A new dracunculus species (Nematoda: Dracunculoidea) in neotropical otters (Lontra longicaudis) from Argentina: morphological and molecular characterization.

A new species of Dracunculus is described in wild neotropical otters, Lontra longicaudis, occurring in Corrientes, Argentina, based on morphological and molecular characteristics. Worms were located in the subcutaneous tissue from two of five investigated otters. Dracunculus jaguape n. sp. is differentiated from the 14 species of Dracunculus described from mammals and reptiles by the prominent dorsal and ventral papillae on the head; deirids posterior to nerve ring; male with long needlelike spicules and presence of gubernaculum; and long first stage larva. Phylogenetic analysis using the 18S rRNA positioned Dracunculus jaguape n. sp. in an anterior position to the rest of Dracunculus sequences available and COI positioned it in a separated clade sister to Dracunculus lutrae sequences. This is the first report on the presence of this nematode in Lontra longicaudis in Argentina.

Human and Non-Human Primate Coexistence in Argentina: Conflicts and Solutions.

There are five different primate species inhabiting widely distinct ecoregions in Argentina. Each of them faces various threats in terms of conservation and conflicts that hamper their ability to coexist with human populations. We present here some of the drivers known to be the causes of conflicts between humans and primates in the southernmost area of distribution of Latin American primates. We focus our synthesis on two of the biggest sources of conflict: the effects of different anthropogenic disturbances, and human misconceptions concerning the role of primates in the ecosystem. In each section, we briefly characterize the conflicts worldwide and then provide specific cases and examples from Argentina. In the last part of the manuscript, we further describe some ongoing national and regional educational, research, and conservation approaches to mitigate those effects.

Best practices are never best: Evaluating primate conservation education programs (PCEPs) with a decolonial perspective.

Who do we aim to educate with primate conservation education programs (PCEPs)? In a commentary published in a recent AJP, Annette Lanjouw suggested that many efforts to "educate" habitat-country communities can be neocolonial in their approaches. Forest destruction and habitat loss are a result of global consumption and expansion. We therefore need to approach conservation education from many angles including local stakeholders, policy makers, government officials, and the humans living in industrialized nations who are major consumers of the items that shrink primate habitats. In this review, we investigate PCEPs to determine if the conservation education goals, education methods, and assessment processes are proceeding within a neocolonial context. We reviewed the last 20 years of primate conservation literature and looked for publications that were focused on education programs. We found that in 50 of 52 publications published between 2001 and 2021, the education programs take place in habitat-country local communities. We also reviewed primate field researcher and field site websites, and in most cases, education programs were also focused on educating local communities living near or in nonhuman primate habitats. Exceptions were student clubs, zoo programs, and a high school outreach program. Many PCEP providers presented a list of "lessons learned" and we compiled their wisdom in combination with our experience to provide a framework for moving forward. We conclude that as conservation primatologists, we must think beyond our field sites to create opportunities for educational outreach. We can reach global consumers by linking to zoos, television/motion picture, print media, social media, and working with schools on curricula. Primatologists can engage our undergraduates to establish clubs and create meaningful assignments that reach beyond the classroom. We encourage primatologists from the Global North to consider their positionality and the history of conservation exclusion in their attempts to conserve primates.

Parasites and Other Infectious Agents in Non-human Primates of Argentina.

Purpose of Review: In Argentina, there are five non-human primate (NHP) species: Sapajus nigritus cucullatus, Sapajus cay, Alouatta caraya, Alouatta guariba clamitans, and Aotus azarae. All of them inhabit protected and non-protected areas and face severe threats due anthropization. We aim to summarize the information available about parasites and infectious diseases of these NHPs and suggest further research on primate diseases in Argentina. Recent Findings: NHPs of Argentina are hosts of several parasites and pathogens important for conservation as well as public health. Alouatta species are lethally susceptible to yellow fever virus, which makes them suitable health sentinels of possible outbreaks. For other primate species, few parasite surveys have been carried out. Summary: Assessing the presence of infectious diseases and long-term surveillance on NHP allow the development of strategies to help in the early detection of pathogens that may threat public health. Increasing the knowledge about parasites and infectious diseases and their consequences in NHP of Argentina is needed, considering a One Health approach.

