- Email: [email protected]
The oxytocin receptor gene (OXTR) polymorphism in cats (Felis catus) is associated with “Roughness” assessed by owners
Accepted Manuscript The oxytocin receptor gene (OXTR) polymorphism in cats (Felis catus) is associated with “Roughness” assessed by owners Minori Arahori, Yusuke Hori, Atsuko Saito, Hitomi Chijiiwa, Saho Takagi, Yuki Ito, Arii Watanabe, Miho Inoue-Murayama, Kazuo Fujita PII:
S1558-7878(15)00139-2
DOI:
10.1016/j.jveb.2015.07.039
Reference:
JVEB 904
To appear in:
Journal of Veterinary Behavior
Received Date: 28 March 2015 Revised Date:
14 June 2015
Accepted Date: 30 July 2015
Please cite this article as: Arahori, M., Hori, Y., Saito, A., Chijiiwa, H., Takagi, S., Ito, Y., Watanabe, A., Inoue-Murayama, M., Fujita, K., The oxytocin receptor gene (OXTR) polymorphism in cats (Felis catus) is associated with “Roughness” assessed by owners, Journal of Veterinary Behavior (2015), doi: 10.1016/j.jveb.2015.07.039. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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The oxytocin receptor gene (OXTR) polymorphism in cats (Felis catus) is associated
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with “Roughness” assessed by owners.
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Yusuke Hori1, 2
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Atsuko Saito3
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Hitomi Chijiiwa1
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Saho Takagi1
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Yuki Ito3
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Minori Arahori1
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Arii Watanabe3
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Miho Inoue-Murayama4, 5
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Kazuo Fujita1
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606-8501, Japan.
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Japan Society for the Promotion of Science, Chiyoda, Tokyo 102-0083, Japan.
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Department of Cognitive and Behavioral Science, Graduate School of Arts and Sciences,
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The University of Tokyo, 3-8-1 Komaba, Meguro, Tokyo 153-8902, Japan.
Department of Psychology, Graduate School of Letters, Kyoto University, Sakyo, Kyoto
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Wildlife Research Center, Kyoto University, Sakyo, Kyoto 606-8203, Japan.
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Wildlife Genome Collaborative Research Group, National Institute for Environmental
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Studies, Onogawa, Tsukuba 305-8506, Japan.
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*Address for reprint requests and correspondence: Minori Arahori, Department of
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Psychology, Graduate School of Letters, Kyoto University, Sakyo, Kyoto 606-8501,
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Japan.
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Tel: +81757532759.
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E-mail address: [email protected] (M Arahori).
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Abstract:
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Polymorphisms of the oxytocin receptor gene (OXTR) have been hypothesized to
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correlate with social behavior in humans and other mammals. We examined the
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relationships between owner-assessed personality and OXTR polymorphisms in cats,
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which have long a history of domestication by humans. We analyzed the exon1 region of
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OXTR in 94 DNA samples (57 males and 37 females), and found 3 SNPs (single
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nucleotide polymorphisms). We focused on the G738A polymorphism. Questionnaires
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containing 30 questions with 6-point scales were completed by the cats’ owners. Factor
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analysis for the scores identified four factors, which we named “Openness”,
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“Friendliness”, “Roughness” and “Neuroticism”. A GLMM analysis revealed that cats
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with the A allele in the SNP G738A showed significantly higher “Roughness” scores than
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cats without the A allele. After dividing cats into four sex x neutering groups, only
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neutered females showed the significant difference between the A allele carriers and non
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A allele carriers in “Roughness” scores. Age also affected personality: younger cats
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showed much higher “Openness” scores whereas older cats showed higher “Roughness”
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scores. This study shows that genetic variation in cats may be linked to their personality
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traits, a finding that may be useful from the viewpoint of companion animal welfare; for
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example, to match potential good owners and cats using genetic information.
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Key words:
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domestic cats; genetic polymorphism; oxytocin receptor gene; personality
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INTRODUCTION In recent years, researchers have become increasingly interested in oxytocin, the
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peptide hormone, which is believed to be related to various social behavior in humans
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such as pair bonding, nursing for children, and coping with stressors (for review; Carter,
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2014). Many studies have also focused on oxytocin receptor gene (OXTR)
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polymorphisms in humans, which underlie individual variation in behavioral tendencies
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including empathy (e.g. Wu et al., 2012), cooperation (e.g. Haas et al., 2013), psychiatric
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disorder such as autism (e.g. Lerer et al., 2008), and prosociality (e.g. Israel et al., 2009;
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Kogan et al., 2011). OXTR polymorphisms are also associated with individual differences
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in nonhuman primates and other mammals (e.g. Kis et al., 2014; Staes et al., 2014). In
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particular, recent studies suggest that the oxytocin system of dogs (Canis familiaris),
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which show human-like communicative skills due to their long history of domestication,
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correlates with their behavior toward humans. For example, dogs’ behavior (e.g.
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proximity seeking) to both strangers and owners in unfamiliar situations were different
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based on their genotype of OXTR (Kis et al., 2014). Dogs taking oxytocin spray showed
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more affiliation behavior to their owners (Romero et al., 2014), and showed positive
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expectation bias (Kis et al., 2015) than dogs with placebo spray.
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By contrast, little research has been done on cats (Felis catus), another companion
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animal with a long history of domestication (Driscoll et al., 2007; Hu et al., 2014). Like 5
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dogs, cats show several human-like cognitive skills, including social referencing (Merola
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et al., 2015), following human pointing (Kraus et al., 2014), and vocal recognition (Saito
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and Shinozuka, 2013). To reveal genetic influences on cat behavior could further reveal
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the effects of domestication on social behavior as a result of genetic change. However, as
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yet it is unknown whether genetic polymorphisms in cats influence their social behavior
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or personality.
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We examined whether OXTR polymorphisms in domestic cats was linked to their
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owner-assessed personality. We conducted a preliminary study to investigate how genetic
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variation in the oxytocin system affects cats’ personality, as assessed by cat owners.
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MATERIAL AND METHODS
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Subjects and DNA collection
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We collected buccal cell samples from 94 cats in Japan (57 males, 37 females;
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mean age = 4 years 6.99 months. SD = 47.90 months) after obtaining permission from
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the owners. Seventy-two cats were neutered (46 males and 26 females). Thirty-eight cats
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belonged to six “cat cafés,” where customers can freely interact with cats, and fifty-six
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cats were pets in their owner’s house. The numbers of cats in each cat café were 13, 6, 6,
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5, 5 and 4, respectively. In Japan, more than 80% of cats are reported as mixed breed
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(mongrel/moggy) by the owners or as of unidentified breed (Japan Pet Food Association,
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2014). Sixty-six cats (70.20%) in the present were described as mixed breed. The samples
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included one mother-offspring pair, one pair of sisters, and a trio of brothers.
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Genetic analysis
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DNA was extracted from buccal cells using QIAamp blood and tissue kit
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(QIAGEN, Valencia, CA, USA). We amplified exon1 region in OXTR by polymerase
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chain reaction (PCR) with 10 μl mixture for each sample, containing LA taq, dNTPs, GC
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buffer I (TaKaRa, Otsu, Shiga, Japan), template DNA, and the forward (OXTR_cat1F:
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5’-GCAGCGCCATTCCTTAAA-3’,
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5’-CATGTTCGCCTCCACCTACT-3’)
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5’-CGTGATCCACGTGATGTAGG
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OXTR_cat2F: (OXTR_cat1R: OXTR_cat2R:
5’-ATTCCAAGGCCCACCTCT-3’) primers. PCR condition consisted of 95 ℃
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preheating for 2 min, 35 cycling of 95℃ for 30s, 60℃ for 30s, 74℃ for 1 min, and 74℃
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for 10 min as last extension. After PCR, we purified for PCR products using High Pure
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PCR Product Purification Kit (Roche Diagnostics K.K., Minato, Tokyo, Japan). After
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checking the purification status by electrophoresing on 1.5% agarose gel, we conducted
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the sequence reaction with the condition of preheating of 96℃ for 1 min, 25 cycling of
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96℃ for 10s, 50℃ for 5s, and 60℃ for 1 min. After that, we conducted ethanol
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precipitation, after which we sequenced the genome using the Applied Biosystems
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3130/3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).
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Rating of cat personality
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We collected questionnaires that was used to identify personality in Japanese Akita
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dogs (Konno et al., 2011). The questionnaire contained 30 questions with 6-point scales
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asking about the personality of the cat, and owners answered it about their cats. We
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performed parallel analysis and factor analysis to reveal the personality structure of cats,
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and used a generalized linear mixed model (GLMM) to examine the effects of the target
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genotype, age, breed, sex x neutering (because neutering effect was predicted to be
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different
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neutered/non-neutered) and environment (cat café or home) on scale-scores on each
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factor. Because 70.20% of the sample was mixed, we included breed as a random factor.
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We used R (ver. 3.0.3) for all statistical analysis (R Development Core Team: R, 2009),
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and also used the libraries such as “psych (Revelle, 2015)”, “GPArotation (Bernaards and
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Jennrich, 2005)”, “lme4 (Bates et al., 2014)”, “car (Fox and Weisberg, 2011)” and
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“genetics (Warnes et al., 2013).”
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sex,
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RESULTS
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Genotyping Among sequenced 1056bp, dog and human sequences were different from those of
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cats in 127bp (12.03%) and 132bp (12.50%), respectively. Among 307 amino acids, dogs
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and humans differed from cats in 19 (6.19%) and 20 (6.51%) amino acids, respectively
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(Figure S1). We found 3 synonymous SNPs (single nucleotide polymorphisms, C474T,
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G723A, and G738A) in exon1. Allele frequencies of the SNP G723A were extremely
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high, and C474T and G738A were strongly linked (Pairwise Linkage Disequilibrium:
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= 59.87, p < 0.001), so we focused on G738A (Table 1). Allele frequencies did not
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overstep in G738A as a result of Hardy-Weinberg equilibrium (Fisher’s exact test; p =
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1.00). The OXTR sequence in cats in our study was deposited in the DDBJ database
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(Accession No. LC029887). We divided genotypes into 2 groups for analysis; A allele
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carriers and non A allele carriers.
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Personality structure of cats
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The number of factors was determined via parallel analysis and the scree plot.
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Factor analysis with maximum likelihood estimation and a varimax rotation detected four
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factors (Table S1), which we named “Openness” (highest positive loadings: playful,
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inquisitive, and curious), “Friendliness” (adaptable, calm, and friendly), “Roughness”
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(irritable, ‘dominant’, forceful, and moody) and “Neuroticism” (vigilant, nervous, and
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fearful). We defined factor loadings ≥|0.5| as salient items. No item had salient loading on
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more than one factor. We calculated scale-score of each factor for each cat. The internal
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consistencies of the four factors assessed by Cronbach’s α were 0.85 for “Neuroticism”,
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0.87 for “Roughness”, 0.92 for “Openness”, and 0.90 for “Friendliness”; hence the
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reliability of the questionnaire was sufficiently high. To evaluate test-retest reliability and
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consistency of individual personality scores across time, we collected a second
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questionnaire randomly from the same owners for almost 25% of the sample (12 males
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and 12 females). The lag between the first and second questionnaire varied from 1 to 19
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months. Pearson correlation coefficients (r) were 0.63 for “Openness”, 0.65 for
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“Friendliness”, 0.70 for “Neuroticism”, and 0.76 for “Roughness”, and were all
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significant at p < 0.01, indicating that personality scores of each cat were stable according
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to Konno et al. (2011).
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Association between the genotype and scale-scores
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A GLMM analysis and type II Wald chi-square tests revealed no significant effect
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for “Friendliness” and “Neuroticism”, but there was an effect of age on “Openness”:
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younger cats showed higher “Openness” scores ( = 0.06, = 15.36, standard error
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= 0.003, 95% Cl=0.99, 1.00, p < 0.001). GLMM showed an effect of OXTR genotype
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( = 0.07, = 5.10, standard error = 0.24, 95% Cl=0.38, 0.97, p = 0.02) and age
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( = 0.06, = 4.89, standard error = 0.002, 95% Cl=1.00, 1.01, p = 0.03) on
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“Roughness”, with older cats and those with the A allele showing higher “Roughness”
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scores. When dividing samples into four groups (neutered male, neutered female, intact
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male and intact female) and conducting GLMM for each group, neutered cats of both
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sexes showed the same tendency in “Roughness” scores, but only neutered females
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showed a statistically significant effect of genotype (male: = 0.58, =
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1.16, standard error = 0.33, 95% Cl = 0.36, 1.34, p = 0.28; female: = 0.40,
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= 8.35, standard error = 0.47, 95% Cl = 0.10, 0.64, p < 0.01; Figure 1). In
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intact cats of both sexes the tendency was reversed, although the sample size was small
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and
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(male: = 0.02, = 0.51, standard error = 0.57, 95% Cl = 0.22, 2.04, p = 0.47;
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female: = 0.58, = 2.35, standard error = 0.72, 95% Cl = 0.08, 1.36, p =
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0.13; Figure 1). To test whether differences between subgroups statistically significant,
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we included sex x genotype, neutering x genotype and sex x neutering x genotype
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interaction terms for “Roughness” score, but there was no significant effect.
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DISCUSSION We found three SNPs in OXTR in cats, and showed that one of these SNPs had an
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effect on owner-assessed “Roughness” scores. To our knowledge, this is the first
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demonstration of a link between genetic polymorphisms and personality variation in cats.
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The present study also gives new information on personality structure in cats. In a
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previous study that included an analysis of domestic cat personality (Gartner et al., 2014),
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three factors emerged “Neuroticism” (highest positive loadings: anxious, insecure, and
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tense), “Impulsiveness” (excitable, active, and playful), “Dominance” (aggressive to
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conspecifics, bullying, and not ‘submissive’). These factors are similar to those that we
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found, but we also found a factor that was not identified in the previous study (Gartner et
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al., 2014). It was possibly because of procedural differences between these two studies
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(e.g. the number of raters, items of the questionnaires, living environment such as home,
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cat cafés, or shelters, and whether cats are pets).
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In recent years, felids other than domestic cats (e.g. Gartner and Powell, 2012;
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Gartner and Weiss, 2013; Gartner et al., 2014) have been being examined from the
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viewpoint of personality. Our finding about OXTR in cats may be an indicator for
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personality evolution of felids and its genetic basis. For example, several studies have
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compared genetic polymorphisms in dopamine D4 receptor gene between dogs and
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wolves (Canis lupus), and reveal that wolves often have polymorphisms which show high
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activity-impulsivity in dogs, suggesting natural selection through domestication (e.g.
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Hejjas et al., 2007; Inoue-Murayama, 2012). Comparing our results to those from wild
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cats and other felids may also reveal whether the mutation in these SNPs took place in
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cats during domestication, as an adaptation to living in close contact with people.
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There were limitations on this study. First, the G738A SNP is a synonymous SNP
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coding the same amino acid, but the role of synonymous SNPs are unknown. Several
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studies reported that synonymous SNPs (e.g. rs2228485, rs237902) have an effect on
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social behavior in humans (Stankova et al., 2012; Lucht et al., 2013). Silent SNPs may
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sometimes change the process of protein folding (Komar, 2007), but the mechanism
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underlying this phenomenon reported in our study is a remaining issue. Second, whether
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the oxytocin system in cats is generally sex-dependent remains unknown although the
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significant differences in “Roughness” depending on genotypes in neutered female cats in
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this study are noteworthy. There is evidence of a significant sex x genotype of OXTR in
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“Harm Avoidance” scores in humans with the effect of the genotype being stringer in
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women than in men (Stankova et al., 2012). No significant interaction among sex,
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neutering and genotype was found in this study, and sex and neutering effect are also
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unclear, so we need to add sample especially to females to sufficiently conduct subgroup
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analysis. Third, we found age effect, but we cannot discuss it because of the small sample
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size and several very old cats (more than 10 years old) in subjects. One possibility for the
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relationship of age and “Roughness” is that a few items (e.g. “moody”) were strongly
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related to old cats. In previous studies showing a link between oxytocin system and dogs’ social
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behavior (Kis et al., 2014; Romero et al., 2014; Kis et al., 2015), the main dependent
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variable was the dogs’ responses toward humans. In the questionnaire used here, owners
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were asked about their cats’ everyday activities. Therefore the object of the cats’
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interactions varied across questions (e.g. stranger, owner’s family, other cats). Future
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researches may wish to conduct a test battery measuring cats’ behavior toward owners
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will be needed to focus on human-cat relationships, to allow comparison of results with
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those reported in dogs.
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CONCLUSION
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This study that cats have four personality factors: “Openness”, “Friendliness”,
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“Roughness”, and “Neuroticism”. This study also demonstrated the relationships
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between genetic polymorphisms and personality traits in cats. G738A, one of three SNPs
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in exon1 of OXTR found in this study, was associated with “Roughness” scores assessed
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by owners. This finding may have implications for animal welfare, for example, in
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predicting compatibility in cat-cat or cat-owner relationships, developing data that shows
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what genetic types of cats would match potential owners.
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Acknowledgements We thank all cats and their owners who participated in our study. We also thank
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Akitsugu Konno (Teikyo University of Science) for the questionnaire, Mana Tsuzuki and
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Ayami Hyuga (Kyoto University) for data collection, and James R. Anderson for English
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correction. This work was supported by the Cooperation Research Program of the
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Wildlife Research Center, Kyoto University. This work was supported by the Japan
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Society for the Promotion of Science (JSPS) Grant-in-Aid for Scientific Research No.
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25118003 to AS, Nos. 25118005 and 25290082 to MI-M, Nos. 25118002 and 25240020
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to KF.
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Authorship statement
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MA designed this study, collected DNA sample and questionnaires, conducted
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genotyping, analyzed data, and drafted the manuscript. AS, HC, ST, YI, and AW
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contributed to data collection. YH and MI-M conducted data analysis, and MI-M and KF
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provided critical discussion regarding the analyses and the manuscript. All authors have
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approved the final article.
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Conflict of Interest We have no competing interests.
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Ethics statement
This study adhered to the ethical guidelines of Kyoto University, and was approved
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by the Animal Experiments Committee of the Graduate School of Letters of Kyoto
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University.
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Associations between the oxytocin receptor gene (OXTR) and “mind-reading” in
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356
polymorphism (OXTR) and trait empathy. J Affect. Disord. 138, 468-472. (doi:
357
10.1016/j.jad.2012.01.009)
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Figure and table caption
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Figure 1. “Roughness” score for each group of sex x neutering x the SNP G738A
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genotype (±s.e.).
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Table 1. Genotype frequencies of 3 SNPs in exon1 of OXTR in cats.
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Table 1.
G723A
G738A
C/C
C/T
0.28 (26)
0.38 (36)
G/G
G/A
0.87 (82)
0.09 (8)
G/G 0.53 (50)
0.34 (32) A/A
0.04 (4)
G/A
A/A
0.40 (38)
0.06 (6)
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T/T
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C474T
Frequency (n)
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Highlights
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Arahori et al.: The oxytocin receptor gene (OXTR) polymorphism in cats (Felis
3
catus) is associated with dominance assessed by owners.
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・Three synonymous SNPs were found in exon1 of oxytocin receptor gene in cats.
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・Questionnaires completed by cat owners showed four factors in cat personality.
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・Cats with A allele in G738A showed higher “Roughness” than non A allele carriers.
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Electronic Supplementary Materials
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The oxytocin receptor gene (OXTR) polymorphism in cats (Felis catus) is associated
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with “Roughness” assessed by owners.
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Minori Arahori1
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Yusuke Hori1, 2
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Atsuko Saito3
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Hitomi Chijiiwa1
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Saho Takagi1
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Yuki Ito3
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Arii Watanabe3
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Miho Inoue-Murayama4, 5
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Kazuo Fujita1
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606-8501, Japan.
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Department of Psychology, Graduate School of Letters, Kyoto University, Sakyo, Kyoto
Japan Society for the Promotion of Science, Chiyoda, Tokyo 102-0083, Japan. 1
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3
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Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro, Tokyo 153-8902, Japan.
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Wildlife Research Center, Kyoto University, Sakyo, Kyoto 606-8203, Japan.
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Wildlife Genome Collaborative Research Group, National Institute for Environmental
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Studies, Onogawa, Tsukuba 305-8506, Japan.
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Department of Cognitive and Behavioral Science, Graduate School of Arts and
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*Address for reprint requests and correspondence: Minori Arahori, Department of
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Psychology, Graduate School of Letters, Kyoto University, Sakyo, Kyoto 606-8501, Japan.
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Tel: +81757532759.
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E-mail address: [email protected] (M Arahori).
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Figure S1. (a)
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Figure S1. (b)
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Figure legend
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Figure S1. (a) Sequence alignment of exon1 region of OXTR of cat described in this study,
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and dog and human. The sequence in cat was deposited in the DDBJ database (Accession
40
No. LC029887). Accession numbers of dog and human are NM_001198659 and
41
NM_000916, respectively. The intron region is shadowed. Locations of primers are
42
surrounded by squares. Square brackets indicate protein coding regions. The numbers
43
above sequence alignment begin from the translation start codon. Dots in dog and human
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sequences mean that the bases are identical to those of cat. Gaps indicate sequence is
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deleted. In this study, three new SNPs were found in cat sequence; C474T, G723A,
46
G738A, indicated by squares and bold letters. The human SNPs related to social behavior
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(Wu et al., 2012; Stankova et al., 2012; Lucht et al., 2013) are indicated by squares and
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bold letters. (b) Deduced amino acid alignment of oxytocin receptor of cat described in
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this study, and dog and human. In human, one amino acid change happens because of the
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SNP rs4686302, indicated by a square.
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Table S1. Factor loadings of items for each of four factors. Factor
Item
“Friendliness”
Playful
0.87
0.15
-0.17
Curious
0.84
0.28
0.01
Inquisitive
0.83
0.11
0.01
Active
0.79
0.23
0.00
Inventive
0.73
0.15
-0.10
Focused
0.60
0.11
-0.26
Mischievous
0.41
-0.03
0.20
0.01
0.21
Adaptable
0.14
0.82
0.03
-0.44
0.89
Friendly
0.17
0.75
-0.17
-0.40
0.78
Calm
0.08
0.74
-0.34
-0.29
0.75
Sociable
0.20
0.70
-0.17
-0.29
0.64
Gentle
0.06
0.57
-0.49
0.05
0.57
Affectionate
0.16
0.35
0.04
-0.03
0.15
-0.11
0.87
0.13
0.80
-0.07
0.75
0.12
0.60
0.13
“Neuroticism”
communalities
-0.12
0.82
-0.17
0.81
-0.26
0.77
-0.11
0.69
0.05
0.57
-0.04
0.44
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Dominant
-0.10
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Irritable
“Roughness”
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“Openness”
-0.15
-0.09
0.73
0.20
0.60
Defiant
0.09
-0.21
0.71
0.09
0.56
-0.22
-0.08
0.67
0.09
0.51
0.21
-0.01
0.54
0.15
0.36
-0.07
-0.03
0.39
-0.12
0.17
-0.02
-0.01
0.29
0.16
0.11
0.28
-0.06
0.29
-0.04
0.17
Aggressive Excitable Distractible Restless Impulsive
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Moody
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Quitting
-0.03
0.01
0.27
-0.11
0.09
Vigilant
-0.23
-0.15
0.14
0.78
0.70
Fearful
-0.11
-0.33
0.12
0.69
0.61
Timid
-0.16
-0.33
0.04
0.67
0.59
Nervous
0.01
-0.15
0.04
0.66
0.46
Cautious
-0.29
-0.35
-0.05
0.55
0.51
Anxious
-0.04
-0.16
0.30
0.44
0.31
Attentive
0.12
0.06
-0.04
0.15
0.04
0.15
0.14
0.11
0.11
Variance explained (%)
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Note: The questionnaire was the same as in a previous study with Japanese Akita dogs
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(Konno et al., 2011). We rounded off to two decimal places of loadings. Salient loadings
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(|>.5|) are in boldface. The right-hand side refers to communalities (h2). The left-hand
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side refers to items.
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References
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Konno, A., Inoue-Murayama, M., Hasegawa, T., 2011. Androgen receptor gene
73
polymorphisms are associated with aggression in Japanese Akita Inu. Biol Lett. 7,
74
658-660. (doi: 10.1098/rsbl.2011.0087)
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Lucht, M.J., Barnow, S., Sonnenfeld, C., Ulrich, I., Grabe, H.J., Schroeder, W., Volzke,
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H., Freyberger, H.J., John, U., Herrmann, F.H., Kroemer, H., Rosskopf, D., 2013.
77
Associations between the oxytocin receptor gene (OXTR) and “mind-reading” in
78
humans-an
79
10.3109/08039488.2012.700731)
exploratory
M AN U
SC
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study.
Nord
J
Psychiat.
67,
15-21.
(doi:
Stankova, T., Eichhammer, P., Langguth, B., Sand, P.G., 2012. Sexually dimorphic effects
81
of oxytocin receptor gene (OXTR) variants on Harm Avoidance. Biol. Sex Differ.
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3, 17. (doi: 10.1186/2042-6410-3-17)
84 85
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Wu, N., Li, Z., Su, Y., 2012. The association between oxytocin receptor gene
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polymorphism (OXTR) and trait empathy. J Affect Disord. 138, 468-472. (doi: 10.1016/j.jad.2012.01.009)
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S1558-7878(15)00139-2
DOI:
10.1016/j.jveb.2015.07.039
Reference:
JVEB 904
To appear in:
Journal of Veterinary Behavior
Received Date: 28 March 2015 Revised Date:
14 June 2015
Accepted Date: 30 July 2015
Please cite this article as: Arahori, M., Hori, Y., Saito, A., Chijiiwa, H., Takagi, S., Ito, Y., Watanabe, A., Inoue-Murayama, M., Fujita, K., The oxytocin receptor gene (OXTR) polymorphism in cats (Felis catus) is associated with “Roughness” assessed by owners, Journal of Veterinary Behavior (2015), doi: 10.1016/j.jveb.2015.07.039. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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The oxytocin receptor gene (OXTR) polymorphism in cats (Felis catus) is associated
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with “Roughness” assessed by owners.
5
Yusuke Hori1, 2
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Atsuko Saito3
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Hitomi Chijiiwa1
8
Saho Takagi1
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Yuki Ito3
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Minori Arahori1
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Arii Watanabe3
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Miho Inoue-Murayama4, 5
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Kazuo Fujita1
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606-8501, Japan.
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Japan Society for the Promotion of Science, Chiyoda, Tokyo 102-0083, Japan.
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Department of Cognitive and Behavioral Science, Graduate School of Arts and Sciences,
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The University of Tokyo, 3-8-1 Komaba, Meguro, Tokyo 153-8902, Japan.
Department of Psychology, Graduate School of Letters, Kyoto University, Sakyo, Kyoto
1
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Wildlife Research Center, Kyoto University, Sakyo, Kyoto 606-8203, Japan.
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Wildlife Genome Collaborative Research Group, National Institute for Environmental
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Studies, Onogawa, Tsukuba 305-8506, Japan.
22
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*Address for reprint requests and correspondence: Minori Arahori, Department of
24
Psychology, Graduate School of Letters, Kyoto University, Sakyo, Kyoto 606-8501,
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Japan.
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Tel: +81757532759.
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E-mail address: [email protected] (M Arahori).
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Abstract:
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Polymorphisms of the oxytocin receptor gene (OXTR) have been hypothesized to
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correlate with social behavior in humans and other mammals. We examined the
33
relationships between owner-assessed personality and OXTR polymorphisms in cats,
34
which have long a history of domestication by humans. We analyzed the exon1 region of
35
OXTR in 94 DNA samples (57 males and 37 females), and found 3 SNPs (single
36
nucleotide polymorphisms). We focused on the G738A polymorphism. Questionnaires
37
containing 30 questions with 6-point scales were completed by the cats’ owners. Factor
38
analysis for the scores identified four factors, which we named “Openness”,
39
“Friendliness”, “Roughness” and “Neuroticism”. A GLMM analysis revealed that cats
40
with the A allele in the SNP G738A showed significantly higher “Roughness” scores than
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cats without the A allele. After dividing cats into four sex x neutering groups, only
42
neutered females showed the significant difference between the A allele carriers and non
43
A allele carriers in “Roughness” scores. Age also affected personality: younger cats
44
showed much higher “Openness” scores whereas older cats showed higher “Roughness”
45
scores. This study shows that genetic variation in cats may be linked to their personality
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traits, a finding that may be useful from the viewpoint of companion animal welfare; for
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example, to match potential good owners and cats using genetic information.
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Key words:
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domestic cats; genetic polymorphism; oxytocin receptor gene; personality
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INTRODUCTION In recent years, researchers have become increasingly interested in oxytocin, the
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peptide hormone, which is believed to be related to various social behavior in humans
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such as pair bonding, nursing for children, and coping with stressors (for review; Carter,
56
2014). Many studies have also focused on oxytocin receptor gene (OXTR)
57
polymorphisms in humans, which underlie individual variation in behavioral tendencies
58
including empathy (e.g. Wu et al., 2012), cooperation (e.g. Haas et al., 2013), psychiatric
59
disorder such as autism (e.g. Lerer et al., 2008), and prosociality (e.g. Israel et al., 2009;
60
Kogan et al., 2011). OXTR polymorphisms are also associated with individual differences
61
in nonhuman primates and other mammals (e.g. Kis et al., 2014; Staes et al., 2014). In
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particular, recent studies suggest that the oxytocin system of dogs (Canis familiaris),
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which show human-like communicative skills due to their long history of domestication,
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correlates with their behavior toward humans. For example, dogs’ behavior (e.g.
65
proximity seeking) to both strangers and owners in unfamiliar situations were different
66
based on their genotype of OXTR (Kis et al., 2014). Dogs taking oxytocin spray showed
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more affiliation behavior to their owners (Romero et al., 2014), and showed positive
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expectation bias (Kis et al., 2015) than dogs with placebo spray.
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By contrast, little research has been done on cats (Felis catus), another companion
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animal with a long history of domestication (Driscoll et al., 2007; Hu et al., 2014). Like 5
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dogs, cats show several human-like cognitive skills, including social referencing (Merola
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et al., 2015), following human pointing (Kraus et al., 2014), and vocal recognition (Saito
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and Shinozuka, 2013). To reveal genetic influences on cat behavior could further reveal
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the effects of domestication on social behavior as a result of genetic change. However, as
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yet it is unknown whether genetic polymorphisms in cats influence their social behavior
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or personality.
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We examined whether OXTR polymorphisms in domestic cats was linked to their
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owner-assessed personality. We conducted a preliminary study to investigate how genetic
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variation in the oxytocin system affects cats’ personality, as assessed by cat owners.
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MATERIAL AND METHODS
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Subjects and DNA collection
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We collected buccal cell samples from 94 cats in Japan (57 males, 37 females;
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mean age = 4 years 6.99 months. SD = 47.90 months) after obtaining permission from
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the owners. Seventy-two cats were neutered (46 males and 26 females). Thirty-eight cats
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belonged to six “cat cafés,” where customers can freely interact with cats, and fifty-six
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cats were pets in their owner’s house. The numbers of cats in each cat café were 13, 6, 6,
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5, 5 and 4, respectively. In Japan, more than 80% of cats are reported as mixed breed
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(mongrel/moggy) by the owners or as of unidentified breed (Japan Pet Food Association,
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2014). Sixty-six cats (70.20%) in the present were described as mixed breed. The samples
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included one mother-offspring pair, one pair of sisters, and a trio of brothers.
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Genetic analysis
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DNA was extracted from buccal cells using QIAamp blood and tissue kit
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(QIAGEN, Valencia, CA, USA). We amplified exon1 region in OXTR by polymerase
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chain reaction (PCR) with 10 μl mixture for each sample, containing LA taq, dNTPs, GC
96
buffer I (TaKaRa, Otsu, Shiga, Japan), template DNA, and the forward (OXTR_cat1F:
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5’-GCAGCGCCATTCCTTAAA-3’,
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5’-CATGTTCGCCTCCACCTACT-3’)
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5’-CGTGATCCACGTGATGTAGG
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5’-ATTCCAAGGCCCACCTCT-3’) primers. PCR condition consisted of 95 ℃
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preheating for 2 min, 35 cycling of 95℃ for 30s, 60℃ for 30s, 74℃ for 1 min, and 74℃
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for 10 min as last extension. After PCR, we purified for PCR products using High Pure
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PCR Product Purification Kit (Roche Diagnostics K.K., Minato, Tokyo, Japan). After
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checking the purification status by electrophoresing on 1.5% agarose gel, we conducted
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the sequence reaction with the condition of preheating of 96℃ for 1 min, 25 cycling of
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96℃ for 10s, 50℃ for 5s, and 60℃ for 1 min. After that, we conducted ethanol
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precipitation, after which we sequenced the genome using the Applied Biosystems
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3130/3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).
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Rating of cat personality
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We collected questionnaires that was used to identify personality in Japanese Akita
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dogs (Konno et al., 2011). The questionnaire contained 30 questions with 6-point scales
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asking about the personality of the cat, and owners answered it about their cats. We
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performed parallel analysis and factor analysis to reveal the personality structure of cats,
114
and used a generalized linear mixed model (GLMM) to examine the effects of the target
115
genotype, age, breed, sex x neutering (because neutering effect was predicted to be
116
different
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neutered/non-neutered) and environment (cat café or home) on scale-scores on each
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factor. Because 70.20% of the sample was mixed, we included breed as a random factor.
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We used R (ver. 3.0.3) for all statistical analysis (R Development Core Team: R, 2009),
120
and also used the libraries such as “psych (Revelle, 2015)”, “GPArotation (Bernaards and
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Jennrich, 2005)”, “lme4 (Bates et al., 2014)”, “car (Fox and Weisberg, 2011)” and
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“genetics (Warnes et al., 2013).”
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sex,
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Genotyping Among sequenced 1056bp, dog and human sequences were different from those of
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cats in 127bp (12.03%) and 132bp (12.50%), respectively. Among 307 amino acids, dogs
128
and humans differed from cats in 19 (6.19%) and 20 (6.51%) amino acids, respectively
129
(Figure S1). We found 3 synonymous SNPs (single nucleotide polymorphisms, C474T,
130
G723A, and G738A) in exon1. Allele frequencies of the SNP G723A were extremely
131
high, and C474T and G738A were strongly linked (Pairwise Linkage Disequilibrium:
132
= 59.87, p < 0.001), so we focused on G738A (Table 1). Allele frequencies did not
133
overstep in G738A as a result of Hardy-Weinberg equilibrium (Fisher’s exact test; p =
134
1.00). The OXTR sequence in cats in our study was deposited in the DDBJ database
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(Accession No. LC029887). We divided genotypes into 2 groups for analysis; A allele
136
carriers and non A allele carriers.
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Personality structure of cats
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The number of factors was determined via parallel analysis and the scree plot.
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Factor analysis with maximum likelihood estimation and a varimax rotation detected four
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factors (Table S1), which we named “Openness” (highest positive loadings: playful,
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inquisitive, and curious), “Friendliness” (adaptable, calm, and friendly), “Roughness”
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(irritable, ‘dominant’, forceful, and moody) and “Neuroticism” (vigilant, nervous, and
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fearful). We defined factor loadings ≥|0.5| as salient items. No item had salient loading on
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more than one factor. We calculated scale-score of each factor for each cat. The internal
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consistencies of the four factors assessed by Cronbach’s α were 0.85 for “Neuroticism”,
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0.87 for “Roughness”, 0.92 for “Openness”, and 0.90 for “Friendliness”; hence the
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reliability of the questionnaire was sufficiently high. To evaluate test-retest reliability and
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consistency of individual personality scores across time, we collected a second
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questionnaire randomly from the same owners for almost 25% of the sample (12 males
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and 12 females). The lag between the first and second questionnaire varied from 1 to 19
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months. Pearson correlation coefficients (r) were 0.63 for “Openness”, 0.65 for
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“Friendliness”, 0.70 for “Neuroticism”, and 0.76 for “Roughness”, and were all
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significant at p < 0.01, indicating that personality scores of each cat were stable according
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to Konno et al. (2011).
