The growth factor EPIREGULIN promotes basal progenitor cell proliferation in the developing neocortex.

Neocortex expansion during evolution is linked to higher numbers of neurons, which are thought to result from increased proliferative capacity and neurogenic potential of basal progenitor cells during development. Here, we show that EREG, encoding the growth factor EPIREGULIN, is expressed in the human developing neocortex and in gorilla cerebral organoids, but not in the mouse neocortex. Addition of EPIREGULIN to the mouse neocortex increases proliferation of basal progenitor cells, whereas EREG ablation in human cortical organoids reduces proliferation in the subventricular zone. Treatment of cortical organoids with EPIREGULIN promotes a further increase in proliferation of gorilla but not of human basal progenitor cells. EPIREGULIN competes with the epidermal growth factor (EGF) to promote proliferation, and inhibition of the EGF receptor abrogates the EPIREGULIN-mediated increase in basal progenitor cells. Finally, we identify putative cis-regulatory elements that may contribute to the observed inter-species differences in EREG expression. Our findings suggest that species-specific regulation of EPIREGULIN expression may contribute to the increased neocortex size of primates by providing a tunable pro-proliferative signal to basal progenitor cells in the subventricular zone.

Traces of dietary patterns in saliva of hominids: Profiling salivary amino acid fingerprints in great apes and humans.

Herbivorous animals that regularly consume tannin-rich food are known to secrete certain tannin-binding salivary proteins (TBSPs), especially proline-rich proteins and histidine-rich proteins, as an effective measure to counteract the antinutritive effects of dietary tannins. Due to their high binding capacity, TBSPs complex with tannins in the oral cavity, and thereby protect dietary proteins and digestive enzymes. Although the natural diet of great apes (Hominidae) is biased toward ripe fruits, analyses of food plants revealed that their natural diet contains considerable amounts of tannins, which is raising the question of possible counter-measures to cope with dietary tannins. In our study, we investigated the salivary amino acid profiles of zoo-housed Pan paniscus, Pan troglodytes, Gorilla gorilla, and Pongo abelii, and compared their results with corresponding data from Homo sapiens. Individual saliva samples of 42 apes and 17 humans were collected and quantitated by amino acid analysis, using cation-exchange chromatography with postcolumn derivatization, following acid hydrolysis. We found species-specific differences in the salivary amino acid profiles with average total salivary protein concentration ranging from 308.8 mg/dL in Po. abelii to 1165.6 mg/dL in G. gorilla. Total salivary protein was consistently higher in ape than in human saliva samples (174 mg/dL). All apes had on average also higher relative proline levels than humans did. Histidine levels had the highest concentration in the samples from Po. abelii followed by P. paniscus. In all ape species, the high salivary concentrations of proline and histidine are considered to be indicative of high concentrations of TBSPs in hominids. Given that the species differences in salivary composition obtained in this study correspond with overall patterns of secondary compound content in the diet of wild populations, we assume that salivary composition is resilient to acute and long-lasting changes in diet composition in general and tannin content in particular.

Human and Nonhuman Primate Lineage-Specific Footprints in the Salivary Proteome.

Proteins in saliva are needed for preprocessing food in the mouth, maintenance of tooth mineralization, and protection from microbial pathogens. Novel insights into human lineage-specific functions of salivary proteins and clues to their involvement in human disease can be gained through evolutionary studies, as recently shown for salivary amylase AMY1 and salivary agglutinin DMBT1/gp340. However, the entirety of proteins in saliva, the salivary proteome, has not yet been investigated from an evolutionary perspective. Here, we compared the proteomes of human saliva and the saliva of our closest extant evolutionary relatives, chimpanzees and gorillas, using macaques as an outgroup, with the aim to uncover features in saliva protein composition that are unique to each species. We found that humans produce a waterier saliva, containing less than half total protein than great apes and Old World monkeys. For all major salivary proteins in humans, we could identify counterparts in chimpanzee and gorilla saliva. However, we discovered unique protein profiles in saliva of humans that were distinct from those of nonhuman primates. These findings open up the possibility that dietary differences and pathogenic pressures may have shaped a distinct salivary proteome in the human lineage.

Quadratic relationships between group size and foraging efficiency in a herbivorous primate.

