Behavioral, physiological, and genetic drivers of coping in a non-human primate.

Animals experience stressful situations, from predation to social conflicts, but mostly deal with them successfully. This adaptive mechanism, coping, reduces the adverse effects of stressors, and its failure may result in reduced fitness. Substantial inter-individual variation in coping is observed, yet little is known about how behavioral, physiological and genetic drivers regulate coping holistically and contribute to such variations. We assessed behavioral coping styles (n=30), emotional arousal (n=12), and personalities (n=32) of long-tailed macaques (Macaca fascicularis) and also investigated the association of coping with a valine/methionine polymorphism encoded by a critical human stress regulatory gene, catechol-O-methyltransferase (COMT) (n=26). Personality and the human equivalent COMT Val/Met polymorphism were associated with "nonaggression-based" and "aggression-based" coping styles. Compared to nonaggression-based, aggression-based copers maintained higher average facial temperatures, indicating potentially lower emotional arousal, as measured using infrared thermography. These findings demonstrate a complex interplay of various proximate mechanisms governing coping in a non-human primate.

Intense Body Contact Increases Homosexual Pair Bond Stability in Female Japanese Macaques (Macaca fuscata).

The Japanese macaque (Macaca fuscata) has become a key species for studying homosexual behavior over recent decades. With the non-conceptive nature, their same-sex consortships illustrate that individual partner preferences can exist beyond direct reproductive benefits or apparent sociosexual strategies. An open question is whether the behavior shared between partners in consortship directly affects their choice to remain with a partner. With this study, we examined behavioral aspects underlying consortship temporal patterns in these promiscuous and bisexual primates. While these patterns could be relevant in both homo- and heterosexual consortships, our study primarily focused on female-female pairs. We hypothesized that the stability of consortships (duration and occurrence) is influenced by a pair's sexual behavior, mutual sexual stimulation, and close affiliative inter-mount behaviors involving high-intensity body contact. A semi-free population of Japanese macaques was observed over one mating season. In total, 40 h of focal data on female-female consortship behaviors were analyzed. Forty-six percent of all sexually mature females engaged in homosexual interactions. Our behavioral analyses of female-female pairs found that close body contact, rather than grooming or sexual interactions, was correlated with the stability of homosexual consortships. The greater the amount of huddling and embracing a pair engaged in, the more likely they were to stay together and reunite again. However, the frequency of mounting, rubbing or thrusting had no discernable effect on consortship stability. The results of this study thus add important knowledge to partner qualities in promiscuous primates as well as to inter-group differences of homosexual behavior in Japanese macaques.

Metabolic rates in female guinea pigs during different reproductive stages.

Reproduction in female mammals is characterized by major changes in steroid hormone concentrations, which can be linked to fluctuations in energy expenditure (EE). Estradiol and cortisol can increase EE and metabolic rates (MRs), but knowledge on MR changes during the estrous cycle and gestation is scarce for many species. This also applies to the domestic guinea pig, a species exhibiting an exceptional estrous cycle among rodents. In this study, MRs were measured through oxygen (O2) consumption in female guinea pigs during different reproductive stages. Mean O2 consumption over 2.5 h, resting metabolic rate (RMR, lowest and most stable O2 consumption over 3 min), body mass, fecal estrogen and progesterone, and saliva cortisol concentrations were measured in twelve female guinea pigs in a repeated measurements design during diestrus, estrus, and the second trimester of gestation. In estrus, body mass was significantly lower and estrogen and cortisol concentrations were significantly higher compared to diestrus and gestation. Mean O2 consumption and RMR both were significantly increased in estrus compared to diestrus. Additionally, a positive effect of body mass on MRs detected during diestrus and gestation was not found during estrus. Mean O2 consumption was also higher during gestation compared to diestrus, and a significant increase in cortisol concentrations during the 2.5-h MR measurement was recorded. The results indicate that estrus in guinea pigs is energetically demanding, which probably reflects catabolic effects of estrogens and cortisol that uncoupled MRs from body mass. Knowledge on the energetic requirements associated with different reproductive stages is important for future physiological and behavioral studies on female guinea pigs.

Corrigendum: Identification of extremely GC-rich micro RNAs for RT-qPCR data normalization in human plasma.

[This corrects the article DOI: 10.3389/fgene.2022.1058668.].

