Whole-exome sequencing analysis of patent ductus arteriosus in a Japanese macaque (Macaca fuscata).

We performed whole-exome sequencing using a human exome capture kit to analyze the potential genetic factors related to patent ductus arteriosus in Japanese macaques. Compared with the reference sequences of other primates, we identified potential missense variants in five genes: ADAM15, AZGP1, CSPG4, TNFRSF13B, and EPOR.

Play face in Japanese macaques reflects the sender's play motivation.

Animals often initiate social interactions by exchanging signals. Especially when initiating amicable interactions, signaling one's friendly stance toward others in advance may be important to avoid being misunderstood as having hostile intentions. We used data on dyadic play fighting in a group of Japanese macaques, Macaca fuscata, to examine the function of "play face" at the opening of a play session. We found no support for the previously proposed hypothesis that play face expression is likelier before entering risky situations (e.g., before gaining an undue advantage over the partner) to avoid being misunderstood. The results showed that play face expression was likelier in male juveniles before initiating play with other males than in females before initiating play with males or other females and that juveniles were likelier to express play face before initiating play with others closer in age. As male Japanese macaques play more frequently than females, and juveniles prefer to play with individuals closer in age, play face expression before play initiation may reflect the individual's motivation for subsequent play interactions. This interpretation is supported by our observation that play bouts lasted longer when initiated with bidirectional play face by both participants than when initiated without play face. We also argued that since there was no tendency that play face was likelier to be expressed toward individuals with low play propensity (e.g., females) or infrequent partners to play with (e.g., individuals more distant in age), Japanese macaques may not tactically deploy this signal to recruit reluctant partners.

Early neurogenic properties of iPSC-derived neurosphere formation in Japanese macaque monkeys.

Non-human primates are important models for investigations of neural development and evolution, and the use of Japanese macaque monkeys has especially contributed to the advancement of neuroscience studies. However, these studies are restricted by the number of animals able to be evaluated and the invasiveness of the methodologies. Induced pluripotent stem cells (iPSCs) can provide an alternative strategy for investigating neural development in vitro. We have established direct neurosphere (dNS) formation cultures of primate iPSCs as an in vitro model of early neurodevelopment in primate species. Here, we used dNS formation and neuronal differentiation cultures established from Japanese macaque iPSCs (jm-iPSCs) to investigate the usefulness of these cells as an in vitro model of early neural development. Time-course analyses of developmental potency and gene expression kinetics were performed during dNS formation culture of jm-iPSCs. During a 1-week culture, jm-iPSC-derived dNSs became neurogenic by day 3 and underwent stepwise expression changes of key developmental regulators along early neural development in a similar manner to chimpanzee dNS formation previously reported. Meanwhile, a subset of genes, including CYP26A1 and NPTX1, showed differential expression propensity in Japanese macaque, chimpanzee, and human iPSC-derived dNSs. Spontaneous upregulation of NOTCH signaling-associated genes HES5 and DLL1 was also observed in neuronal differentiation cultures of Japanese macaque but not chimpanzee dNSs, possibly reflecting the earlier neurogenic competence in Japanese macaques. The use of jm-iPSCs provides an alternative approach to neurological studies of primate development. Furthermore, jm-iPSCs can be used to investigate species differences in early neural development that are key to primate evolution.

Hip medial rotator action of gluteus medius in Japanese macaque (Macaca fuscata) and implications to adaptive significance for quadrupedal walking in primates.

The gluteus medius (GM) muscle in quadrupedal primates has long been thought to mainly act as a hip extensor. However, previous reports argue that it may be a prime hip medial rotator and functions to rotate the pelvis in the horizontal plane, suggesting the functional differentiation between the GM and other hip extensors as hamstrings. In this study, we aim to quantify the muscle actions of the GM and hamstrings using muscle moment arm lengths and discuss the functional differentiation among hip extensors. Muscle attachment sites of eight specimens of Japanese macaque (Macaca fuscata) were digitized, and musculoskeletal models were constructed. Flexor-extensor, abductor-adductor, and medial-lateral rotator moment arms were calculated as the models were moved following the experimentally acquired kinematic data during walking on a pole substrate. Using electromyography, we also recorded the pattern of muscle activation. The GM showed a larger medial rotator moment arm length than the extensor moment arm length when it was activated, suggesting this muscle acts mainly as a hip medial rotator rather than as a hip extensor. The medial rotator action of the GM in the early support phase may rotate the pelvis in the horizontal plane and function to help contralateral forelimb reaching as a previous study suggested and facilitate contralateral hindlimb swinging to place the foot near the ipsilateral forelimb's hand.

