Genetic characterization of a captive marmoset (Callithrix jacchus) colony using genotype-by-sequencing.

The marmoset is a fundamental nonhuman primate model for the study of aging, neurobiology, and many other topics. Genetic management of captive marmoset colonies is complicated by frequent chimerism in the blood and other tissues, a lack of tools to enable cost-effective, genome-wide interrogation of variation, and historic mergers and migrations of animals between colonies. We implemented genotype-by-sequencing (GBS) of hair follicle derived DNA (a minimally chimeric DNA source) of 82 marmosets housed at the Southwest National Primate Research Center (SNPRC). Our primary goals were the genetic characterization of our marmoset population for pedigree verification and colony management and to inform the scientific community of the functional genetic makeup of this valuable resource. We used the GBS data to reconstruct the genetic legacy of recent mergers between colonies, to identify genetically related animals whose relationships were previously unknown due to incomplete pedigree information, and to show that animals in the SNPRC colony appear to exhibit low levels of inbreeding. Of the >99,000 single-nucleotide variants (SNVs) that we characterized, >9800 are located within gene regions known to harbor pathogenic variants of clinical significance in humans. Overall, we show the combination of low-resolution (sparse) genotyping using hair follicle DNA is a powerful strategy for the genetic management of captive marmoset colonies and for identifying potential SNVs for the development of biomedical research models.

Timing of the luteal-placental shift is delayed with additional fetuses in litter-bearing callitrichid monkeys, Saguinus oedipus and Callithrix jacchus.

The luteal-placental shift is an important milestone of mammalian pregnancy signifying when endocrine control of pregnancy shifts from the corpus luteum of the ovary to the placenta. The corpus luteum is maintained by chorionic gonadotropin (CG). Upon sufficient placental maturation, CG production wanes, the corpus luteum involutes, and control is shifted to the placenta, one consequence of which is a midgestational rise in glucocorticoid production, especially cortisol and cortisone, by both mother and fetus. Glucocorticoids are involved in initiating parturition, prenatal programming of offspring phenotype, and maturing fetal organs. Limited evidence from human pregnancy suggests that the timing of this shift is delayed in twin pregnancies, but little is known about the timing of the luteal-placental shift in litter-bearing monkeys from the primate family Callitrichidae. Here we provide evidence from cotton-top tamarins (Saguinus oedipus) and common marmosets (Callithrix jacchus) of longer duration of elevated CG associated with multiple infant births compared to single births. Urinary profiles from cotton-top tamarins demonstrate that the decline of the extended elevation of CG precedes the onset of the midpregnancy sustained rise in glucocorticoids; this shift occurs later with an increase from one to two fetuses carried to term. In the common marmoset, the onset of the sustained rise of glucocorticoids in maternal urine is also delayed with an increase in infant number. Total urinary glucocorticoid levels during the last half of gestation increase monthly but do not differ by infant number. The significant delay in the luteal-placental shift suggests a longer period of placental maturation is needed to support a greater number of fetuses.

Current practices in nutrition management and disease incidence of common marmosets (Callithrix jacchus).

BACKGROUND: A survey was developed to characterize disease incidence, common pathology lesions, environmental characteristics, and nutrition programs within captive research marmoset colonies. METHODS: Seventeen research facilities completed the electronic survey. RESULTS: Nutritional management programs varied amongst research institutions housing marmosets; eight primary base diets were reported. The most common clinical syndromes reported were gastrointestinal disease (i.e. inflammatory bowel disease like disease, chronic lymphocytic enteritis, chronic malabsorption, chronic diarrhea), metabolic bone disease or fracture, infectious diarrhea, and oral disease (tooth root abscesses, gingivitis, tooth root resorption). The five most common pathology morphologic diagnoses were colitis, nephropathy/nephritis, enteritis, chronic lymphoplasmacytic enteritis, and cholecystitis. Obesity was more common (average 20% of a reporting institution's population) than thin body condition (average 5%). CONCLUSIONS: Through review of current practices, we aim to inspire development of evidence-based practices to standardize husbandry and nutrition practices for marmoset research colonies.

Maternal weight affects placental DNA methylation of genes involved in metabolic pathways in the common marmoset monkey (Callithrix jacchus).