A novel methodological approach for group classification during fission of a semi-free-ranging group of Japanese macaques (Macaca fuscata).

The self-initiated split of a social group, known as fission, is a challenge faced by many group-living animals. The study of group fission and the social restructuring process in real time provides insights into the mechanism of this biologically important process. Previous studies on fission in Japanese macaques (Macaca fuscata) assigned individuals to newly reorganized groups mainly using behavioral observations and group attendance records based on periods before or after fission itself. Here, we present a novel framework for group classification during the process of fission that uses quantifiable behavioral variables and statistical analyses. The framework was tested on a group fission process at Affenberg Landskron (Austria), a park that housed around 160 semi-free-ranging Japanese macaques. The behavioral data were collected for 26 days during fission. We analyzed three behavioral developments recurrent in fissions in Japanese macaques, that is, independence of behavior, participation in group movements, and separation of nomadic ranges. These analyses were combined to assign individuals to different groups. Our study resulted in one main group (N = 33), one subgroup (N = 36) and 56 individuals whose group membership was still undefined. The demographic characteristics of these newly formed groups were comparable with those of fissioned groups in wild populations. Furthermore, we found that these newly forming groups showed early social dynamics of fission five months before group level movements, that is: grouping based on spatial proximity and spatial withdrawal of the subgroup to the periphery. These results underline the validity of our novel framework to study social dynamics in Japanese macaques during the process of fission. It represents an important addition to existing methods, and we recommend testing its scope in other primate societies.

Identification of extremely GC-rich micro RNAs for RT-qPCR data normalization in human plasma.

We aimed at extending the repertoire of high-quality miRNA normalizers for reverse transcription-quantitative PCR (RT-qPCR) of human plasma with special emphasis on the extremely guanine-cytosine-rich portion of the miRNome. For high-throughput selection of stable candidates, microarray technology was preferred over small-RNA sequencing (sRNA-seq) since the latter underrepresented miRNAs with a guanine-cytosine (GC) content of at least 75% (p = 0.0002, n = 2). miRNA abundances measured on the microarray were ranked for consistency and uniformity using nine normalization approaches. The eleven most stable sequences included miRNAs of moderate, but also extreme GC content (45%-65%: miR-320d, miR-425-5p, miR-185-5p, miR-486-5p; 80%-95%: miR-1915-3p, miR-3656-5p, miR-3665-5p, miR-3960-5p, miR-4488-5p, miR-4497 and miR-4787-5p). In contrast, the seven extremely GC-rich miRNAs were not found in the two plasma miRNomes screened by sRNA-seq. Stem-loop RT-qPCR was employed for stability verification in 32 plasma samples of healthy male Caucasians (age range: 18-55 years). In general, inter-individual variance of miRNA abundance was low or very low as indicated by coefficient of variation (CV) values of 0.6%-8.2%. miR-3665 and miR-1915-3p outperformed in this analysis (CVs: 0.6 and 2.4%, respectively). The eight most stable sequences included four extremely GC-rich miRNAs (miR-1915-3p, miR-3665, miR-4787-5p and miR-4497). The best-performing duo normalization factor (NF) for the condition of human plasma, miR-320d and miR-4787-5p, also included a GC-extreme miRNA. In summary, the identification of extremely guanine-cytosine-rich plasma normalizers will help to increase accuracy of PCR-based miRNA quantification, thus raise the potential that miRNAs become markers for psychological stress reactions or early and precise diagnosis of clinical phenotypes. The novel miRNAs might also be useful for orthologous contexts considering their conservation in related animal genomes.

Twenty-three-year demographic history of the Affenberg Japanese macaques (Macaca fuscata), a translocated semi-free-ranging group in southern Austria.