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Association between the genotype and scale-scores
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A GLMM analysis and type II Wald chi-square tests revealed no significant effect
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for “Friendliness” and “Neuroticism”, but there was an effect of age on “Openness”:
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younger cats showed higher “Openness” scores ( = 0.06, = 15.36, standard error
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= 0.003, 95% Cl=0.99, 1.00, p < 0.001). GLMM showed an effect of OXTR genotype
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( = 0.07, = 5.10, standard error = 0.24, 95% Cl=0.38, 0.97, p = 0.02) and age
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( = 0.06, = 4.89, standard error = 0.002, 95% Cl=1.00, 1.01, p = 0.03) on
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“Roughness”, with older cats and those with the A allele showing higher “Roughness”
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scores. When dividing samples into four groups (neutered male, neutered female, intact
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male and intact female) and conducting GLMM for each group, neutered cats of both
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sexes showed the same tendency in “Roughness” scores, but only neutered females
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showed a statistically significant effect of genotype (male: = 0.58, =
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1.16, standard error = 0.33, 95% Cl = 0.36, 1.34, p = 0.28; female: = 0.40,
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= 8.35, standard error = 0.47, 95% Cl = 0.10, 0.64, p < 0.01; Figure 1). In
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intact cats of both sexes the tendency was reversed, although the sample size was small
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and
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(male: = 0.02, = 0.51, standard error = 0.57, 95% Cl = 0.22, 2.04, p = 0.47;
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female: = 0.58, = 2.35, standard error = 0.72, 95% Cl = 0.08, 1.36, p =
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0.13; Figure 1). To test whether differences between subgroups statistically significant,
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we included sex x genotype, neutering x genotype and sex x neutering x genotype
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interaction terms for “Roughness” score, but there was no significant effect.
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DISCUSSION We found three SNPs in OXTR in cats, and showed that one of these SNPs had an
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effect on owner-assessed “Roughness” scores. To our knowledge, this is the first
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demonstration of a link between genetic polymorphisms and personality variation in cats.
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The present study also gives new information on personality structure in cats. In a
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previous study that included an analysis of domestic cat personality (Gartner et al., 2014),
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three factors emerged “Neuroticism” (highest positive loadings: anxious, insecure, and
184
tense), “Impulsiveness” (excitable, active, and playful), “Dominance” (aggressive to
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conspecifics, bullying, and not ‘submissive’). These factors are similar to those that we
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found, but we also found a factor that was not identified in the previous study (Gartner et
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al., 2014). It was possibly because of procedural differences between these two studies
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(e.g. the number of raters, items of the questionnaires, living environment such as home,
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cat cafés, or shelters, and whether cats are pets).
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In recent years, felids other than domestic cats (e.g. Gartner and Powell, 2012;
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Gartner and Weiss, 2013; Gartner et al., 2014) have been being examined from the
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viewpoint of personality. Our finding about OXTR in cats may be an indicator for
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personality evolution of felids and its genetic basis. For example, several studies have
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compared genetic polymorphisms in dopamine D4 receptor gene between dogs and
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wolves (Canis lupus), and reveal that wolves often have polymorphisms which show high
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activity-impulsivity in dogs, suggesting natural selection through domestication (e.g.
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Hejjas et al., 2007; Inoue-Murayama, 2012). Comparing our results to those from wild
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cats and other felids may also reveal whether the mutation in these SNPs took place in
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cats during domestication, as an adaptation to living in close contact with people.
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There were limitations on this study. First, the G738A SNP is a synonymous SNP
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coding the same amino acid, but the role of synonymous SNPs are unknown. Several
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studies reported that synonymous SNPs (e.g. rs2228485, rs237902) have an effect on
203
social behavior in humans (Stankova et al., 2012; Lucht et al., 2013). Silent SNPs may
204
sometimes change the process of protein folding (Komar, 2007), but the mechanism
205
underlying this phenomenon reported in our study is a remaining issue. Second, whether
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the oxytocin system in cats is generally sex-dependent remains unknown although the
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significant differences in “Roughness” depending on genotypes in neutered female cats in
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this study are noteworthy. There is evidence of a significant sex x genotype of OXTR in
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“Harm Avoidance” scores in humans with the effect of the genotype being stringer in
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women than in men (Stankova et al., 2012). No significant interaction among sex,
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neutering and genotype was found in this study, and sex and neutering effect are also
212
unclear, so we need to add sample especially to females to sufficiently conduct subgroup
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analysis. Third, we found age effect, but we cannot discuss it because of the small sample
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size and several very old cats (more than 10 years old) in subjects. One possibility for the
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relationship of age and “Roughness” is that a few items (e.g. “moody”) were strongly
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related to old cats. In previous studies showing a link between oxytocin system and dogs’ social
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behavior (Kis et al., 2014; Romero et al., 2014; Kis et al., 2015), the main dependent
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variable was the dogs’ responses toward humans. In the questionnaire used here, owners
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were asked about their cats’ everyday activities. Therefore the object of the cats’
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interactions varied across questions (e.g. stranger, owner’s family, other cats). Future
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researches may wish to conduct a test battery measuring cats’ behavior toward owners
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will be needed to focus on human-cat relationships, to allow comparison of results with
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those reported in dogs.
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CONCLUSION
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This study that cats have four personality factors: “Openness”, “Friendliness”,
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“Roughness”, and “Neuroticism”. This study also demonstrated the relationships
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between genetic polymorphisms and personality traits in cats. G738A, one of three SNPs
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in exon1 of OXTR found in this study, was associated with “Roughness” scores assessed
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by owners. This finding may have implications for animal welfare, for example, in
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predicting compatibility in cat-cat or cat-owner relationships, developing data that shows
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what genetic types of cats would match potential owners.
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Acknowledgements We thank all cats and their owners who participated in our study. We also thank
236
Akitsugu Konno (Teikyo University of Science) for the questionnaire, Mana Tsuzuki and
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Ayami Hyuga (Kyoto University) for data collection, and James R. Anderson for English
238
correction. This work was supported by the Cooperation Research Program of the
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Wildlife Research Center, Kyoto University. This work was supported by the Japan
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Society for the Promotion of Science (JSPS) Grant-in-Aid for Scientific Research No.
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25118003 to AS, Nos. 25118005 and 25290082 to MI-M, Nos. 25118002 and 25240020
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to KF.
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Authorship statement
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MA designed this study, collected DNA sample and questionnaires, conducted
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genotyping, analyzed data, and drafted the manuscript. AS, HC, ST, YI, and AW
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contributed to data collection. YH and MI-M conducted data analysis, and MI-M and KF
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provided critical discussion regarding the analyses and the manuscript. All authors have
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approved the final article.
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Conflict of Interest We have no competing interests.
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Ethics statement
This study adhered to the ethical guidelines of Kyoto University, and was approved
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by the Animal Experiments Committee of the Graduate School of Letters of Kyoto
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University.
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Figure and table caption
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Figure 1. “Roughness” score for each group of sex x neutering x the SNP G738A
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genotype (±s.e.).
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Table 1. Genotype frequencies of 3 SNPs in exon1 of OXTR in cats.
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Table 1.
G723A
G738A
C/C
C/T
0.28 (26)
0.38 (36)
G/G
G/A
0.87 (82)
0.09 (8)
G/G 0.53 (50)
0.34 (32) A/A
0.04 (4)
G/A
A/A
0.40 (38)
0.06 (6)
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T/T
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C474T
Frequency (n)
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Highlights
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Arahori et al.: The oxytocin receptor gene (OXTR) polymorphism in cats (Felis
3
catus) is associated with dominance assessed by owners.
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・Three synonymous SNPs were found in exon1 of oxytocin receptor gene in cats.
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・Questionnaires completed by cat owners showed four factors in cat personality.
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・Cats with A allele in G738A showed higher “Roughness” than non A allele carriers.
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Electronic Supplementary Materials
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The oxytocin receptor gene (OXTR) polymorphism in cats (Felis catus) is associated
4
with “Roughness” assessed by owners.
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Minori Arahori1
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Yusuke Hori1, 2
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Atsuko Saito3
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Hitomi Chijiiwa1
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Saho Takagi1
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Yuki Ito3
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Arii Watanabe3
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Miho Inoue-Murayama4, 5
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Kazuo Fujita1
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1
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606-8501, Japan.
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2
Department of Psychology, Graduate School of Letters, Kyoto University, Sakyo, Kyoto
Japan Society for the Promotion of Science, Chiyoda, Tokyo 102-0083, Japan. 1
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3
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Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro, Tokyo 153-8902, Japan.
21
4
Wildlife Research Center, Kyoto University, Sakyo, Kyoto 606-8203, Japan.
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5
Wildlife Genome Collaborative Research Group, National Institute for Environmental
23
Studies, Onogawa, Tsukuba 305-8506, Japan.
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Department of Cognitive and Behavioral Science, Graduate School of Arts and
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*Address for reprint requests and correspondence: Minori Arahori, Department of
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Psychology, Graduate School of Letters, Kyoto University, Sakyo, Kyoto 606-8501, Japan.
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Tel: +81757532759.
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E-mail address: [email protected] (M Arahori).
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Figure S1. (a)
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Figure S1. (b)
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Figure legend
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Figure S1. (a) Sequence alignment of exon1 region of OXTR of cat described in this study,
39
and dog and human. The sequence in cat was deposited in the DDBJ database (Accession
40
No. LC029887). Accession numbers of dog and human are NM_001198659 and
41
NM_000916, respectively. The intron region is shadowed. Locations of primers are
42
surrounded by squares. Square brackets indicate protein coding regions. The numbers
43
above sequence alignment begin from the translation start codon. Dots in dog and human
44
sequences mean that the bases are identical to those of cat. Gaps indicate sequence is
45
deleted. In this study, three new SNPs were found in cat sequence; C474T, G723A,
46
G738A, indicated by squares and bold letters. The human SNPs related to social behavior
47
(Wu et al., 2012; Stankova et al., 2012; Lucht et al., 2013) are indicated by squares and
48
bold letters. (b) Deduced amino acid alignment of oxytocin receptor of cat described in
49
this study, and dog and human. In human, one amino acid change happens because of the
50
SNP rs4686302, indicated by a square.
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Table S1. Factor loadings of items for each of four factors. Factor
Item
“Friendliness”
Playful
0.87
0.15
-0.17
Curious
0.84
0.28
0.01
Inquisitive
0.83
0.11
0.01
Active
0.79
0.23
0.00
Inventive
0.73
0.15
-0.10
Focused
0.60
0.11
-0.26
Mischievous
0.41
-0.03
0.20
0.01
0.21
Adaptable
0.14
0.82
0.03
-0.44
0.89
Friendly
0.17
0.75
-0.17
-0.40
0.78
Calm
0.08
0.74
-0.34
-0.29
0.75
Sociable
0.20
0.70
-0.17
-0.29
0.64
Gentle
0.06
0.57
-0.49
0.05
0.57
Affectionate
0.16
0.35
0.04
-0.03
0.15
-0.11
0.87
0.13
0.80
-0.07
0.75
0.12
0.60
0.13
“Neuroticism”
communalities
-0.12
0.82
-0.17
0.81
-0.26
0.77
-0.11
0.69
0.05
0.57
-0.04
0.44
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-0.10
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Irritable
“Roughness”
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“Openness”
-0.15
-0.09
0.73
0.20
0.60
Defiant
0.09
-0.21
0.71
0.09
0.56
-0.22
-0.08
0.67
0.09
0.51
0.21
-0.01
0.54
0.15
0.36
-0.07
-0.03
0.39
-0.12
0.17
-0.02
-0.01
0.29
0.16
0.11
0.28
-0.06
0.29
-0.04
0.17
Aggressive Excitable Distractible Restless Impulsive
EP
Moody
AC C
52
Quitting
-0.03
0.01
0.27
-0.11
0.09
Vigilant
-0.23
-0.15
0.14
0.78
0.70
Fearful
-0.11
-0.33
0.12
0.69
0.61
Timid
-0.16
-0.33
0.04
0.67
0.59
Nervous
0.01
-0.15
0.04
0.66
0.46
Cautious
-0.29
-0.35
-0.05
0.55
0.51
Anxious
-0.04
-0.16
0.30
0.44
0.31
Attentive
0.12
0.06
-0.04
0.15
0.04
0.15
0.14
0.11
0.11
Variance explained (%)
6
ACCEPTED MANUSCRIPT
Note: The questionnaire was the same as in a previous study with Japanese Akita dogs
54
(Konno et al., 2011). We rounded off to two decimal places of loadings. Salient loadings
55
(|>.5|) are in boldface. The right-hand side refers to communalities (h2). The left-hand
56
side refers to items.
RI PT
53
SC
57 58
M AN U
59 60
64 65 66 67
EP
63
AC C
62
TE D
61
68 69 70
7
ACCEPTED MANUSCRIPT
References
72
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73
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RI PT
71
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76
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M AN U
SC
75
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84 85
EP
Wu, N., Li, Z., Su, Y., 2012. The association between oxytocin receptor gene
AC C
83
TE D
80
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86
8