Ethics statement
The permission for trapping Formosan macaques in Mt. Longevity was obtained from the Forest Bureau (Permit no.: COA, Forestry Bureau, 1041701001). The procedures for trapping Formosan macaques in Mt. Longevity, administering anesthesia, and collecting samples were approved by Institutional Animal Care and Use Committee (IACUC) of National Pingtung University of Science and Technology (Approval no.: NPUST-104-021). All the procedures that involve live animals were performed in compliance with the IACUC regulations and Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. No other permit was needed for collecting fecal samples. All details on sampling methods are provided in the following sections.
Study site
This study was conducted in Mt. Longevity, which is located in SNNP (latitude: 22.650 and longitude: 120.260) on the west side of Kaohsiung city (Fig. 2). A city and an ocean isolate Mt. Longevity from other natural habitats. The terrain of Mt. Longevity is characterized by uplifted coral reefs and limestone covered with natural forest. Formosan macaques are endemic to Mt. Longevity. The estimated population size of Formosan macaques ranged from 1200 to 1500 in the sampling area from 2012 to 201527. The density of Formosan macaques in Mt. Longevity was high for a long period owing to food provision for a long term history28. The average density of Formosan macaques on Taiwan island was 0.72 troops/km229; however, its population density in Mt. Longevity was 5.72 troops/km230. Food provision to macaques is prohibited, and a law was strongly enforced against food provision after the establishment of SNNP in 2011, which may have limited the food for macaques in Mt. Longevity.
Histopathological evaluation of alopecia
Free-ranging Formosan macaques with symmetrical alopecia were trapped for the histopathological evaluation of skin tissues with alopecia. We used steel-mesh box traps (Tomahawk Live Trap, LLC., Hazelhurst, WI, USA) modified for remote access, and a banana was used as a bait. Individuals with severe symmetrical alopecia were trapped. The trapped macaques were anesthetized by veterinarians with a mixture of dexmedetomidine hydrochloride (25 µg/kg) and tiletamine HCl/zolazepam HCl (2 mg/kg). Skin tissues of the back area with alopecia of approximately 1 cm2 were surgically collected and fixed in 10% neutral buffered formalin for further histopathological evaluation. Vertical and transversal sections at the reticular dermis layer of skin biopsy were obtained and stained with hematoxylin and eosin stain. We examined the histopathological changes in skin sections to evaluate the possible etiology of alopecia. In addition, transversal skin sections were used for obtaining follicular counts and determining follicular stages, namely anagen, catagen, and telogen, based on Whiting 31. We estimated follicular density per square millimeter by dividing the total count of follicles by the area covered in the image by using ImageJ software 32. The follicles on the edge of the image were not counted. Moreover, we grouped catagen and telogen follicles together and estimated the anagen to catagen/telogen ratio 33.
EIA validation and FGM screening for macaques with and without alopecia
Stress manipulation of Formosan macaques in captivity
We adopted a biological validation procedure to induce GC secretion through restraint and anesthesia procedures25,34,35 for routine health evaluation in captivity. We then assessed whether the increased GC secretion could be detected based on different EIA FGM quantities. In addition, we evaluated the capacity and specificity of different antibodies to quantify FGM abundance through integration with high-performance liquid chromatography (HPLC) analysis. Lastly, we compiled the results of the EIA and HPLC to evaluate the suitability of four EIAs in assessing adrenocortical activity in Formosan macaques.
For validating the EIAs, fresh fecal samples were collected for 5 consecutive days from one male and one female captive adult Formosan macaque housed individually in the Pingtung Rescue Center for Endangered Wild Animals (PTRC), National Pingtung University of Science and Technology. The individual cages used for housing the macaques were 2 m high and 1 m wide and deep; the upper area of the cage had a platform for resting.
Pooled fecal samples of one day of each individual were collected by the PTRC staff before and after an anesthesia procedure for routine health evaluation of each individual. Anesthesia was administered to stimulate stress (physical challenge) in the macaques on the third day of sample collection. Collected feces were stored at −20°C immediately until analysis. The feces collected before the health check were used for baseline analysis, and the samples collected after the health check were used for evaluating the adrenal responses to stress.
Fecal sample collection from Formosan macaques in Mt. Longevity
We collected fecal samples of Formosan macaques with and without alopecia by following troops with alopecia individuals in Mt. Longevity. When field crews observed a macaque defecating, a fecal sample was collected and stored in a coolbox immediately. The feces were then stored at −20°C. We recorded the age, sex, and status of individuals with alopecia while collecting fecal samples. Age was recorded as juvenile, subadult, and adult according to the classification by Hsu and Lin 36. We did not collect sample from infant in this study due to the limitation of availability.
Extraction of fecal hormone metabolites
Collected feces were transferred to 15-mL centrifuge tubes and covered with parafilm. Holes were made on the parafilm to allow evaporation. Lyophilization was then performed for 72 h. Dehydrated feces were filtered to exclude large particles, and the rest were ground to powder. The fecal powder of 50 mg was transferred to new 15-mL centrifuge tubes, and 3 mL of 80% methanol was then added. After vortexing for 10 min and centrifuging at 3000 rpm for 10 min, the supernatant was transferred to a 5-mL vial, labeled, and stored at −20°C until further analysis.