Demographic studies on translocated primate groups provide a unique opportunity to study population dynamics, social strategies, and reproductive parameters of a species adapting to new environments. In 1996, 38 Japanese macaques (Macaca fuscata) of the Minoo-H group (Osaka Prefecture, Japan) were translocated to Affenberg Landskron, a four-hectare naturally forested park in southern Austria. By January 2020, the population had increased to 160 individuals, and a total of 223 births were recorded. Births peaked in late April to late May, and the timing was influenced by neither offspring sex nor parity status of the mother. Infant mortality was low (8.97%), mostly involving primiparous females, and the average interbirth intervals were shorter following the death of an infant (1.10 years) than a surviving infant (1.77 years). Females rarely had offspring with the same males repeatedly, and the reproductive success among males declined with increasing years of presence in the group. The main aspects of reproduction, mortality, and mate choice are consistent with published data on natural and provisioned populations in Japan and those translocated to other countries. The life expectancy for females, however, was relatively high (11.72% chance of reaching the age of 20), whereas birth control prevented them from using their lifetime reproductive potential. By January 2020, the number of old individuals (> 18 years; 17.5%) was close to that of juveniles (< 4 years; 22.5%). The specific group composition, along with the inability of males to emigrate out of their natal group, may affect the social dynamics of the population, which merits further attention in future studies.

Flaxseed oil as omega-3 polyunsaturated fatty acid source modulates cortisol concentrations and social dominance in male and female guinea pigs.

Flaxseed oil is an excellent source of the essential omega-3 polyunsaturated fatty acid (PUFA) alpha-linolenic acid (ALA). Omega-3 PUFAs are important neuronal components and can counteract aggressive, depressive, and anxiety-like behavior, reduce glucocorticoid (e.g. cortisol) concentrations under chronic stress but also increase acute glucocorticoid responses. As glucocorticoids per se and glucocorticoid responsiveness can modulate the establishment of dominance hierarchies, we investigated if flaxseed oil high in ALA can promote social dominance through effects on glucocorticoid concentrations. Two male and two female groups of domestic guinea pigs (n = 9 per group) were maintained on a control or a 5% (w/w) flaxseed oil diet for four weeks. Social behaviors, hierarchy indices, locomotion, and saliva cortisol concentrations were determined during basal group housing conditions and stressful social confrontations with unfamiliar individuals of the other groups. Flaxseed groups had increased basal cortisol concentrations and showed no cortisol increase during social confrontations. Cortisol concentrations in control groups significantly increased during social confrontations. Such higher cortisol responses positively affected individual hierarchy indices in control males. However, flaxseed males became dominant irrespective of cortisol concentrations. In females, the opposite was detected, namely a higher dominant status in control compared to flaxseed females. Open-field- and dark-light-tests for anxiety-like behavior revealed no pronounced differences, but flaxseed males showed the highest locomotor activity. Flaxseed oil as an ALA source sex-specifically promoted social dominance irrespective of cortisol concentrations and responses. The underlying neuronal mechanisms remain to be determined, but a sex-specific energetic advantage may have accounted for this effect.

Dietary fatty acids modulate cortisol concentrations and social dominance during social confrontations in adolescent male guinea pigs.

The hypothalamic-pituitary-adrenal (HPA)-axis and related glucocorticoid concentrations regulate physiology and behavior, which can be modulated by nutritional conditions, particularly by the dietary fatty acid composition. Omega-3 polyunsaturated fatty acids (PUFAs) have been shown to promote hypothalamic-pituitary-adrenal (HPA)-axis functions, whereas saturated fatty acids (SFAs) in general produce adverse effects and even increase baseline glucocorticoid concentrations. Glucocorticoids (e.g. cortisol) were further documented to modulate the establishment of dominance relationships, while the involvement of dietary fatty acids remains understudied. This study focused on different effects of PUFAs and SFAs on cortisol concentrations and social dominance in male guinea pigs. Three groups of animals were maintained on diets high in PUFAs (10 % w/w walnut oil), SFAs (10 % w/w coconut fat), or on an untreated control diet starting already prenatally. During adolescence, at an age of 60, 90, and 120 days, each individual's saliva cortisol concentrations and hierarchy index (calculated by initiated and received agonistic behavior) were measured during basal group housing conditions and stressful social confrontations with unfamiliar individuals of the other groups. SFA males showed highest baseline cortisol concentrations, lowest cortisol responses to social confrontations, and became subdominant. PUFA and control males showed significant cortisol responses. However, while control males became dominant during social confrontations, the hierarchy index in PUFA males decreased with age. Individual hierarchy indices during consecutive social confrontations revealed a high consistency. The findings presented here indicate that dietary fatty acids differently affect HPA-axis functions and social dominance but the underlying mechanisms remain to be determined.

Effects of dietary fatty acids on the social life of male Guinea pigs from adolescence to adulthood.

