The critical role of dietary foliage in maintaining the gut microbiome and metabolome of folivorous sifakas.

The gut microbiome (GMB) of folivores metabolizes dietary fiber into nutrients, including short-chain fatty acids (SCFAs); however, experiments probing the consequences of foliage quality on host GMBs are lacking. We therefore examined GMB structure and function via amplicon sequencing and Nuclear Magnetic Resonance spectroscopy in 31 captive sifakas (Propithecus coquereli) during dietary manipulations associated with husbandry. Supplementing standard diets with diverse foliage blends, versus with a single plant species, promoted more diverse GMBs, enriched for taxa implicated in plant-fiber metabolism, but depleted in taxa implicated in starch metabolism and bile tolerance. The consumption of diverse blends was associated with greater concentrations of colonic SCFAs. Abundant foliage, via forest access, promoted compositionally distinct and more stable GMBs, but reduced concentrations of SCFAs, possibly reflecting selection of high-quality leaves. In 11 subjects denied forest access, we examined the temporal pace of microbial shifts when supplemental foliage was abruptly switched between diverse blends and single species. The sifaka GMB responded within days, with community diversity and composition closely tracking foliage diversity. By providing experimental evidence that the folivore GMB is sensitive to minor changes in dietary foliage, we reveal the fragility of specialist GMBs, with implications for managing the wellbeing of endangered wildlife.

Feeding strategy shapes gut metagenomic enrichment and functional specialization in captive lemurs.

Many studies have demonstrated the effects of host diet on gut microbial membership, metagenomics, and fermentation individually; but few have attempted to interpret the relationship among these biological phenomena with respect to host features (e.g. gut morphology). We quantitatively compare the fecal microbial communities, metabolic pathways, and fermentation products associated with the nutritional intake of frugivorous (fruit-eating) and folivorous (leaf-eating) lemurs. Our results provide a uniquely multidimensional and comparative perspective on the adaptive dynamics between host and microbiome. Shotgun metagenomic sequencing revealed significant differential taxonomic and metabolic pathway enrichment, tailored to digest and detoxify different diets. Frugivorous metagenomes feature pathways to degrade simple carbohydrates and host-derived glycosaminoglycans, while folivorous metagenomes are equipped to break down phytic acid and other phytochemical compounds in an anaerobic environment. We used nuclear magnetic resonance based metabolic profiling of fecal samples to link metabolic pathways to fermentation products, confirming that the dissimilar substrates provided in each diet select for specific microbial functions. Fecal samples from frugivorous lemurs contained significantly different profiles of short chain fatty acids, alcohol fermentation products, amino acids, glucose, and glycerol compared to folivorous lemurs. We present the relationships between these datasets as an integrated visual framework, which we refer to as microbial geometry. We use microbial geometry to compare empirical gut microbial profiles across different feeding strategies, and suggest additional utility as a tool for hypothesis-generation.

The Nutritional Geometry of Resource Scarcity: Effects of Lean Seasons and Habitat Disturbance on Nutrient Intakes and Balancing in Wild Sifakas.

Animals experience spatial and temporal variation in food and nutrient supply, which may cause deviations from optimal nutrient intakes in both absolute amounts (meeting nutrient requirements) and proportions (nutrient balancing). Recent research has used the geometric framework for nutrition to obtain an improved understanding of how animals respond to these nutritional constraints, among them free-ranging primates including spider monkeys and gorillas. We used this framework to examine macronutrient intakes and nutrient balancing in sifakas (Propithecus diadema) at Tsinjoarivo, Madagascar, in order to quantify how these vary across seasons and across habitats with varying degrees of anthropogenic disturbance. Groups in intact habitat experience lean season decreases in frugivory, amounts of food ingested, and nutrient intakes, yet preserve remarkably constant proportions of dietary macronutrients, with the proportional contribution of protein to the diet being highly consistent. Sifakas in disturbed habitat resemble intact forest groups in the relative contribution of dietary macronutrients, but experience less seasonality: all groups' diets converge in the lean season, but disturbed forest groups largely fail to experience abundant season improvements in food intake or nutritional outcomes. These results suggest that: (1) lemurs experience seasonality by maintaining nutrient balance at the expense of calories ingested, which contrasts with earlier studies of spider monkeys and gorillas, (2) abundant season foods should be the target of habitat management, even though mortality might be concentrated in the lean season, and (3) primates' within-group competitive landscapes, which contribute to variation in social organization, may vary in complex ways across habitats and seasons.