The effect of feeding competition on foraging efficiency is an important link between ecological factors and the social organization of gregarious species. We examined the effects of group size on daily travel distances, activity budgets, and energy intake of mountain gorillas in Rwanda. We measured daily travel distances of five groups, activity budgets of 79 gorillas in nine groups, and energy intake data for 23 adult females in three groups over a 16-month period. Travel distances and the proportion of time spent traveling increased with size for most groups, which would be expected if their foraging efficiency is limited by intragroup feeding competition. However, travel distances and times decreased for the largest group, which also had higher energy intake rates than intermediate sized groups. The improved foraging efficiency of the largest group may be explained by advantages in intergroup contest competition. The largest group had much lower home range overlap than the other study groups which may be due to groups avoiding one another as a result of male mating competition. Collectively, our results indicate that intermediate sized groups had the lowest foraging efficiency and provide a new twist on the growing evidence of non-linear relationships between group size and foraging efficiency in primates.

Metabolic acceleration and the evolution of human brain size and life history.

Humans are distinguished from the other living apes in having larger brains and an unusual life history that combines high reproductive output with slow childhood growth and exceptional longevity. This suite of derived traits suggests major changes in energy expenditure and allocation in the human lineage, but direct measures of human and ape metabolism are needed to compare evolved energy strategies among hominoids. Here we used doubly labelled water measurements of total energy expenditure (TEE; kcal day(-1)) in humans, chimpanzees, bonobos, gorillas and orangutans to test the hypothesis that the human lineage has experienced an acceleration in metabolic rate, providing energy for larger brains and faster reproduction without sacrificing maintenance and longevity. In multivariate regressions including body size and physical activity, human TEE exceeded that of chimpanzees and bonobos, gorillas and orangutans by approximately 400, 635 and 820 kcal day(-1), respectively, readily accommodating the cost of humans' greater brain size and reproductive output. Much of the increase in TEE is attributable to humans' greater basal metabolic rate (kcal day(-1)), indicating increased organ metabolic activity. Humans also had the greatest body fat percentage. An increased metabolic rate, along with changes in energy allocation, was crucial in the evolution of human brain size and life history.

Temporal variation selects for diet-microbe co-metabolic traits in the gut of Gorilla spp.

Although the critical role that our gastrointestinal microbes play in host physiology is now well established, we know little about the factors that influenced the evolution of primate gut microbiomes. To further understand current gut microbiome configurations and diet-microbe co-metabolic fingerprints in primates, from an evolutionary perspective, we characterized fecal bacterial communities and metabolomic profiles in 228 fecal samples of lowland and mountain gorillas (G. g. gorilla and G. b. beringei, respectively), our closest evolutionary relatives after chimpanzees. Our results demonstrate that the gut microbiomes and metabolomes of these two species exhibit significantly different patterns. This is supported by increased abundance of metabolites and bacterial taxa associated with fiber metabolism in mountain gorillas, and enrichment of markers associated with simple sugar, lipid and sterol turnover in the lowland species. However, longitudinal sampling shows that both species' microbiomes and metabolomes converge when hosts face similar dietary constraints, associated with low fruit availability in their habitats. By showing differences and convergence of diet-microbe co-metabolic fingerprints in two geographically isolated primate species, under specific dietary stimuli, we suggest that dietary constraints triggered during their adaptive radiation were potential factors behind the species-specific microbiome patterns observed in primates today.

Socioecological correlates of energy balance using urinary C-peptide measurements in wild female mountain gorillas.

Maintaining a balanced energy budget is important for survival and reproduction, but measuring energy balance in wild animals has been fraught with difficulties. Female mountain gorillas are interesting subjects to examine environmental correlates of energy balance because their diet is primarily herbaceous vegetation, their food supply shows little seasonal variation and is abundant, yet they live in cooler, high-altitude habitats that may bring about energetic challenges. Social and reproductive parameters may also influence energy balance. Urinary C-peptide (UCP) has emerged as a valuable non-invasive biomarker of energy balance in primates. Here we use this method to investigate factors influencing energy balance in mountain gorillas of the Virunga Volcanoes, Rwanda. We examined a range of socioecological variables on energy balance in adult females in three groups monitored by the Karisoke Research Center over nine months. Three variables had significant effects on UCP levels: habitat (highest levels in the bamboo zone), season (highest levels in November during peak of the bamboo shoot availability) and day time (gradually increasing from early morning to early afternoon). There was no significant effect of reproductive state and dominance rank. Our study indicates that even in species that inhabit an area with a seemingly steady food supply, ecological variability can have pronounced effects on female energy balance.

Detecting intraannual dietary variability in wild mountain gorillas by stable isotope analysis of feces.