Dietary intake of polyunsaturated fatty acids (PUFAs) or saturated fatty acids (SFAs) differently modulates neurophysiological and behavioral functions in response to altered hypothalamic-pituitary-adrenal (HPA)-axis activity and an individual's development. In this context, an individual's social environment, including social interactions and social hierarchies, is closely related to hormone concentrations and possibly interacts with dietary fatty acid effects. We investigated if dietary supplementation with walnut oil (high in PUFAs) and coconut fat (high in SFAs), compared to a control group, affects body mass gain, cortisol and testosterone concentrations, plasma fatty acids, and social behavior in male domestic guinea pigs from adolescence to adulthood. For analyses of cortisol and testosterone concentrations, social interactions were included as covariates in order to consider effects of social behavior on hormone concentrations. Our results revealed that SFAs increased escalated conflicts like fights and stimulated cortisol and testosterone concentrations, which limited body mass gain and first-year survival. PUFAs did not remarkably affect social behavior and hormone concentrations, but enabled the strongest body mass gain, which probably resulted from an energetic advantage. Neither sociopositive nor agonistic behaviors explained age-specific differences in hormone concentrations between groups. However, a high number of subdominant individuals and lower testosterone concentrations were related to increased cortisol concentrations in adult PUFA males. Our findings demonstrate the importance of dietary fatty acids regarding behavioral and endocrine developmental processes and adaptations to the social environment by modulating HPA-axis function and body homeostasis.

Population-specific, recent positive directional selection suggests adaptation of human male reproductive genes to different environmental conditions.

BACKGROUND: Recent human transcriptomic analyses revealed a very large number of testis-enriched genes, many of which are involved in spermatogenesis. This comprehensive transcriptomic data lead us to the question whether positive selection was a decisive force influencing the evolution and variability of testis-enriched genes in humans. We used two methodological approaches to detect different levels of positive selection, namely episodic positive diversifying selection (i.e., past selection) in the human lineage within primate phylogeny, potentially driven by sperm competition, and recent positive directional selection in contemporary human populations, which would indicate adaptation to different environments. RESULTS: In the human lineage (after correction for multiple testing) we found that only the gene TULP2, for which no functional data are yet available, is subject to episodic positive diversifying selection. Using less stringent statistical criteria (uncorrected p-values), also the gene SPATA16, which has a pivotal role in male fertility and for which episodes of adaptive evolution have been suggested, also displays a putative signal of diversifying selection in the human branch. At the same time, we found evidence for recent positive directional selection acting on several human testis-enriched genes (MORC1, SLC9B1, ROPN1L, DMRT1, PLCZ1, RNF17, FAM71D and WBP2NL) that play important roles in human spermatogenesis and fertilization. Most of these genes are population-specifically under positive selection. CONCLUSION: Episodic diversifying selection, possibly driven by sperm competition, was not an important force driving the evolution of testis-enriched genes in the human lineage. Population-specific, recent positive directional selection suggests an adaptation of male reproductive genes to different environmental conditions. Positive selection acts on eQTLS and sQTLs, indicating selective effects on important gene regulatory functions. In particular, the transcriptional diversity regulated by sQTLs in testis-enriched genes may be important for spermatocytes to respond to environmental and physiological stress.

On the sunny side of (new) life: Effect of sunshine duration on age at first reproduction in Japanese macaques (Macaca fuscata).

To produce offspring early in life is energetically demanding and depends greatly on environmental conditions. In female primates, age at first reproduction (AFR) has been associated with social parameters (e.g., population density and social rank), food availability and meteorological conditions (e.g., photoperiod, rainfall patterns, and temperature). Regarding the latter, less attention has been given to the influence of sunshine. In nonhuman primates, including the northern-most distributed Japanese macaque (Macaca fuscata), sunbathing is an effective thermoregulatory strategy to maintain sufficient energy intake during harsh winter months. Furthermore, the energetic value of sunshine and its role in the synthesis of essential vitamins important for sexual development and overall fertility is well investigated using human and animal models. In the present study, we hypothesized that female's AFR is influenced by the amount of sunshine in a semi-free-ranging, provisioned a group of Japanese macaques. To test this, we gathered data on sunshine duration in the year females theoretically experienced the onset of puberty. This phase of the female life cycle is particularly prone to the effects of environmental conditions. In addition to the investigation of sunshine duration and other meteorological conditions (i.e., rainfall and temperature) we controlled for social parameters (i.e., group size and sex ratio) as potential covariates. We found a clear effect of sunshine duration on female AFR: Females who entered puberty in years with more sunshine reproduced for the first time at significantly younger ages than females who experienced less sunshine during this specific period of their development. Possible mechanisms for how the sunshine influences sexual maturation in Japanese macaques are discussed.