Molecular characterization of Giardia duodenalis and evidence for cross-species transmission in Northern Argentina.

Anthropogenic activities, such as human population expansion and land-use change, create ecological overlap between humans, domesticated animals, and wildlife and can exacerbate the zoonotic transmission of parasites. To improve our understanding of this dynamic, we employed multi-locus genotyping to conduct a cross-sectional study of the potential for zoonotic transmission of the protozoan parasite Giardia duodenalis among humans, household associated livestock and dogs, and black and gold howler monkeys (Alouatta caraya) in the Corrientes Province of Argentina. We found Giardia prevalence to be highest in howler monkeys (90.3% (47/52)), followed by humans (61.1% (22/36)), dogs (44.4% (16/36)), and cattle (41.9% (18/43)). We further established that howler monkeys exclusively harbored strains of assemblage B (100%) while humans were infected with either assemblage A (13.3%) or B (80%) or A and B (6.7%), and cattle and dogs were infected with either assemblage A (cattle, 94.1%; dogs, 80%)), A and C (10%), or their host-adapted assemblage (cattle, 5.9%; dogs, 10%). Our finding of G. duodenalis in both humans and domesticated animals (assemblage A) and humans and wild primates (assemblage B) suggests that cross-species transmission of multiple assemblages of G. duodenalis may occur in rural complexes such as northern Argentina where people, domesticated animals, and wildlife overlap. We further highlight the need to investigate the implications of these results for human health, the economics of livestock production, and wildlife conservation in this and similar systems.

Comparative Stress Response of Black and Gold Howler Monkey (Alouatta caraya) in Urban and Rural Environments of Northern Argentina.

The expansion of human populations associated with urbanization results in extensive modification of natural habitats. While many species cannot persist in these highly modified environments, some species adopt new strategies, which contribute to their survival. Several primate species have persisted in altered habitats, including members of the genus Alouatta. To improve our understanding of this interplay, we compared glucocorticoid (GCC) levels in male and female black and gold howler monkeys (Alouatta caraya) in urban and rural areas in northeastern Argentina. Fecal samples (n = 60) were collected from adults and hormone extracts were analyzed by enzyme immunoassays (EIA). Monkeys living in urban areas (females: 109.15 ± 18.83; males: 106.15 ± 10.48) had lower GCClevels than monkeys living in rural areas (females: 152.01 ± 19.50; males:139,82 ± 10.85). Interestingly, males living in urban areas had lower GCC levels compared to those living in rural areas, whereas no differences were observed in GCC levels between females living in urban and rural areas. While these results suggest that urban areas may provide a release from intergroup competition for male howler monkeys, future work is needed to better understand the dynamics of this association to best inform management and conservation of this vulnerable species.

Use of genetic tools to assess predation on reintroduced howler monkeys (Alouatta caraya) in Northeastern Argentina.

Despite strong support from the media, the reintroduction of animals into natural environments does not always achieve its goal. Alouatta caraya is the primate species facing the greatest hunting pressure due to the illegal pet trade in Argentina. Confiscations of this species are common, as is the voluntary surrender of animals by owners no longer able or willing to care for them. These animals ultimately arrive at rehabilitation centers and, in many cases, are released into natural environments that may differ from the original sites where they were captured. Until recently, the lack of genetic analysis of the individuals involved led to biased relocation decisions. We followed the reintroduction of 12 A. caraya individuals in a protected area (Isla Palacio, Misiones, Argentina). The presence of potential predators such as pumas (Puma concolor) and jaguars (Panthera onca) in this area was confirmed by camera traps, footprints and feces. After the disappearance of four A. caraya at the reintroduction site, we investigated the applicability of genetic assignment tests based on genotypic data to accurately identify predated individuals. Genetic analyses allowed us to determine the predator species (P. onca) and to identify the predated individuals as two of the reintroduced animals. This procedure is promising for identifying the remains of predated individuals, and can contribute to the design of reintroduction policies based on scientific evidence.