Costs and benefits of living in a vegetated, compared with non-vegetated, enclosure in male Japanese macaques (Macaca fuscata).

Improving captive environments for primates has been an important tool to enhance animal welfare. One method has been to provide primates with naturalistic vegetated enclosures to promote species-specific behaviors, enhancing interactions with their social and natural environment, such as an increase in feeding, foraging, and positive social behaviors such as play. To investigate the benefits in which living in naturalistic environments promote general animal well-being, we observed immature and adult males living in two outdoor housed groups of Japanese macaques (Macaca fuscata) in the Kyoto University Primate Research Institute (KUPRI), Inuyama, Japan, from June of 2015 until June 2016. One enclosure was naturally vegetated and the other was not. We recorded male activity budgets, affiliative (groom, play) behaviors, and rates of agonistic interactions. To examine health status, we recorded and compared coat conditions for both groups. We found that males in the vegetated enclosure spent more time in social play than males in the non-vegetated enclosure, while males in the non-vegetated enclosure displayed more stereotypic behaviors and agonistic interactions. We recorded better coat conditions in the vegetated enclosure males while rates of social grooming or self-grooming were no different between males in the two enclosures. The males in the vegetated enclosure did not have activity budgets more similar to their wild counterparts; but they spent more time in feeding-related activities and less time resting, which was more similar to their wild counterparts than males in the non-vegetated enclosure. Our findings suggest that individuals housed in naturalistic environments have significantly greater behavioral and physical markers of wellbeing than those housed in unnatural, large outdoor enclosures. Although we found that males in both types of enclosures overall had similar time budgets to males in the wild, the detailed behavioral and health results suggest that the welfare benefits to males were greater in the vegetated enclosure, compared with non-vegetated enclosures. Efforts to mimic more natural environments should promote the well-being of primates.

Urothelial carcinoma in the urinary bladder of a Japanese macaque (Macaca fuscata).

Tumors of urinary origin are infrequently reported in non-human primates. Urothelial carcinoma involving the urinary bladder was diagnosed in an adult female Japanese macaque that extended transmurally to the uterus and cervix. To our knowledge, this is the first report of a primary cystic urothelial carcinoma in a Japanese macaque.

Effects of diet and age-sex class on the fecal particle size of wild Japanese macaques (Macaca fuscata yakui).

Fecal particle size provides important information on the feeding and digestion of herbivores. Understanding the effects of the potential proximate determinants on fecal particle size helps us interpret this widely used measurement. In folivores, previous studies found that diet composition, dietary toughness, and age-sex-related factors, such as body size and tooth wear, influenced fecal particle size. However, the role of these factors remains unknown in frugivorous and omnivorous primates. This study aims to clarify how age-sex class and diet influence fecal particle size in omnivorous Japanese macaques in Yakushima. We expected that their variable diet and differences among age-sex classes would cause variations in fecal particle size. We simultaneously documented Japanese macaques' diet, dietary toughness, and fecal particle size in the lowland area of Yakushima in the period from March 2018 to April 2019. Unexpectedly, fecal particle size showed limited differences across months and no difference among age-sex classes. Dietary toughness showed no effects on fecal particle size, while the consumption of fruits showed only a marginally significant negative effect. Our data indicate that the results of chewing were not affected by dietary toughness in our study subjects, while age-sex classes showed no difference in food comminution. This lack of variation might derive from a diet with low dietary toughness. We also found that the physical structure of preferred foods played an important role in fecal particle size variations. These results suggest that food comminution is less variable in frugivorous and omnivorous primates compared to highly specialized species (e.g., geladas). Factors other than what we examined in this study, such as food physical structure and chewing behavior, should also be taken into consideration.