Accumulating evidence suggests that dysregulation of placental DNA methylation (DNAm) is a mechanism linking maternal weight during pregnancy to metabolic programming outcomes. The common marmoset, Callithrix jaccus, is a platyrrhine primate species that has provided much insight into studies of the primate placenta, maternal condition, and metabolic programming, yet the relationships between maternal weight and placental DNAm are unknown. Here, we report genome-wide DNAm from term marmoset placentas using reduced representation bisulfite sequencing. We identified 74 genes whose DNAm pattern is associated with maternal weight during gestation. These genes are predominantly involved in energy metabolism and homeostasis, including the regulation of glycolytic and lipid metabolic processes pathways.

Marmosets as a translational aging model-Introduction.

The life history of the common marmoset (Callithrix jacchus) points to this species as a premiere nonhuman primate aging model. In order to take advantage of these features, we require an expanded and refined understanding of aging in this species. The papers in this special issue move this field forward substantially by providing exciting new findings about the aging of the common marmoset and the potential this species offers for revealing aging's secrets and improving the lives of aging humans.

Age-related changes in myelin of axons of the corpus callosum and cognitive decline in common marmosets.

Executive control is a higher-level cognitive function that involves a range of different processes that are involved in the planning, coordination, execution, and inhibition of responses. Many of the processes associated with executive control, such as response inhibition and mental flexibility, decline with age. Degeneration of white matter architecture is considered to be the one of the key factors underlying cognitive decline associated with aging. Here we investigated how white matter changes of the corpus callosum were related to cognitive aging in common marmosets (Callithrix jacchus). We hypothesized that reduction in myelin thickness, myelin density, and myelin fraction of axonal fibers in the corpus callosum would be associated with performance on a task of executive function in a small sample of geriatric marmosets (n = 4) and young adult marmosets (n = 2). Our results indicated declines in myelin thickness, density, and myelin fraction with age. Considerable variability was detected on these characteristics of myelin and cognitive performance assessed via the detoured reach task. Age-related changes in myelin in Region II of the corpus callosum were predictive of cognitive performance on the detoured reach task. Thus the detoured reach task appears to also measure aspects of corticostriatal function in addition to prefrontal cortical function.

The metabolome as a biomarker of mortality risk in the common marmoset.

Recently, the common marmoset has been proposed as a non-human primate model of aging. Their short lifespan coupled with pathologies that are similar to humans make them an ideal model to understand the genetic, metabolic, and environmental factors that influence aging and longevity. However, many of the underlying physiological changes that occur with age in the marmoset are unknown. Here, we attempt to determine if individual metabolites are predictive of future death and to recapitulate past metabolomic results after a change in environment (move across the country) was imposed on a colony of marmosets. We first determined that low levels of tryptophan metabolism metabolites were associated with risk of death in a 2-year follow-up in the animals, suggesting these metabolites may be used as future biomarkers of mortality. We also discovered that betaine metabolism and methionine metabolism are associated with aging regardless of environment for the animals, or of metabolomic assay technique. These two metabolic pathways are therefore of particular interest to examine as future targets for health and lifespan extending interventions. Many of the pathways associated with age in our first study of marmoset metabolomics were not found to have significant age effects in our second study, suggesting more work is needed to understand the reproducibility of large scale metabolomic studies in mammalian models. Overall, we were able to show that while several metabolomics markers show promise in understanding health and lifespan relationships with aging, it is possible that choice of technique for assay and reproducibility in these types of studies are still issues that need to be examined further.

Comparative genomics of Bifidobacterium species isolated from marmosets and humans.