Locomotor energetics in primates: gait mechanics and their relationship to the energetics of vertical and horizontal locomotion.

All primates regularly move within three-dimensional arboreal environments and must often climb, but little is known about the energetic costs of this critical activity. Limited previous work on the energetics of incline locomotion suggests that there may be differential selective pressures for large compared to small primates in choosing to exploit a complex arboreal environment. Necessary metabolic and gait data have never been collected to examine this possibility and biomechanical mechanisms that might explain size-based differences in the cost of arboreal movement. Energetics and kinematics were collected for five species of primate during climbing and horizontal locomotion. Subjects moved on a treadmill with a narrow vertical substrate and one with a narrow horizontal substrate at their maximum sustainable speed for 10­20 min while oxygen consumption was monitored. Data during climbing were compared to those during horizontal locomotion and across size. Results show that climbing energetic costs were similar to horizontal costs for small primates (<0.5 kg) but were nearly double for larger species. Spatio-temporal gait characteristics suggest that the relationship between the cost of locomotion and the rate of force production changes between the two locomotor modes. Thus, the main determinants of climbing costs are fundamentally different from those during horizontal locomotion. These new results combining spatiotemporal and energetic data confirm and expand on our previous argument (Hanna et al.: Science 320 (2008) 898) that similar costs of horizontal and vertical locomotion in small primates facilitated the successful occupation of a fine-branch arboreal milieu by the earliest primates.

Maximum ingested food size in captive strepsirrhine primates: scaling and the effects of diet.

Little is known about ingested food size (V(b)) in primates, even though this variable has potentially important effects on food intake and processing. This study provides the first data on V(b) in strepsirrhine primates using a captive sample of 17 species. These data can be used for generating and testing models of feeding energetics. Strepsirrhines are of interest because they are hypometabolic and chewing rate and daily feeding time do not show a significant scaling relationship with body size. Using melon, carrot, and sweet potato we found that maximum V(b) scales isometrically with body mass and mandible length. Low dietary quality in larger strepsirrhines might explain why V(b) increases with body size at a greater rate than does resting metabolic rate. Relative to body size, V(b) is large in frugivores but small in folivores; furthermore scaling slopes are higher in frugivores than in folivores. A gross estimate of dietary quality explains much of the variation in V(b) that is not explained by body size. Gape adaptations might favor habitually large bites for frugivores and small ones for folivores. More data are required for several feeding variables and for wild populations.

A comparison of salivary pH in sympatric wild lemurs (Lemur catta and Propithecus verreauxi) at Beza Mahafaly Special Reserve, Madagascar.

