Integrating behavior, hormones and genes associated with the primate HPA-axis
by Daria Raffaella Gutleb
Date of Examination:2018-12-03
Date of issue:2019-04-17
Advisor:Prof. Dr. Julia Ostner
Referee:Prof. Dr. Julia Ostner
Referee:PD Dr. Christian Roos
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Abstract
English
For zoologists, and especially primatologists, it has been a longstanding aim to decipher the causes of individual variability. Phenotypes associated with the hypothalamic-pituitary-adrenal (HPA)-axis, a central physiological pathway activated in response to stress, have been in the focus of research on wild primates. Scientists working on humans have gathered convincing evidence for a major role of genetics in explaining individual variation in HPA-axis-related phenotypes. Up to 50 % and more of the variation in levels of the end product of the HPA-axis (cortisol), aggression and social behavior can be attributed to the underlying genotype. One non-synonymous substitution in a catecholamine degrader has been particularly well studied with regard to human aggression: Val158Met in the catechol-o-methyltransferase gene (COMT). Causing different activities of the enzyme that is substantially involved in catabolizing dopamine in the prefrontal cortex, the Val158Met polymorphism has been repeatedly associated with aggressive behavior and has been assumed to be unique to humans probably due to species-specific cognitive abilities. Concerning the link between sociality and HPA-axis activity, first evidence from human literature indicates that the attenuating effect of social bonds on HPA-axis activity, a phenomenon called social buffering, is partly moderated by genetic variation. The overall aim of this thesis was to promote and shed more light on the behavioral genetics of the primate HPA-axis under natural conditions. Studies linking genotype data to behavioral patterns in the wild are generally scarce and progress in this field has been hampered by a lack of convenient genetic high-throughput methods applicable to low-quality DNA samples. Further shortcomings concern that studies on non-human primates investigated only one or a few loci instead of screening several HPA-related target regions, broad-scale interspecific contrasts in behavioral patterns instead of actual observed individual behaviors and only one or two individuals of different primate species to conclude the absence of a polymorphism – as in the case of COMT Val158Met. In this thesis, I specifically addressed how genetic variation contributes to a better understanding of the following aspects of the primate HPA-axis: aggression rates (study 2), immunoreactive urinary cortisol levels, risk-taking, i.e. rate of initiating aggression, social bond strength, social bond maintenance and the social buffering effect (study 3). In order to carry out these behavioral genetic association studies, I firstly designed a multi-locus next-generation sequencing panel including 46 target regions in 21 HPA-axis genes applicable to low-quality DNA samples (study 1). I chose wild Assamese macaques (Macaca assamensis) as a study species, in which variation in cortisol levels and aggression as well as causal effects of social bonds have been previously demonstrated under natural selection pressures. In this species, the strength of male bonds predicts coalition formation and recruitment during fights, which in turn predict future social dominance relating to reproductive success. Data for this thesis were collected from adult male and female Assamese macaques living in four habituated groups in Phu Khieo Wildlife Sanctuary, Thailand. The non-invasive data collection covered standardized behavioral observations as well as the collection of fecal samples for DNA analyses and urine samples to quantify immunoreactive cortisol. Specifically, I designed a multi-locus sequencing panel (46 target regions in 21 genes, including COMT Val158Met), genotyped all adults from four study groups (37 males, 39 females), collected focal animal behavioral data (5756 focal hours) and 366 urine samples from 23 males for quantification of immunoreactive cortisol via enzyme immunoassays. The results of study 1 are (i) the compilation of a list of presumably functional target regions in genes that are involved in the HPA-axis, (ii) the design of a high-throughput genotyping panel useful when investigating the factors contributing to HPA-axis-related phenotypes, (iii) the demonstration that the panel is applicable to low-quality DNA samples such as feces, which is often the only available sample material from wild animal populations, and (iv) the demonstration that polymorphisms at purportedly functional HPA-axis loci exist in a natural primate population. By targeting 46 target regions in 21 genes 159 single nucleotide polymorphisms were detected. Applying the genotyping panel designed in study 1, I identified the COMT Val158Met polymorphism and associated it with aggression rates of male and female macaques in study 2. The results are that (i) the widely studied human COMT Val158Met polymorphism occurs in a non-human primate species with similar genotype frequencies (14 Met/Met, 40 Val/Met, 22 Val/Val), (ii) macaques’ aggression rates increased with dominance rank in Val/Val individuals, but decreased in individuals carrying other genotypes, and (iii) when changing from a lower to a higher dominance rank position, Val/Val individuals decreased, whereas Met/Met individuals increased their aggression rate. In study 3, I focused on 15 non-synonymous polymorphisms detected among the 159 single nucleotide polymorphisms and calculated a genetic risk score as the proportion of missense variants an individual carries. In the investigated males an increasing genetic risk score was associated with (i) increasing levels of immunoreactive urinary cortisol, (ii) decreasing risk-taking behavior, (iii) a decreasing social buffering effect, (iv) a trend to have stronger social bonds, and (v) an increasing maintenance of close proximity with strong partners. These results contribute to our understanding of phenotypic consequences of individual variation in HPA-axis genotypes. Study 1 served as the basic methodology necessary to conduct study 2 and 3 and shall motivate field biologists to include multi-locus genotype data in future studies on wild, non-model populations more frequently. As COMT Val158Met is not unique to humans and yields similar behavioral phenotypes in another primate species, follow-up studies on this polymorphism can be conducted in several species to investigate the distribution of this polymorphism among taxa and help to decipher its evolutionary roots and contribution to individual variation. The investigation of the social buffering hypothesis revealed a significant interaction effect between social bond strength and genetic risk on HPA-axis activity. The association between strong social bonds and low cortisol levels seemed to diminish when moving from low to high genetic risk, indicating that social buffering is in effect in individuals at the lower, but not the higher end of genetic risk. These results depict that future studies considering genotype as a mediator of social buffering in a wide range of animal taxa are essential. However, as the study included potential relatives, further analyses including relatedness data are necessary to decipher whether the associations between HPA-axis genotypes and phenotypes remain in effect when controlling for kinship. In conclusion, I have performed the first comprehensive analysis of behavioral genetics associated with the HPA-axis in Assamese macaques. Combining ethological and molecular methods, my thesis suggests that genetics is a significant source of variability in a range of primate HPA-axis phenotypes. Thereby I was able to improve our knowledge of factors contributing to individual variation in HPA-axis activity, aggression rates, risk-taking, social bonding behavior and social buffering. Variation in the genetic constitution of macaques may allow individuals to adapt differently to social situations and stressors. Over evolutionary times the balance of different traits may preserve polymorphisms for different phenotypes in a population. This thesis aids the advancement of multi-locus methods and the appreciation of wild animal populations in behavioral genetics. It broadens the spectrum of behavioral ecology and primatology in particular by addressing how inherent factors contribute to individual patterns and social mechanisms of animals in the wild. The fact that polymorphisms in HPA-axis genes cumulatively explain individual variation should have strong implications for primatological studies which in large parts neglected the genetic contribution to the investigated phenotypes. Primates can serve as valuable animal models which help to shed light on some of the ambiguous findings from human behavioral genetics by providing the chance to investigate naturalistic phenotypes using comparable measures. The incorporation of genotype in field primatology contributes to the current discussions about ambiguous findings in classical primatology and might have the potential to resolve some of them in the future.
Keywords: stress; aggression; HPA-axis; next-generation sequencing; behavioral genetics; single nucleotide polymorphism; catechol-O-methyltransferase; COMT Val158Met; dominance hierarchy; gene-environment; Macaca assamensis; macaque; non-human primate; rs4680; social rank; Val157Met; social buffering