HPLC analysis
The samples collected at the PTRC from one male and one female macaque were used for reverse-phased high-performance liquid chromatography. To purify the fecal extractions, 1 mL of the fecal extract was mixed with 3 mL of sodium acetate buffer (0.2M, pH 4.2) and filtered using solid-phase extraction (SPE) cartridges (Sep-pak C18 cartridge 1 g, Waters, MA, USA). The SPE cartridge was activated by pumping 10 mL of 100% methanol and 10 mL of double-distilled water slowly with a syringe before addition of the extraction mixture. After the total volume of the extraction mixture was passed, 10 mL of double-distilled water was pumped using a syringe to wash the cartridge. The elution was discarded, and the cartridge was left to dry at room temperature for 3 h. After drying, 10 mL of methanol (100%) was added to elute the extraction from the cartridge. The elution was collected and evaporated to dryness through nitrogen sweeping and then reconstituted in 400 μL of 40% Acetonitrile (ACN) buffer for the HPLC process to separate the steroids. The HPLC settings include a flow speed of 0.4 mL/min and ACN:H2O ratio of 40:60 as mobile phase before injection of 100 μL of the purified extraction. Fractions (n = 80, 400 μL each) were collected every minute and were evaporated to dryness. An assay buffer (1 mL) was then added to each fraction and stored at −20℃ before the EIA analysis. Moreover, the standards of the four selected FGMs (cortisol, corticosterone, 11-oxoaetiocholanolone, and 11β-hydroxyetiocholanolone) were separated into fractions with HPLC to obtain elution positions for reference.
Enzyme immunoassays
Four competing EIAs, namely cortisol, corticosterone, 11-oxoaetiocholanolone, and 11β-hydroxyetiocholanolone, were used to detect targeted steroids and metabolites of HPLC elution and fecal extractions, as described by Heistermann, et al. 25. The antibody, standard, and biotin-labeled steroid of each target used in this study were kindly provided by Dr. Palme from the Department of Biomedical Sciences, University of Veterinary Medicine, Vienna, Austria. The cross-reactivities of all antibodies applied in this study were described by Heistermann, et al. 25. The EIAs were performed using 96-well microtiter plates (MTPs) coated with protein A (P-7837, Sigma-Aldrich, Vienna, Austria), according to procedures described by Palme and Möstl 37. In brief, samples were diluted 20 times with an EIA assay buffer before reaction. Biotin-labeled steroids (100 μL) and diluted samples or standards (50 μL), as well as antibodies (100 μL), were added to each well and incubated overnight at 4°C. An enzyme solution (250 μL/well) containing streptavidin–horseradish peroxidase conjugate (RPN1231, GE Healthcare) was added to the plate after four washes with 0.02% Tween 20 (VE-V900548, Vetec) and then incubated with mild shaking in the dark at 4°C for 45 min. The plate was washed again before the addition of 250 μL of substrate solution containing tetramethylbenzidine (SI-T2885, Sigma-Aldrich) and incubated with mild shaking in the dark for another 45 min at 4°C. After incubation, 50 μL of 10% H2SO4 was added to each well to stop the reaction, and the plate was read using a multimode microplate reader (Corona Electric Co., Ltd, Japan) within 30 min to measure absorbance at 450 and 620 nm as measuring and reference filters, respectively (Fig. S1). The differences in both readings were used for obtaining the standard curve formula, which was then used for the concentration calculation of each sample. Intra- and interassay coefficients of variance (CVs) of low and high concentration pool samples were calculated for quality control in EIA analysis. Only MTPs with less than 15% CV were considered valid results 38.
Suitability of EIAs
The suitability of EIAs for analyzing the adrenal activity response of Formosan macaque to stress was assessed based on four criteria according to Heistermann, et al. 25: (1) tremendous FGM response after physiological challenge, (2) substantial immunoreactivity in HPLC elution, (3) no indication of comeasurement of fecal androgen metabolites, and (4) low variation in baseline levels.
Data analysis
We used a multivariate logistic regression analysis to evaluate the relationship between alopecia, stress, and other possible covariates, such as age and sex. The analysis was conducted using the Generalized Liner Model (GLM) module in the computing environment R (R Development Core Team, 2010). The alopecia status during fecal sample collection was treated as the dependent variable. The fecal concentrations of 11-oxoaetiocholanolone and 11β-hydroxyetiocholanolone were treated as explanatory variables. Furthermore, age and sex variables were treated as possible confounders for data analysis. We first transformed all the explanatory variables to dummy variables and centered or standardized the numerical variables. Multicollinearity between the explanatory variables was evaluated using the variance inflation factor (VIF) 39. The VIF threshold was set as 10 to avoid the problematic effect of multicollinearity on parameter estimations 40. Variables were discarded from model construction if the VIF value was more than 10. The explanatory variables selected in the models were based on the Wald test with a p threshold value of 0.05 41,42. We compared the model fit based on the Akaike information criterion (AIC), with lower AIC values indicating a better model fit to the dataset.