Protein measurements in venous plasma, earlobe capillary plasma and in plasma stored on filter paper.

In this study, levels of inflammatory protein biomarkers in venous plasma, plasma derived from capillary blood from the earlobe, and capillary plasma stored as dried plasma spots (DPS) were compared. Samples from 12 male individuals were assessed with a panel of 92 inflammation-related proteins using multiplex proximity extension assay. Correlations between sample types varied greatly between analytes. A high correlation of ρ > 0.8 was observed between capillary plasma and DPS for 32 analytes. At this level of correlation, 13 analytes correlated between venous and capillary plasma and 5 analytes in the comparison of venous blood with DPS.

Saliva cortisol responses to altered plasma PUFA patterns in guinea pigs.

PUFA modulate hypothalamic-pituitary-adrenal (HPA) axis activity and cortisol concentrations and therefore affect physiological stress responses and the regulation of energy balance in the short- and long-term. Especially dietary intake of n-3 PUFA and a lowered n-6:n-3 ratio are highly encouraged due to beneficial and diminishing effects on basal cortisol secretions. However, the time of such effects to occur and how plasma PUFA patterns affect cortisol concentrations in the short-term was rarely investigated. In order to address this, we supplemented forty male and forty female guinea pigs with diets high in the essential PUFA α-linolenic acid (ALA, 18 : 3n-3) and linoleic acid (LA, 18 : 2n-6) for 20 d. Saliva cortisol concentrations in relation to altering plasma PUFA patterns during this time span were analysed in a repeated measurement design both during basal conditions (individual housing) in 5-d intervals and during stressful social confrontations. We detected very fast plasma PUFA accumulation rates, corresponding to the major dietary PUFA, which resulted in plasma PUFA plateau phases after 10 d. ALA negatively and LA positively affected saliva cortisol concentrations throughout the study. A positive effect of the plasma n-6:n-3 ratio on saliva cortisol concentrations was detected during peak plasma PUFA accumulations and social confrontations, while no effects were detected in relation to plasma PUFA plateau phases. These results suggest that the plasma n-6:n-3 ratio diminishes HPA axis activity during altered physiological conditions only and highlights the importance of altering plasma PUFA patterns for HPA axis functions and the control of energy balance and physiological stress.

Fight or flight? Effects of vaginal oestrus on cortisol, testosterone, and behaviour in guinea pig female-female interaction.

It is accepted that social stress in relation to confrontation and competition can elicit behavioural and hormonal changes in social mammals. These effects have, however, been less frequently studied among female-female interactions. In the present study female-female confrontation experiments were carried out to monitor socio-positive and agonistic behaviour by controlling for the oestrus cycles of 12 individuals. Additionally, plasma cortisol (CORT) and testosterone (T) levels were determined before and after the experiments. During non-oestrus conditions a significant increase in CORT levels from pre- to post confrontation was registered and females spent more time to sit side by side. During vaginal oestrus the confrontation experiments revealed avoiding of a conspecific female by showing increased flight behaviour. However, during that period no changes in CORT levels were found. But, a non-significant increase in T was measured from pre- to post confrontation in both cycle phases, while no differences in the display of aggressive behaviours were found. These findings indicate considerable influences of different oestrus cycle phases on social stress-induced CORT secretion and the modulation of socio-positive and agonistic behaviour in female guinea pigs.

Dietary fatty acids sex-specifically modulate guinea pig postnatal development via cortisol concentrations.