The genus Bifidobacterium is purported to have beneficial consequences for human health and is a major component of many gastrointestinal probiotics. Although species of Bifidobacterium are generally at low relative frequency in the adult human gastrointestinal tract, they can constitute high proportions of the gastrointestinal communities of adult marmosets. To identify genes that might be important for the maintenance of Bifidobacterium in adult marmosets, ten strains of Bifidobacterium were isolated from the feces of seven adult marmosets, and their genomes were sequenced. There were six B. reuteri strains, two B. callitrichos strains, one B. myosotis sp. nov. and one B. tissieri sp. nov. among our isolates. Phylogenetic analysis showed that three of the four species we isolated were most closely related to B. bifidum, B. breve and B. longum, which are species found in high abundance in human infants. There were 1357 genes that were shared by at least one strain of B. reuteri, B. callitrichos, B. breve, and B. longum, and 987 genes that were found in all strains of the four species. There were 106 genes found in B. reuteri and B. callitrichos but not in human bifidobacteria, and several of these genes were involved in nutrient uptake. These pathways for nutrient uptake appeared to be specific to Bifidobacterium from New World monkeys. Additionally, the distribution of Bifidobacterium in fecal samples from captive adult marmosets constituted as much as 80% of the gut microbiome, although this was variable between individuals and colonies. We suggest that nutrient transporters may be important for the maintenance of Bifidobacterium during adulthood in marmosets.

Cross-sectional comparison of health-span phenotypes in young versus geriatric marmosets.

The development of the marmoset as a translational model for healthspan and lifespan studies relies on the characterization of health parameters in young and geriatric marmosets. This cross-sectional study examined health phenotypes in marmosets for five domains of interest for human health and aging: mobility, cognition, metabolism, homeostasis, and immune function. Geriatric marmosets were found to have significant executive function impairment when compared to young animals. While geriatric animals did not show gross abnormalities in mobility and measures of locomotion, their types of movement were altered from young animals. Geriatric marmosets had alterations in cardiac function, with significantly increased mean arterial pressures; metabolism, with significantly lower VO2 ; and suppressed immune function. Further, this study sought to characterize and describe histopathology for both young and geriatric healthy marmosets. Overall this study provides a characterization of health parameters for young and geriatric marmosets which will greatly enhance future aging and interventional testing in marmosets.

A comparative study of litter size and sex composition in a large dataset of callitrichine monkeys.

In many birds and mammals, the size and sex composition of litters can have important downstream effects for individual offspring. Primates are model organisms for questions of cooperation and conflict, but the factors shaping interactions among same-age siblings have been less-studied in primates because most species bear single young. However, callitrichines (marmosets, tamarins, and lion tamarins) frequently bear litters of two or more, thereby providing the opportunity to ask whether variation in the size and sex composition of litters affects development, survival, and reproduction. To investigate these questions, we compiled a large dataset of nine species of callitrichines (n = 27,080 individuals; Callithrix geoffroyi, Callithrix jacchus, Cebuella pygmaea, Saguinus imperator, Saguinus oedipus, Leontopithecus chrysomelas, Leontopithecus chrysopygus, Leontopithecus rosalia, and Callimico goeldii) from zoo and laboratory populations spanning 80 years (1938-2018). Through this comparative approach, we found several lines of evidence that litter size and sex composition may impact fitness. Singletons have higher survivorship than litter-born peers and they significantly outperform litter-born individuals on two measures of reproductive performance. Further, for some species, individuals born in a mixed-sex litter outperform isosexually-born individuals (i.e., those born in all-male or all-female litters), suggesting that same-sex competition may limit reproductive performance. We also document several interesting demographic trends. All but one species (C. pygmaea) has a male-biased birth sex ratio with higher survivorship from birth to sexual maturity among females (although this was significant in only two species). Isosexual litters occurred at the expected frequency (with one exception: C. pygmaea), unlike other animals, where isosexual litters are typically overrepresented. Taken together, our results indicate a modest negative effect of same-age sibling competition on reproductive output in captive callitrichines. This study also serves to illustrate the value of zoo and laboratory records for biological inquiry.

The common marmoset monkey: avenues for exploring the prenatal, placental, and postnatal mechanisms in developmental programming of pediatric obesity.

Animal models have been critical in building evidence that the prenatal experience and intrauterine environment are capable of exerting profound and permanent effects on metabolic health through developmental programming of obesity. However, despite physiological and evolutionary similarities, nonhuman primate models are relatively rare. The common marmoset monkey ( Callithrix jacchus) is a New World monkey that has been used as a biomedical model for well more than 50 years and has recently been framed as an appropriate model for exploring early-life impacts on later health and disease. The spontaneous, multifactorial, and early-life development of obesity in the common marmoset make it a valuable research model for advancing our knowledge about the role of the prenatal and placental mechanisms involved in developmental programming of obesity. This paper provides a brief overview of obesity in the common marmoset, followed by a discussion of marmoset reproduction and placental characteristics. We then discuss the occurrence and utility of variable intrauterine environments in developmental programming in marmosets. Evidence of developmental programming of obesity will be given, and finally, we put forward future directions and innovations for including the placenta in developmental programming of obesity in the common marmoset.