Chemical deterioration of teeth is common among modern humans, and has been suggested for some extinct primates. Dental erosion caused by acidic foods may also obscure microwear signals of mechanical food properties. Ring-tailed lemurs at the Beza Mahafaly Special Reserve (BMSR), Madagascar, display frequent severe tooth wear and subsequent tooth loss. In contrast, sympatric Verreaux's sifaka display far less tooth wear and infrequent tooth loss, despite both species regularly consuming acidic tamarind fruit. We investigated the potential impact of dietary acidity on tooth wear, collecting data on salivary pH from both species, as well as salivary pH from ring-tailed lemurs at Tsimanampesotse National Park, Madagascar. We also collected salivary pH data from ring-tailed lemurs at the Indianapolis Zoo, none of which had eaten for at least 12 hr before data collection. Mean salivary pH for the BMSR ring-tailed lemurs (8.098, n=41, SD=0.550) was significantly more alkaline than Verreaux's sifaka (7.481, n=26, SD=0.458). The mean salivary pH of BMSR (8.098) and Tsimanampesotse (8.080, n=25, SD=0.746) ring-tailed lemurs did not differ significantly. Salivary pH for the Indianapolis Zoo sample (8.125, n=16, SD=0.289) did not differ significantly from either the BMSR or Tsimanampesotse ring-tailed lemurs, but was significantly more alkaline than the BMSR Verreaux's sifaka sample. Regardless of the time between feeding and collection of pH data (from several minutes to nearly 1 hr), salivary pH for each wild lemur was above the "critical" pH of 5.5, below which enamel demineralization occurs. Thus, the high pH of lemur saliva suggests a strong buffering capacity, indicating the impact of acidic foods on dental wear is short-lived, likely having a limited effect. However, tannins in tamarind fruit may increase friction between teeth, thereby increasing attrition and wear in lemurs. These data also suggest that salivary pH varies between lemur species, corresponding to broad dietary categories.

Influence of reproductive season and rank on fecal glucocorticoid levels in free-ranging male Verreaux's sifakas (Propithecus verreauxi).

Studies in anthropoid primates and other mammals suggest that reproductive season, rank, reproductive skew, aggression received, and social support are the major factors influencing glucocorticoid output. In which way these are also affecting adrenal function in lemurid primates has been studied rarely. Here, we examine the influence of reproductive season and rank on glucocorticoid output in male sifakas (Propithecus verreauxi), a species characterized by high breeding seasonality, a hierarchy among males and extreme reproductive skew towards dominant males. We established a fecal assay for non-invasively monitoring adrenal activity and collected 315 fecal samples during the reproductive and birth season from 10 male sifakas living in 5 groups in Western Madagascar. We found a significant effect of season on glucocorticoid output, with males exhibiting higher fecal glucocorticoid levels during the reproductive compared to the birth season in conjunction with an increase in overall aggression rates during the former period. Moreover, our data indicate a significant effect of rank on adrenocortical activity with dominant males exhibiting higher glucocorticoid levels than subordinate males in the reproductive season. However, dominant males did not differ significantly in rates of initiated or received aggression and rates of affiliative behavior from subordinates but showed significantly lower rates of submission. Given their highly formalized dominance relationships, we conclude that higher glucocorticoid output in dominant males during the 4-month reproductive season is likely related to higher energetic demands necessary to cope with the challenges of male reproduction rather than to physical demands of increased fighting frequency to maintain dominance status. High rank in sifakas may thus carry high costs, which, however, may be outweighed by monopolization of almost all paternities. In sum, our data generally support the findings on the relationship between environmental and social factors and glucocorticoid output found in non-lemurid primates.

Anogenital gland secretions of Lemur catta and Propithecus verreauxi coquereli: a preliminary chemical examination.

Although prosimians are greatly olfaction-oriented, little is known about the specifics of how they use scent to communicate. In this preliminary study we attempted to delineate intra- and interspecific differences among the anogenital gland secretions of two lemur species (Lemur catta and Propithecus verreauxi coquereli) using gas chromatography-mass spectrometry (GC-MS). The results indicate that the two species are discernible through scent. Furthermore, we were able to identify reproductive status using this technique. The anogenital secretions of the different sexes in L. catta, though perhaps not P. v. coquereli, are chemically distinguishable. Given this information, it appears that at least some lemur species can use scent marks to determine species, sex, and reproductive status.

Vitamin C biosynthesis in prosimians: evidence for the anthropoid affinity of Tarsius.

This report examines the taxonomic distribution of the in vitro biosynthesis of ascorbic acid in the Prosimii (Order: Primates). Liver and kidney samples of 15 prosimian taxa, including Tarsius bancanus, were quantitatively tested for the enzyme L-gulono-1,4-lactone oxidase. Liver samples from all taxa except Tarsius had substantial levels of the enzyme. Furthermore, unlike other eutherian mammals, kidney tissue from members of the family Lemuridae showed low but consistent levels of enzyme activity. The result for Tarsius, by fitting with the pattern exhibited by the monkeys, apes, and man, adds significant independent evidence for this animal's relatively close genetic relationship with the Anthropoidea.

Serum concentrations of calcium and vitamin D metabolites in prosimians.

The concentrations of total calcium, 25-hydroxyvitamin D [25-(OH)D] and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] were measured in serum obtained from prosimians, the brown lemurs. The mean serum calcium level was 10.6 mg/dl in male and female lemurs. The mean serum mean 25-(OH)D concentration in serum from male and female lemurs was 27.1 and 31.0 ng/ml, respectively. The mean serum level of 1,25-(OH)2D3 in the female and male lemurs was 65.2 and 65.9 pg/ml, respectively. A small segment of the lemurs had hypercalcemia and elevated serum concentrations of 25-(OH)D or 1,25-(OH)2D3, suggesting the idea that the episodic ingestion of a large quantity of the calcium- and vitamin D-enriched diet normally provided ad libitum might cause hypercalcemia.

The similarity of chorionic gonadotrophin and its subunits in term placentae from man, apes, old and New World monkeys and a prosimian.

Chorionic gonadotrophin (CG) was estimated, by bioassay and radioimmunoassay (RIA), in placental extracts from 11 ape and monkey species. There was a significant correlation between the results of the two assay systems (r = 0.903, p less than 0.001). The concentration of CG in most primate term placentae was the same as that in the human placenta at term. Extracts from all placentae cross-reacted with antiserum to ovine LH-beta subunit, and those of the chimpanzee and gorilla also had a significant cross-reaction with an antiserum to the carboxyl terminal peptide of the HCG-beta subunit. Primate placentae chromatographed on Sephadex G-200 had components active in the RIA systems for HCG, HCG-alpha HCG-beta subunits. In general, the elution profiles of all ape and monkey placental extracts resemble those made from human term placentae and of purified HCG and its subunits. The shape of the elution patterns from human and non-human material suggests that there was more than one molecular form of CG-alpha subunit activity. A second, more retarded molecular form having beta subunit activity was found in extracts made from human, gorilla, gibbon and rhesus monkey placentae. The similarity between the structure of ape and monkey placental CG with HCG and its subunits implies a function similar to that of HCG in late pregnancy.

Acetylcholinesterase enzyme localization in the amygdala: a comparative histochemical and ultrastructural study.

The distribution of acetylcholinesterase enzyme was studied in the amygdala of some rodents, subprimates and several primates. The cytoarchitecture of the amygdala has presented various problems to anatomists, including the question as to how many nuclear groups and subgroups should be identified. Among the mammals examined, the arrangement of the amygdaloid nuclei is remarkably uniform and no clear phylogenetic trend can be recognised. Although there are minor differences, there seems to be a general similarity between most mammals examined in so far as the distribution of cholinesterase is concerned. The staining is less intense in the brains of the monkeys examined. The sole exception to the rule, that cholinesterase distribution is slightly different from nucleus to nucleus in different animals, is the magnocellular part of the basal nucleus. This amygdaloid nucleus stains quite strongly in all animals examined. From these findings, and those of others studying the distribution of choline acetyltransferase, it was concluded that the basal amygdaloid nucleus is cholinergic and possible cholinoceptive. The ultrastructural investigations appear to confirm this point. This is particularly applicable to the magnocellular part of the basal amygdaloid nucleus.

Immunofluorescence study of LRH-producing neurons in prosimians (Tupaia and Galago).