We use stable isotope ratios in feces of wild mountain gorillas (Gorilla beringei) to test the hypothesis that diet shifts within a single year, as measured by dry mass intake, can be recovered. Isotopic separation of staple foods indicates that intraannual changes in the isotopic composition of feces reflect shifts in diet. Fruits are isotopically distinct compared with other staple foods, and peaks in fecal δ(13)C values are interpreted as periods of increased fruit feeding. Bayesian mixing model results demonstrate that, although the timing of these diet shifts match observational data, the modeled increase in proportional fruit feeding does not capture the full shift. Variation in the isotopic and nutritional composition of gorilla foods is largely independent, highlighting the difficulty for estimating nutritional intake with stable isotopes. Our results demonstrate the potential value of fecal sampling for quantifying short-term, intraindividual dietary variability in primates and other animals with high temporal resolution even when the diet is composed of C(3) plants.

Relationship between behavior, adrenal activity, and environment in zoo-housed western lowland gorillas (Gorilla gorilla gorilla).

Monitoring adrenal activity through noninvasive fecal hormone sampling is rapidly gaining popularity as a tool to assess zoo animal welfare. However, few studies have sought to investigate the interrelationships between behavior, adrenal activity, and environment, and ask whether both behavioral and adrenal monitoring strategies are required to assess welfare sufficiently. We present the findings of a 9-month study of a small group (one male, two females) of Western lowland gorillas, Gorilla gorilla gorilla. First, we examined the effect of environmental variables on gorilla behavior. Second, we examined the effect of environmental variables on the concentration of fecal glucocorticoid metabolites (FGC) and the relationship between behavior and FGC. Environmental variables had similar effects on all three gorillas. Negative vigilance of visitors (NVV; staring, posturing, and charging at visitors) significantly increased in all subjects as environmental noise levels increased, and food-related behavior significantly decreased in all subjects as crowd size increased. Exhibit modifications had a number of positive effects on behavior. Notably, when privacy screens were used, NVV significantly decreased in two subjects. We found no significant effects of environmental variables on FGC. However, we did find significant relationships between behavior and FGC in one female. Specifically, her NVV was significantly higher one day before, and on the same day as, raised FGC. Also, hair plucking significantly increased in the two days following raised FGC. Overall, this study demonstrates how concurrent noninvasive fecal and behavioral monitoring can be used for gorilla welfare assessment.

Evolution of metamorphism in thymidylate synthases within the primate lineages.

Crystal structures of human thymidylate synthase (hTS) revealed that the protein exists in active and inactive conformations, defined by the position of a loop containing the active site nucleophile. TS is highly homologous among diverse species; however, the residue at position 163 (hTS) differs among species. Arginine at this position is predicted by structural modeling to enable conformational switching. Arginine or lysine is reported at this position in all mammals in the GenBank and Ensembl databases, with arginine reported in only primates. Sequence analysis of the TS gene of representative primates revealed that arginine occurs at this relative position in all primates except a representative of prosimians. Mutant human proteins were created with residues at position 163 that occur in TSs from prokaryotes and eukaryotes. Catalytic constants (k(cat)) of mutant enzymes were 45-149% of hTS, with the lysine mutant (R163K) exhibiting the highest k(cat). The effect of lysine substitution on solution structure and on ligand binding was investigated. R163K exhibited higher intrinsic fluorescence, a more negative molar ellipticity, and higher dissociation constants (K(d)) for ligands that modulate protein conformation than hTS. Temperature effects on intrinsic fluorescence and catalytic activity of hTS and R163K are consistent with proteins populating different conformational states. The data indicate that the enzyme with arginine at the position corresponding to 163 (hTS) evolved after the divergence of prosimians and simians and that substitution of lysine by arginine confers unique structural and functional properties to the enzyme expressed in simian primates.

Perimenopause and menopause: documenting life changes in aging female gorillas.

As our closest living relatives, great apes likely experience physiological patterns associated with reproductive aging that are similar to humans. We present results from a nationwide zoo-based study on female western lowland gorillas during which we evaluated concentrations of progestogens via daily fecal sampling in 30 gorillas, 22 of whom were geriatric (>or=30). Whereas control females cycled regularly, ca. 23% of geriatric females were acyclic (menopausal), and approximately 1/3 showed variable hormonal patterns suggestive of perimenopause. Patterns included increased cycle variability, low luteal phase rises of progestogens - possibly indicative of anovulatory cycling - and peak height variability of progestogens in the luteal phase of the cycle. We discovered a progressive trend toward increased variability in estrous cycle length and toward decreased concentrations of fecal progestogens when we compared control to geriatric cycling and to geriatric noncycling females. Noncycling females had significantly lower overall progestogen concentrations than the cycling females, though differences were not significant when cycle phase was incorporated. Preliminary analyses of follow-up data on 10 perimenopausal females indicated that subjects experienced age-related changes in reproductive function that mirrored those observed in aging human females including a female who transitioned from perimenopause to menopause. To date, maximum longevity in captive female gorillas is 52 years, with poor reproductive prognosis beginning from the age of 37 suggesting a postreproductive lifespan of >25%. Continued study of aging apes is warranted, with emphasis on longitudinal monitoring of aged subjects.