Immunoelectron Microscopic Characterization of Vasopressin-Producing Neurons in the Hypothalamo-Pituitary Axis of Non-Human Primates by Use of Formaldehyde-Fixed Tissues Stored at -25 °C for Several Years.

Translational research often requires the testing of experimental therapies in primates, but research in non-human primates is now stringently controlled by law around the world. Tissues fixed in formaldehyde without glutaraldehyde have been thought to be inappropriate for use in electron microscopic analysis, particularly those of the brain. Here we report the immunoelectron microscopic characterization of arginine vasopressin (AVP)-producing neurons in macaque hypothalamo-pituitary axis tissues fixed by perfusion with 4% formaldehyde and stored at -25 °C for several years (4-6 years). The size difference of dense-cored vesicles between magnocellular and parvocellular AVP neurons was detectable in their cell bodies and perivascular nerve endings located, respectively, in the posterior pituitary and median eminence. Furthermore, glutamate and the vesicular glutamate transporter 2 could be colocalized with AVP in perivascular nerve endings of both the posterior pituitary and the external layer of the median eminence, suggesting that both magnocellular and parvocellular AVP neurons are glutamatergic in primates. Both ultrastructure and immunoreactivity can therefore be sufficiently preserved in macaque brain tissues stored long-term, initially for light microscopy. Taken together, these results suggest that this methodology could be applied to the human post-mortem brain and be very useful in translational research.

Variation in Adult Male-Juvenile Affiliative Behavior in Japanese Macaques (Macaca fuscata).

Adult males of some primate species are known to positively interact with juveniles. In cases where paternal certainty is high, these behaviors have been largely attributed to the paternal investment hypothesis. Males have also been observed to interact with nonkin juveniles, which has often been explained in terms of mating effort. Here, we examined variation in adult male-juvenile affiliation in semi-free ranging Japanese macaques (Macaca fuscata) at the Oregon National Primate Research Center against possible influencing factors such as age, dominance rank, and female affiliation, and we also tested for fitness benefits. We conducted 154 h of focal observations of 14 adult males from June to September 2018. Males differed significantly in their rate of juvenile-directed affiliation, but not in their fitness in terms of number of offspring. There was a significant positive correlation between rank and age in the group, indicating that, in this group, rank does not conform to the classic inverted-U pattern observed elsewhere in this species. Although there was a significant positive correlation between rank and juvenile-directed affiliation, the highest-ranking male had few offspring and exhibited little juvenile-directed affiliation. These results suggest little to no preliminary support for either the paternal investment or mating effort hypotheses as explanations for juvenile-directed affiliation. This study suggests that there are multiple behavioral strategies for older males that may influence reproductive success.

Sex and season predict wounds in zoo-housed Japanese macaques (Macaca fuscata): A multi-institutional study.

Japanese macaque societies are characterized by frequent intragroup conflict both in free-ranging and zoo-housed conditions. In zoos, understanding the factors that contribute to wounding is of interest because this knowledge can aid in proactive husbandry and management planning that can minimize the negative impacts wounding can have on individual welfare. This study sought to determine whether the variables sex, season and age predicted wounding rates. Data were collected for 24 months on 119 Japanese macaques living in 10 zoos, and we analyzed the contribution of variables using generalized linear mixed models. A total of 1,007 wounds were reported, and the best model included the interaction between sex and season. Follow-up analyses revealed that females incurred more wounds than males, and this sex difference was more pronounced during the breeding compared to the nonbreeding season. On average, individuals received 4.67 (±SEM: 0.55) wounds per year and 77.31% of the population incurred at least one wound in the study period. The majority of wounds were superficial and did not require veterinary intervention. Wound locations were not randomly distributed across body regions and were most often reported on the face. Finally, macaques living in larger social groups experienced more wounds, on average. This study represents the first quantification of wounding in this species and may provide insight to help inform husbandry and management strategies in zoos.

Hepatic neuroendocrine carcinoma in a Japanese macaque (Macaca fuscata).

Primary neuroendocrine neoplasm of the liver is extremely rare in both humans and non-human primates. The present report describes the clinical and pathological findings of an aged Japanese macaque (Macaca fuscata) with hepatic neuroendocrine carcinoma. To our knowledge, this is the first report of hepatic neuroendocrine neoplasm in macaques.

Discovery of a CLN7 model of Batten disease in non-human primates.

We have identified a natural Japanese macaque model of the childhood neurodegenerative disorder neuronal ceroid lipofuscinosis, commonly known as Batten Disease, caused by a homozygous frameshift mutation in the CLN7 gene (CLN7-/-). Affected macaques display progressive neurological deficits including visual impairment, tremor, incoordination, ataxia and impaired balance. Imaging, functional and pathological studies revealed that CLN7-/- macaques have reduced retinal thickness and retinal function early in disease, followed by profound cerebral and cerebellar atrophy that progresses over a five to six-year disease course. Histological analyses showed an accumulation of cerebral, cerebellar and cardiac storage material as well as degeneration of neurons, white matter fragmentation and reactive gliosis throughout the brain of affected animals. This novel CLN7-/- macaque model recapitulates key behavioral and neuropathological features of human Batten Disease and provides novel insights into the pathophysiology linked to CLN7 mutations. These animals will be invaluable for evaluating promising therapeutic strategies for this devastating disease.

Hormonal contraceptive affects heterosexual but not homosexual behavior in free-ranging female Japanese macaques over 17 mating seasons.

We assessed the effect of a progestin-based contraceptive treatment (chlormadinone acetate) on female heterosexual and homosexual behaviors in a free-ranging group of Japanese macaques (Macaca fuscata) living at Arashiyama-Kyoto, Central Japan. The data included estimated intensity of fertility cues, sexual solicitations and mounting behaviors collected daily during 17 consecutive mating seasons (1995-2012) from 159 females. Females that were on contraception: (1) exhibited less intense cues of putative fertility and for shorter periods; (2) were solicited by fewer males, and those males that did solicit them did so less often (i.e., lower heterosexual attractivity); (3) solicited fewer males and when they did perform sexual solicitations they did so less often (i.e., lower heterosexual proceptivity); (4) engaged in shorter heterosexual consortships with fewer male partners (i.e., lower heterosexual receptivity), compared with females that were not on contraception. In contrast, contraceptive treatment had no significant effect on the prevalence, occurrence, frequency, or duration of female homosexual behaviors. Even though heterosexual and homosexual behaviors can both be considered sexual in character and under hormonal control, our results suggested they are, to some extent, dissociable. Because females engaging in homosexual interactions showed less intense cues of putative fertility than those engaging in heterosexual interactions, regardless of contraceptive treatment, we argued that the hormonal threshold required for the expression of heterosexual behavior by females was associated with elevated sex hormones levels compared to homosexual behavior. We discussed the hormonal correlates of sexual behavior and partner preferences in Japanese macaques.

LESIONS OF THE FEMALE REPRODUCTIVE TRACT IN JAPANESE MACAQUE ( MACACA FUSCATA) FROM TWO CAPTIVE COLONIES.

Reproductive lesions have been described in various nonhuman primate species, including rhesus macaques ( Macaca mulatta), cynomolgus macaques ( Macaca fascicularis), baboons ( Papio spp.), squirrel monkeys ( Saimiri sciureus), and chimpanzees ( Pan spp.); however, there are few publications describing reproductive disease and pathology in Japanese macaques ( Macaca fuscata). A retrospective evaluation of postmortem reports for two captive M. fuscata populations housed within zoos from 1982 through 2015 was completed, comparing reproductive diseases diagnosed by gross pathology and histopathology. Disease prevalence, organs affected, and median age at death between the two institutions was also compared. Fifteen female captive M. fuscata, ranging in age from 15 to 29 yr were identified with reproductive tract lesions, including endometriosis, endometritis, leiomyoma, leiomyosarcoma, and adenomyosis. No significant differences were identified in disease prevalence, organs affected, and median age of death between the two institutions. Endometriosis was the most common disease process identified and was found in 10 of the 15 cases (66.7%), followed by leiomyoma (4 of 15; 26.7%). In four cases (26.7%), severe endometriosis and secondary hemorrhage was indicated as the cause of death or the primary reason for humane euthanasia. These findings were compared with a separate population of Japanese macaques managed within a research facility in the United States, with a prevalence of endometriosis of 7.6%. This study discusses possible risk factors and potential treatment options for the management of endometriosis in captive M. fuscata.