Evolutionary genetics and implications of small size and twinning in callitrichine primates.

New World monkeys (NWMs) are characterized by an extensive size range, with clawed NWMs (subfamily Callitrichinae, or callitrichines) such as the common marmoset manifesting diminutive size and unique reproductive adaptations. Perhaps the most notable of these adaptations is their propensity toward multiple gestations (i.e., dichorionic twins and trichorionic triplets). Indeed, with the exception of Goeldi's monkey (Callimico), callitrichine singleton pregnancies rarely occur. Multiple gestations seem to have coevolved with a suite of reproductive adaptations, including hematopoetic chimerism of siblings, suppression of reproduction in nondominant females, and cooperative alloparenting. The sequencing of the common marmoset (Callithrix jacchus) genome offers the opportunity to explore the genetic basis of these unusual traits within this primate lineage. In this study, we hypothesized that genetic changes arising during callitrichine evolution resulted in multiple ovulated ova with each cycle, and that these changes triggered adaptations that minimized complications common to multiple gestations in other primates, including humans. Callitrichine-specific nonsynonymous substitutions were identified in GDF9, BMP15, BMP4, and WFIKKN1. WFIKKN1, a multidomain protease inhibitor that binds growth factors and bone morphogenetic proteins, has nonsynonymous changes found exclusively in common marmosets and other tested callitrichine species that twin. In the one callitrichine species that does not produce twins (Callimico), this change has reverted back to the ancestral (nontwinning) primate sequence. Polymorphisms in GDF9 occur among human cohorts with a propensity for dizygotic twins, and polymorphisms in GDF9 and BMP15 are associated with twinning in sheep. We postulate that positive selection affected NWM growth patterns, with callitrichine miniaturization coevolving with a series of reproductive adaptations.

Protective efficacy of a Zika purified inactivated virus vaccine candidate during pregnancy in marmosets.

Zika virus (ZIKV) infection during pregnancy poses significant threats to maternal and fetal health, leading to intrauterine fetal demise and severe developmental malformations that constitute congenital Zika syndrome (CZS). As such, the development of a safe and effective ZIKV vaccine is a critical public health priority. However, the safety and efficacy of such a vaccine during pregnancy remain uncertain. Historically, the conduct of clinical trials in pregnant women has been challenging. Therefore, clinically relevant animal pregnancy models are in high demand for testing vaccine efficacy. We previously reported that a marmoset pregnancy model of ZIKV infection consistently demonstrated vertical transmission from mother to fetus during pregnancy. Using this marmoset model, we also showed that vertical transmission could be prevented by pre-pregnancy vaccination with Zika purified inactivated virus (ZPIV) vaccine. Here, we further examined the efficacy of ZPIV vaccination during pregnancy. Vaccination during pregnancy elicited virus neutralizing antibody responses that were comparable to those elicited by pre-pregnancy vaccination. Vaccination also reduced placental pathology, viral burden and vertical transmission of ZIKV during pregnancy, without causing adverse effects. These results provide key insights into the safety and efficacy of ZPIV vaccination during pregnancy and demonstrate positive effects of vaccination on the reduction of ZIKV infection, an important advance in preparedness for future ZIKV outbreaks.

Body mass growth in common marmosets: toward a model of pediatric obesity.