New insights in insect prey choice by chimpanzees and gorillas in southeast Cameroon: the role of nutritional value.

The insect diet of chimpanzees and gorillas living at the northern periphery of the Dja Biosphere Reserve in southeast Cameroon and its nutritional contribution is described. We analyzed fecal samples and recorded additional evidence of insectivory. A detailed prey species list is presented for both apes. We carried out nutritional analyses (macronutrients, macro- and micro-minerals) on 11 important and eight nonimportant, but accessible, ant and termite prey species, and estimated the average nutrient intake/day through insects. Although gorillas ate insects more frequently, the average prey biomass intake/day by chimpanzees was twice that by gorillas. The lack of tool-use by gorillas cannot be the main reason for the small overlap of important prey species. Both apes did not seem to consume ant prey for one or more specific nutrients. Also other factors, such as medicinal use, should be considered. Termites, on the other hand, seemed to be selected for particular nutrients. Gorilla intake of the important termite prey, Cubitermes and Thoracotermes, met with estimated iron requirements. Their potential role as antidiarrheal treatment is as yet unclear. Chimpanzee intake of the important termite prey, Macrotermes spp., met with estimated manganese requirements and the protein intake/day (mean: 2 g/d) reached significant values (>20 g/d). To fully understand the importance of nutritional contributions of insects to ape diets in Cameroon, the chemical composition and nutrient intake of fruit and foliage in their diets should be investigated.

New insights into the evolution of chromosome 1.

A complex low-repetitive human DNA probe (BAC RP11-35B4) together with two microdissection-derived region-specific probes of the multicolor banding (MCB) probe-set for chromosome 1 were used to re-analyze the evolution of human chromosome 1 in comparison to four ape species. BAC RP11-35B4 derives from 1q21 and contains 143 kb of non-repetitive DNA; however, it produces three specific FISH signals in 1q21, 1p12 and 1p36.1 of Homo sapiens (HSA). Human chromosome 1 was studied in comparison to its homologues in Hylobates lar (HLA), Pongo pygmaeus (PPY), Gorilla gorilla (GGO) and Pan troglodytes (PTR). A duplication of sequences homologous to human 1p36.1 could be detected in PPY plus an additional signal on PPY 16q. The region homologous to HSA 1p36.1 is also duplicated in HLA, and split onto chromosomes 7q and 9p; the region homologous to HSA 1q21/1p12 is present as one region on 5q. Additionally, the breakpoint of a small pericentric inversion in the evolution of human chromosome 1 compared to other great ape species could be refined. In summary, the results obtained here are in concordance with previous reports; however, there is evidence for a deletion of regions homologous to human 1p34.2-->p34.1 during evolution in the Pongidae branch after separation of PPY.

Evidence from urinary cortisol that maternal behavior is related to stress in gorillas.

By studying western lowland gorillas (Gorilla gorilla gorilla, n = 8) in zoological gardens via ethological and non-invasive physiological techniques, we have demonstrated that their postpartum maternal behavior is related negatively to their postpartum urinary titers of cortisol. On the basis of this finding, it is proposed that postpartum stress contributes to disrupted maternal behavior in the gorilla in captivity. Morning urine samples were collected with a mean sampling interval of 1.6 days from Day 14 prepartum to Day 14 postpartum (n = 11 pregnancies). Creatinine-indexed (Cr) urinary cortisol titers declined significantly between Day 9 to 1 prepartum (0.634 +/- 0.014 microg/mg of Cr, mean +/- SEM) and Day 1 to 6 postpartum (0.396 +/- 0.030 microg/mg of Cr, mean +/- SEM; p < 0.01-0.001). For each pregnancy, the relative postpartum decline in urinary cortisol was calculated as (microg of cortisol/mg of Cr Day 1 to 4)/(microg of cortisol/mg of Cr Day -4 to -1). Values ranged from 0.35 to 1.12, were independent of absolute prepartum cortisol titers, and were interpreted as evidence of inter-female differences in postpartum hypothalamo-pituitary-adrenal axis activity and, therefore, postpartum stress. This postpartum stress index was negatively correlated with the amount of time (0-100%) that females carried and supported their 0-14 day-old infants in a ventral position during locomotion (r(s) = -0.68, p < 0.05) and tended to be negatively correlated with the total amount of time (0-100%) they spent in ventro-ventral contact with their infants (r(s) = -0.58; p < 0.10). This study provides the first physiological evidence that postpartum stress is an important etiologic factor in gorilla maternal failure in captive environments.