Reactive LRH neurons were characterized in prosimians (Tupaia and Galago) by immunofluorescence using rabbit immunesera against unconjugated synthetic LRH, or LRH conjugated with bovine serum albumin. These neurons, which vary individually in number in one species, are mainly concentrated in the rostral hypothalamus (medial preoptic area and anterior hypothalamic area) and in the lamina terminalis. In contrast to the simians and man, immunoreactive perikarya were not routinely found in the mediobasal hypothalamus of the prosimians investigated in the present study. Reactive axons of the hypothalamo-hypophyseal tract are more numerous and conspicuous in the retrochiasmatic area and in the postinfundibular eminence. They give rise to radiating collaterals ending mainly around the capillaries of the primary portal plexus of the median eminence and of the infundibular stem (where they are generally more numerous). Reactive axons of the preoptico-terminal tract, originating from the perikarya of the lamina terminalis, end around the capillaries of the vascular organ or below and between the ependymal cells lining its ventricular side. In Galago a small but very distinct tract of reactive axons runs under the optic chiasma, between the lamina terminalis and the ventral labium of the infundibulum. Very fine reactive extrahypothalamic axons were observed in the posterior part of the habenular ganglia, along the preamygdaloid portion of the stria terminalis and along the blood vessels of the parolfactory area.

Tupaias (tree shrews)--a new animal model for gallstone research. II. Influence of fat, sugar, and cholesterol on bile composition.

The lipid composition of bile obtained from the gallbladder and from the liver of tupaias was determined. Compared to the gallbladder bile of man, the gallbladder bile of tupaias contains the same percentage of phospholipids (PL), one third of the cholesterol (CH), and 50% more total bile acids (TBA). The lithogenic index (LI) of the bile is about 0.2--0.3. No significant sex differences or diurnal variations are found in the composition of bile. The concentration of PL, CH, and TBA is found 5 to 8 times higher in the gallbladder bile than in the liver bile. The bile acid spectrum in tupaias equals that in man qualitatively and is similar quantitatively. In a feeding experiment, the influences of butter, cholesterol, butter plus cholesterol and sucrose on bile composition, clinicochemical serum values, and liver and gallbladder histology were studied. Groups of six animals each were fed the different diets over a 10-week period. In group A: 20% butter added to the standard diet, PL content was higher, the LI was unchanged. In group B: 2% cholesterol added to the standard diet, the CH value and the LI were elevated, CH crystals but no stones were found. In group C: 2% cholesterol and 20% butter in the standard diet, stone formation was observed. PL and CH values were higher than in group A and B, TBA values were lower. In group D: 50% sucrose added to the standard diet very low PL and TBA values were determined in the liver bile, resulting in elevated LIs. It is concluded that all three components of a diet, which had proven lithogenic in a preceding experiment (Schwaier, 1979a), contribute to gallstone formation.

Codominant autosomal inheritance of polymorphic red cell acid phosphates of lemurs and some properties of the enzymes.

Red cell acid phosphatase phenotypes of 207 captive animals of the genera Lemur, Hapalemur, and Prophithecus were determined by starch gel electrophoresis and phosphatase-specific staining. In Lemur fulvus, three phenotypes, designated A, B, and AB, were observed. In each of the species L. catta, L. macaco, L. mongoz, and L. variegatus, a single phenotype was observed, In Hapalemur griseus, three phenotypes were found: A,B, and AB. In Propithecus verreauxi, a single phenotype was found. Examination of breeding records in conjunction with the results of the electrophoretic analyses supports the conclusion that the erythrocytic acid phosphatases in this group of nonhuman primates are the products of at least two codominant autosomal alleles. There is a wide range of specific activities of the acid phosphatases as determined by colorimetric assays. The values range from 60.6 micronmoles of p-nitrophenol released per gram of hemoglobin per 30 min in Lemur catta to 429.1 micronmoles in Propithecus verreauxi. The enzymes of L. fulvus and P. vereauxi were purified approximately 400-fold, and Michaelis-Menten constants were determined on the purified preparations. For L. fulvus phenotype A, Km = 0.8 mM; for L. fulvus phenotype B, Km = 0.8 mM; and for P. verreauxi, Km = 0.6 mM; the substrate in each case was p-nitrophenylphosphate.