While much is known about adult obesity in nonhuman primates, very little is known regarding development of childhood adiposity. As small monkeys that are easy to handle and have a relatively fast life history, common marmoset monkeys (Callithrix jacchus) offer interesting opportunities to examine the question of fat versus lean mass growth in a nonhuman primate. This article provides an overview of our understanding of early life growth in mass in marmoset monkeys, based primarily upon our past 20 years of research, culminating in our recent findings on early life obesity in this species. Common marmosets display variance in early life growth patterns that is related to both pre- and postnatal factors and the marmoset uterine environment is exquisitely designed to reflect resources available for the gestation of multiple offspring, making them an interesting model of developmental programming. We have demonstrated that obesity can be generated in very early life in captive marmosets, with excess adiposity evident by one month of age, making this species a potentially valuable model in which to study pediatric obesity and its sequelae. Birth weight is associated with adiposity in animals vulnerable to obesity. Early life exposure to higher fat diets enhances the chances of postweaning obesity development. However, overall higher food consumption is also associated with obesity development at later ages. One unexpected finding in our studies has been the relatively high body fat percentage of neonatal (12-18%) marmosets suggesting that hypotheses regarding the uniqueness of high human neonatal adiposity merit further examination.

Digestive efficiency mediated by serum calcium predicts bone mineral density in the common marmoset (Callithrix jacchus).

Two health problems have plagued captive common marmoset (Callithrix jacchus) colonies for nearly as long as those colonies have existed: marmoset wasting syndrome and metabolic bone disease. While marmoset wasting syndrome is explicitly linked to nutrient malabsorption, we propose metabolic bone disease is also linked to nutrient malabsorption, although indirectly. If animals experience negative nutrient balance chronically, critical nutrients may be taken from mineral stores such as the skeleton, thus leaving those stores depleted. We indirectly tested this prediction through an initial investigation of digestive efficiency, as measured by apparent energy digestibility, and serum parameters known to play a part in metabolic bone mineral density of captive common marmoset monkeys. In our initial study on 12 clinically healthy animals, we found a wide range of digestive efficiencies, and subjects with lower digestive efficiency had lower serum vitamin D despite having higher food intakes. A second experiment on 23 subjects including several with suspected bone disease was undertaken to measure digestive and serum parameters, with the addition of a measure of bone mineral density by dual-energy X-ray absorptiometry (DEXA). Bone mineral density was positively associated with apparent digestibility of energy, vitamin D, and serum calcium. Further, digestive efficiency was found to predict bone mineral density when mediated by serum calcium. These data indicate that a poor ability to digest and absorb nutrients leads to calcium and vitamin D insufficiency. Vitamin D absorption may be particularly critical for indoor-housed animals, as opposed to animals in a more natural setting, because vitamin D that would otherwise be synthesized via exposure to sunlight must be absorbed from their diet. If malabsorption persists, metabolic bone disease is a possible consequence in common marmosets. These findings support our hypothesis that both wasting syndrome and metabolic bone disease in captive common marmosets are consequences of inefficient nutrient absorption.

Development of metabolic function biomarkers in the common marmoset, Callithrix jacchus.

Metabolic assessment of a non-human primate model of metabolic syndrome and obesity requires the necessary biomarkers specific to the species. While the rhesus monkey has a number of specific assays for assessing metabolic syndrome, the marmoset does not. Furthermore, the common marmoset (Callithrix jacchus) has a small blood volume that necessitates using a single blood volume for multiple analyses. The common marmoset holds a great potential as an alternative primate model for the study of human disease but assay methods need to be developed and validated for the biomarkers of metabolic syndrome. Here we report on the adaptation, development, and validation of commercially available immunoassays for common marmoset samples in small volumes. We have performed biological validations for insulin, adiponectin, leptin, and ghrelin to demonstrate the use of these biomarkers in examining metabolic syndrome and other related diseases in the common marmoset.

Impact of prior dengue virus infection on Zika virus infection during pregnancy in marmosets.