Quantitative assessment of placental perfusion by contrast-enhanced ultrasound in macaques and human subjects.

BACKGROUND: The uteroplacental vascular supply is a critical determinant of placental function and fetal growth. Current methods for the in vivo assessment of placental blood flow are limited. OBJECTIVE: We demonstrate the feasibility of the use of contrast-enhanced ultrasound imaging to visualize and quantify perfusion kinetics in the intervillous space of the primate placenta. STUDY DESIGN: Pregnant Japanese macaques were studied at mid second trimester and in the early third trimester. Markers of injury were assessed in placenta samples from animals with or without contrast-enhanced ultrasound exposure (n = 6/group). Human subjects were recruited immediately before scheduled first-trimester pregnancy termination. All studies were performed with maternal intravenous infusion of lipid-shelled octofluoropropane microbubbles with image acquisition with a multipulse contrast-specific algorithm with destruction-replenishment analysis of signal intensity for assessment of perfusion. RESULTS: In macaques, the rate of perfusion in the intervillous space was increased with advancing gestation. No evidence of microvascular hemorrhage or acute inflammation was found in placental villous tissue and expression levels of caspase-3, nitrotyrosine and heat shock protein 70 as markers of apoptosis, nitrative, and oxidative stress, respectively, were unchanged by contrast-enhanced ultrasound exposure. In humans, placental perfusion was visualized at 11 weeks gestation, and preliminary data reveal regional differences in intervillous space perfusion within an individual placenta. By electron microscopy, we demonstrate no evidence of ultrastructure damage to the microvilli on the syncytiotrophoblast after first-trimester ultrasound studies. CONCLUSIONS: Use of contrast-enhanced ultrasound did not result in placental structural damage and was able to identify intervillous space perfusion rate differences within a placenta. Contrast-enhanced ultrasound imaging may offer a safe clinical tool for the identification of pregnancies that are at risk for vascular insufficiency; early recognition may facilitate intervention and improved pregnancy outcomes.

Hygienic tendencies correlate with low geohelminth infection in free-ranging macaques.

Parasites are ubiquitous in nature and can be costly to animal fitness, so hosts have evolved behavioural counter-strategies to mitigate infection risk. We investigated feeding-related infection-avoidance strategies in Japanese macaques via field-experimentation and observation. We first examined risk sensitivity during foraging tasks involving faecally contaminated or debris-covered food items, and then investigated individual tendencies to manipulate food items during natural foraging bouts. We concurrently monitored geohelminth infection in all subjects. We ran a principal component analysis on the observational/experimental data to generate a hygienic index across individuals and found that hygienic tendencies towards faeces avoidance and food manipulation correlated negatively with geohelminth infection. Females scored higher in hygienic tendencies than males, which might contribute to the common vertebrate pattern of male-biased infection. The behavioural tendencies observed may reflect a general form of hygiene, providing a mechanism of behavioural immunity against parasites with implications for the evolution and diversification of health maintenance strategies in humans.

Asbestosis in a Japanese Macaque (Macaca fuscata).