Effects of anthropogenic habitat disturbance and Giardia duodenalis infection on a sentinel species' gut bacteria.

Habitat disturbance, a common consequence of anthropogenic land use practices, creates human-animal interfaces where humans, wildlife, and domestic species can interact. These altered habitats can influence host-microbe dynamics, leading to potential downstream effects on host physiology and health. Here, we explored the effect of ecological overlap with humans and domestic species and infection with the protozoan parasite Giardia duodenalis on the bacteria of black and gold howler monkeys (Alouatta caraya), a key sentinel species, in northeastern Argentina. Fecal samples were screened for Giardia duodenalis infection using a nested PCR reaction, and the gut bacterial community was characterized using 16S rRNA gene amplicon sequencing. Habitat type was correlated with variation in A. caraya gut bacterial community composition but did not affect gut bacterial diversity. Giardia presence did not have a universal effect on A. caraya gut bacteria across habitats, perhaps due to the high infection prevalence across all habitats. However, some bacterial taxa were found to vary with Giardia infection. While A. caraya's behavioral plasticity and dietary flexibility allow them to exploit a range of habitat conditions, habitats are generally becoming more anthropogenically disturbed and, thus, less hospitable. Alterations in gut bacterial community dynamics are one possible indicator of negative health outcomes for A. caraya in these environments, since changes in host-microbe relationships due to stressors from habitat disturbance may lead to negative repercussions for host health. These dynamics are likely relevant for understanding organism responses to environmental change in other mammals.

Postural behavior of howler monkeys (Alouatta palliata, A. macconnelli, and A. caraya) during sleep: an assessment across the genus range.

Sleep is the longest and most continuous behavioral phase in the 24 h cycle of mammals. However, selection of postures, substrates, and tree parts during sleep has not been adequately explored, as well as their evolutionary consequences. The present study investigates postural behavior, substrate, and tree part use during sleep in three howler species (A. palliata, A. macconnelli, and A. caraya) in Nicaragua, French Guiana, and Argentina. All three species were consistent in the use of a crouched ball-like sit-in posture on large, horizontal, unramified, or bifurcated substrates, and in avoiding the periphery of tree crowns. The regularities of these sleeping patterns are very likely functionally associated with protection from potential predators and extreme weather conditions, biomechanical stability, thermoregulation, and enhancement of the digestive process of hard-to-decompose plant material.

Parasitologic examination and associated risk factors of domestic dogs at the domestic-wildlife interface in the Iberá wetlands Ecoregion, Argentina.

The wildlife-domestic interface represents points of contact between wild animals and human-associated domestic animals, and provides opportunities for the dissemination and transmission of parasitic agents. Domestic dogs are reservoirs of parasitic agents, many of which are capable of infecting wild animals. To better investigate this potential risk at the Iberá wetlands eco-region, fecal samples from dogs present at the wildlife-domestic interface of two protected areas in Corrientes province, Argentina were examined. The prevalence of intestinal parasites and overall generic richness were examined and associations with age, sex, body condition, anthelmintic treatments and dog movement restriction were explored. From August-September 2017, questionnaires were carried out and fecal samples collected from 51 dogs at the interface zones. The overall estimated prevalence of gastrointestinal parasites was 63%. Nine genera of helminths and four protozoans were found in the examined dogs, many of which are known to infect wild carnivores and several have zoonotic potential. Ancylostoma and Isospora were the most prevalent genera. Freedom to roam was positively associated with prevalence and richness. From all dogs in our study, only a small proportion (17%) is subjected to some degree of movement restriction, while most dogs were free roaming. Veterinary health care and treatment was also extremely limited. The findings of this study revealed conditions of high prevalence of gastrointestinal parasites, lack of veterinary care and unrestricted roaming; all circumstances that suggest that dogs that live at these interface zones pose an infection risk to sympatric wildlife.

Molecular identification of Leishmania in free-ranging black and gold howler monkeys (Alouatta caraya) in northeastern Argentina.

Parasitological surveys of non-human primates provides an important opportunity to better understand the epidemiology, transmission dynamics and emergence risk of anthropozoonoses such as leishmaniasis, which affect human populations in several regions accross South America. