behavior first evolved in a common felid ancestor and has been retained. As many felids rely on stealth to stalk and ambush their prey, requiring them to remain cryptic and often unmoving, a repellent that reduces their susceptibility to both the irritation of biting mosquitoes and the diseases that these insect vectors carry is likely to provide a strong selective advantage. Stimulation of the μ-opioid system might further help by providing analgesia to reduce irritation where biting arthropods have not been repelled. While this can explain why this characteristic behavior has been retained in many Felidae species, it does not explain why the behavior has evolved only in felids. A specific ability to detect these iridoid chemicals in a common felid ancestor may have been a crucial preadaptation that provided the opportunity for this self-anointing behavior to evolve, allowing animals from multiple species within this family to acquire mosquito repellence.
The catnip response is inherited as an autosomal dominant trait in domestic cats (5), strongly suggesting the presence of one or few genes responsible for the silver vine and catnip response in felids. The felids that were positive responders in this study focused close attention toward nepetalactol and other iridoid stimuli presented, responding even to the low level of nepetalactol recovered by wiping donors that had rubbed this into their fur during the silver vine response. By contrast, dogs, mice, and negative responder cats failed to even stop and sniff nepetalactol stimuli. These findings suggest that domestic cats and the nondomestic felids that also respond might have acquired specific olfactory receptor(s) that detect nepetalactol and other iridoids emitted from some plants with high sensitivity. A genome-wide association study among positive and negative responder cats to identify the olfactory receptor genes and specific neuronal pathways involved in this response could provide invaluable clues for understanding how and why this characteristic response to silver vine and catnip has evolved specifically in felids.
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