Neuropeptide Y in the infundibular nucleus and hypophysis of great apes.

We studied the distribution of neuropeptide Y (NPY) immunoreactivity in the infundibular nucleus and the hypophysis of the chimpanzee, gorilla, and orangutan. Using antibodies developed in rabbit against synthetic porcine NPY, we found numerous NPY-immunoreactive neuronal somata in the infundibular nucleus; this nucleus was also filled with short NPY-positive processes and an abundance of punctate structures that could be indicative of synaptic terminals. Numerous varicose NPY-positive fibers were concentrated in the upper infundibular stem in association with capillary loops of the portal vasculature and with the long portal vessels. Bundles of long varicose fibers ran down the infundibular stem, some appearing to terminate in the lower stem in the vicinity of short portal vessels. The bulbous infundibular process contained only sparsely distributed fibers; they were mostly concentrated near vessels at the border between the infundibular process and the anterior pituitary gland, where the fibers often terminated in a spray-like fashion near blood vessels. No NPY immunoreactivity was seen in the anterior pituitary gland. These results provide anatomical evidence for the release of NPY into the portal vasculature of great apes.

Galanin immunoreactivity within the primate basal forebrain: evolutionary change between monkeys and apes.

Galanin immunoreactivity (GAL-ir) is differentially expressed within the basal forebrain of monkeys and humans. Most monkey magnocellular basal forebrain neurons colocalize GAL-ir. In contrast, virtually no human magnocellular basal forebrain neurons express GAL-ir. Rather, an extrinsic galaninergic fiber plexus innervates these neurons in humans. The present study examined the expression of GAL-ir within the basal forebrain of apes to establish the phylogenetic level at which this transformation occurs. The staining patterns of GAL-ir within the basal forebrain of both lesser (gibbons) and great (chimpanzee and gorilla) apes were compared to that previously observed within monkeys and humans. All apes displayed a pattern of basal forebrain GAL-ir indistinguishable from humans. GAL-ir was not expressed within ape basal forebrain magnocellular neurons as seen in monkeys. Rather like humans, a dense collection of GAL-ir fibers was seen in close apposition to magnocellular perikarya. In addition, a few GAL-ir parvicellular neurons were scattered within the ape basal forebrain. These data indicate that the evolutionary change in the expression of GAL-ir within the primate basal forebrain occurs at the branch point of monkeys and apes.

The interaction of primate transferrins with receptors on bacteria pathogenic to humans.

The binding of primate transferrins by receptors in the human pathogens Neisseria meningitidis, Moraxella (Branhamella) catarrhalis, and Haemophilus influenzae was assessed and compared with the binding of anti-human transferrin monoclonal antibodies by primate transferrins. In competitive binding assays the three pathogens showed identical specificity for primate transferrins. Only human, gorilla, chimpanzee and orangutan sera were capable of blocking binding of labelled human transferrin. Direct binding assays and affinity isolation of receptor proteins confirmed that chimpanzee transferrin, but not rhesus monkey transferrin, was capable of effectively binding to the bacterial receptors. Five distinct patterns of binding were seen when five anti-human transferrin monoclonal antibodies were reacted with the primate transferrins and these patterns reflected phylogenetic relatedness of these species to humans. A monoclonal antibody which showed transferrin-binding specificity identical to that seen with the bacterial receptors was found to block binding of human transferrin by receptors in the three bacterial species.

Amylase levels in the tissues and body fluids of several primate species.

1. Serum amylase levels in the gorilla, orang-utan, chimpanzee and squirrel monkey are similar to man. Serum amylase in the Rhesus macaque is almost a whole order of magnitude higher than man. 2. Of the several species tested, all have appreciable amylase in saliva or the parotid gland except the squirrel monkey. 3. High levels of amylase were found in the pancreas of all species tested. Amylase was found in the livers of all species tested. 4. In in vivo experiments with squirrel monkeys, injection of puromycin did not alter serum amylase levels.

Patterns of oestrogen and testosterone excretion during pregnancy in a gorilla (Gorilla gorilla).

Oestrogen and testosterone concentrations in the urine were determined by radioimmunoassay. Total oestrogen concentrations rose from 25 ng/mg creatinine in the non-pregnant animal to a mean of about 2 microgram/mg creatinine in the 50 days before term. There was a significant (P less than 0.001) correlation between oestrogen and testosterone concentrations until 11 days before parturition.