Asbestosis is a chronic lung disease caused by inhalation of asbestos, a fibrous mineral. It is one of the most severe diseases resulting from environmental contamination. We found asbestosis in a female Japanese macaque over 25 years of age that died from senility. Clear needle-like crystals were deposited throughout the lung lobes, particularly in the perivascular areas. Asbestos bodies were observed in some of these crystals. Fibrosis without inflammation was observed in the periarterial and peribronchiolar regions. The crystals were identified as tremolite, and a total of 16,633,968 asbestos bodies and 465,334,411 tremolite fibers were observed in 1 g of dry lung tissue. No tumors or pleural adhesions were seen. This is the first report of spontaneous asbestosis in a nonhuman animal.

Characterization of spontaneous malignant lymphomas in Japanese macaques (Macaca fuscata).

Lymphomas are common spontaneous tumors in nonhuman primates but remain poorly characterized in Japanese macaques (Macaca fuscata). This study examined 5 cases of spontaneous malignant lymphoma in Japanese macaques, focusing on the immunophenotypes and presence of simian lymphocryptoviruses, which are Epstein-Barr virus-related herpesviruses in nonhuman primates. The macaques with lymphoma were 5 to 28 years old, indicating that lymphomas develop over a wide age range. The common macroscopic findings were splenomegaly and enlargement of lymph nodes. Histologic and immunohistochemical analyses revealed that all cases were non-Hodgkin type and exhibited a T-cell phenotype, positive for CD3 but negative for CD20 and CD79α. The lymphomas exhibited diverse cellular morphologies and were subdivided into 3 types according to the World Health Organization classification. These included 3 cases of peripheral T-cell lymphoma, not otherwise specified; 1 case of T-cell prolymphocytic leukemia; and 1 case of an unclassifiable T-cell lymphoma. Positive signals were detected by in situ hybridization in 2 of the 4 examined cases using probes for the Epstein-Barr virus-encoded small RNA (EBER). Furthermore, the presence of M. fuscata lymphocryptovirus 2, a macaque homolog of Epstein-Barr virus, was demonstrated in EBER-positive cases by polymerase chain reaction amplification followed by direct sequencing. Immunohistochemistry using antibody to the Epstein-Barr virus-encoded nuclear antigen 2 was negative, even in the EBER-positive cases. The present study suggests that T-cell lymphoma is more common than B-cell lymphoma in Japanese macaques and that M. fuscata lymphocryptovirus 2 is present in some cases.

My studies of primates: Sex, affinity, and competition.

In this essay, I summarize my research career, with reference to the early days of the Laboratory of Physical Anthropology (LPA) at Kyoto University led by Kinji Imanishi and Junichiro Itani. When I started conducting research on the sexual behavior of Japanese macaques in 1975, I made some unexpected observations. High-ranking males did not obtain high mating success. Estrous females often rejected the courtships of high-ranking males and chose to mate with lower-ranking males. Some male-female dyads exhibited long-lasting affinitive relations, but they avoided mating. Females frequently showed 'excessive' sexuality. Clear explanations for some of these observations do not exist. After that, I changed my study subjects several times from chimpanzees, Yakushima macaques (a subspecies of Japanese macaque), and ringtailed lemurs. It is difficult to summarize my findings into a consistent story. Instead, I review my research and experiences. Throughout my career, I kept two things in mind. The first was established by Imanishi at the Laboratory of Physical Anthropology at Kyoto University: to explore the evolution of human society. Second, I tried to understand seemingly incomprehensible phenomena using evolutionary theory. Despite adhering to these foundational concepts, things did not always work out as planned.

Molecular identification of Oesophagostomum and Trichuris eggs isolated from wild Japanese macaques.

Natural habitat fragmentation and reducing habitat quality have resulted in an increased appearance of Japanese macaques, Macaca fuscata (Gray, 1870), in suburban areas in Japan. To investigate the risk of zoonotic infections, a coprological survey of helminth eggs passed by wild Japanese macaques was carried out in 2009 and 2010 in Shiga Prefecture, Japan. Microscopic examination found helminth eggs in high prevalence, and nucleotide sequencing of DNA extracted from the eggs identified Oesophagostomum cf. aculeatum and Trichuris trichiura. A fecal culture also detected infective larvae of Strongyloides fuelleborni. These zoonotic nematodes pose a potential health issue to local people in areas frequented by Japanese macaques.