Early ontogenetic periods and postnatal maturation in organisms are sex-specifically sensitive to hypothalamic-pituitary-adrenal (HPA)-axis activities, related glucocorticoid secretions, and their effects on energy balance and homeostasis. Dietary polyunsaturated (PUFAs) and saturated (SFAs) fatty acids potentially play a major role in this context because PUFAs positively affect HPA-axis functions and a shift towards SFAs may impair body homeostasis. Here we show that dietary PUFAs positively affect postnatal body mass gain and diminish negative glucocorticoid-effects on structural growth rates in male guinea pigs. In contrast, SFAs increased glucocorticoid concentrations, which positively affected testes size and testosterone concentrations in males, but limited their body mass gain and first year survival rate. No distinct diet-related effects were detectable on female growth rates. These results highlight the importance of PUFAs in balancing body homeostasis during male's juvenile development, which clearly derived from a sex-specific energetic advantage of dietary PUFA intakes compared to SFAs.

Steroid hormone concentrations and body mass are differently affected by polyunsaturated fatty acids during the oestrous cycle in guinea pigs.

Reproductive functions in female mammals can be significantly affected by the actions of dietary polyunsaturated fatty acids (PUFAs) on steroid hormone secretion rates. Nevertheless, the effects of plasma free PUFAs on the oestrous cycle have seldom been considered. Therefore, in the present study, the diet of domestic guinea pigs was supplemented with high concentrations of different PUFAs and the effects of altered plasma PUFA patterns on steroid hormone concentrations, measured non-invasively, and body mass during oestrus and dioestrus were analysed. The oestrous cycle was characterised by increased oestrogen and cortisol concentrations in oestrus, corroborated by lowest bodyweight, whereas progesterone concentrations were highest in dioestrus. Plasma concentrations of the long-chain PUFAs docosahexaenoic acid (DHA; 22:6 ω3) and arachidonic acid (AA; 20:5 ω6) affected steroid hormone concentrations differently in oestrus and dioestrus. DHA positively affected oestrogen and progesterone concentrations and diminished cortisol concentrations only in oestrus. In contrast, AA negatively affected oestrogen and stimulated cortisol concentrations in oestrus and reduced progesterone concentrations in general. These findings imply selective and opposite contributions of DHA and AA to ovarian functions during different stages of the oestrous cycle, indicating a high biological relevance of plasma free PUFAs in female reproductive function.

MARRIAGE GAP IN CHRISTIANS AND MUSLIMS.

For modern Western societies with a regime of monogamy, it has recently been demonstrated that the socioeconomic status of men is positively associated with being or having been married. This study aims to compare marriage patterns (if a person has been married at least once) for cultures with a tradition of monogamy and polygyny. As no worldwide data on polygyny exist, religion was used as a proxy for monogamy (Christians) vs polygyny (Muslims). The analyses were based on 2000-2011 census data from 39 countries worldwide for 52,339,594 men and women, controlling for sex, sex ratio, age, education, migration within the last 5 years and employment. Overall, a higher proportion of Muslims were married compared with Christians, but the difference in the fraction of married men compared with married women at a certain age (the 'marriage gap') was much more pronounced in Muslims than in Christians, i.e. compared with Christians, a substantially higher proportion of Muslim women than men were married up to the age of approximately 31 years. As expected for a tradition of polygyny, the results indicate that the socioeconomic threshold for entering marriage is higher for Muslim than Christian men, and Muslim women in particular face a negative effect of socioeconomic status on the probability of ever being married. The large 'marriage gap' at a certain age in Muslim societies leads to high numbers of married women and unmarried young men, and may put such polygenic societies under pressure.

Status, Stress and Performance in Track and Field Athletes during the European Games in Baku (Azerbaijan).

This study analyzes the relationship between salivary cortisol and testosterone levels and performance in track and field athletes. In addition, we analyzed the influence of status among athletes (measured based on previous athletic achievement) on hormone levels. Nineteen members of the Austrian track and field team (eleven males, eight females, 25.9 ± 3.9 years of age, 74.9 ± 20.1 kg, and 179.3 ± 10 cm) participated in this study. Data was collected during the European Games in Baku. Athletes delivered saliva samples at various time-points including morning samples and samples directly before and after the competition. Scoring points of the International Association of Athletics Federation were used as an individual measure of relative performance. We found that performance was negatively correlated with rise in testosterone concentrations in the last 24 h prior to the competition. A similar trend was found for cortisol levels, but only when the three least competitive athletes were removed from analysis. Pre-competition cortisol levels were significantly increased compared to measurements 24 h earlier. No effect of status on cortisol or testosterone increase in the same timeframe was found. We conclude that the tournament represented a stressor and that excessive endocrine response was associated with reduced performance.