Zika virus (ZIKV) infection during pregnancy causes severe developmental defects in newborns, termed congenital Zika syndrome (CZS). Factors contributing to a surge in ZIKV-associated CZS are poorly understood. One possibility is that ZIKV may exploit the antibody-dependent enhancement of infection mechanism, mediated by cross-reactive antibodies from prior dengue virus (DENV) infection, which may exacerbate ZIKV infection during pregnancy. In this study, we investigated the impact of prior DENV infection or no DENV infection on ZIKV pathogenesis during pregnancy in a total of four female common marmosets with five or six fetuses per group. The results showed that negative-sense viral RNA copies increased in the placental and fetal tissues of DENV-immune dams but not in DENV-naïve dams. In addition, viral proteins were prevalent in endothelial cells, macrophages, and neonatal Fc receptor-expressing cells in the placental trabeculae and in neuronal cells in the brains of fetuses from DENV-immune dams. DENV-immune marmosets maintained high titers of cross-reactive ZIKV-binding antibodies that were poorly neutralizing, raising the possibility that these antibodies might be involved in the exacerbation of ZIKV infection. These findings need to be verified in a larger study, and the mechanism involved in the exacerbation of ZIKV infection in DENV-immune marmosets needs further investigation. However, the results suggest a potential negative impact of preexisting DENV immunity on subsequent ZIKV infection during pregnancy in vivo.

Establishing meal patterns by lickometry in the marmoset monkey (Callithrix jacchus): translational applications from the bench to the field and the clinic.

The ability to measure and interpret variables associated with feeding behavior and food intake is essential to a variety of nonhuman primate study modalities. The development of a technique to accurately and efficiently measure food intake and meal patterning in captivity will enhance both the interpretation of foraging behavior in the wild as well as our ability to model clinically relevant human feeding pathologies. In this study, we successfully developed the use of a rodent lickometer system to monitor meal patterning in captive common marmosets. We describe the modifications necessary for this type of instrumentation to be used successfully with marmosets. We define variables of interest that relate to both previous rodent literature and human clinical measures. Finally, we relate our findings to potential translational value for both primate field research and biomedical applications.

The development of obesity begins at an early age in captive common marmosets (Callithrix jacchus).

Animal models to study the causes and consequences of obesity during infancy in humans would be valuable. In this study, we examine the patterns of fat mass gain from birth to 12 months in common marmosets (Callithrix jacchus). Lean and fat mass was measured by quantitative magnetic resonance at 1, 2, 6, and 12 months for 31 marmosets, 15 considered Normal and 16 considered Fat (> 14% body fat) at 12 months. Animals were fed either the regular colony diet mix or a high-fat variation. Subjects classified as Fat at 12 months already had greater lean mass (198.4 +/- 6.2 g vs. 174.0 +/- 6.8 g, P = 0.013) and fat mass (45.5 +/- 5.0 g vs. 24.9 +/- 3.4 g, P = .002) by 6 months. Body mass did not differ between groups prior to 6 months, however, by 1 month, Fat infants had greater percent body fat. Percent body fat decreased between 1 and 12 months in Normal subjects; in Fat subjects, it increased. The high-fat diet was associated with body fat > 14% at 6 months (P = 0.049), but not at 12 months. This shift was due to three subjects on the normal diet changing from Normal to Fat between 6 and 12 months. Although maternal prepregnancy adiposity did not differ, overall, between Normal and Fat subjects, the subjects Normal at 6 and Fat at 12 months all had Fat mothers. Therefore, diet and maternal obesity appear to have potentially independent effects that may also vary with developmental age. Although birth weight did not differ between groups, it was associated with fat mass gain from 1 to 6 months in animals with > 14% body fat at 6 months of age (r = 0.612, P = 0.026); but not in 6-month-old animals with < 14% body fat (r = -0.012, P = 0.964). Excess adiposity in captive marmosets develops by 1 month. Birth weight is associated with adiposity in animals vulnerable to obesity.

After short interbirth intervals, captive callitrichine monkeys have higher infant mortality.

Life history theory predicts a trade-off between the quantity and quality of offspring. Short interbirth intervals-the time between successive births-may increase the quantity of offspring but harm offspring quality. In contrast, long interbirth intervals may bolster offspring quality while reducing overall reproductive output. Further research is needed to determine whether this relationship holds among primates, which have intensive parental investment. Using Cox proportional hazards models, we examined the effects of interbirth intervals (short, normal, or long) on infant survivorship using a large demographic dataset (n = 15,852) of captive callitrichine monkeys (marmosets, tamarins, and lion tamarins). In seven of the nine species studied, infants born after short interbirth intervals had significantly higher risks of mortality than infants born after longer interbirth intervals. These results suggest that reproduction in callitrichine primates may be limited by physiologic constraints, such that short birth spacing drives higher infant mortality.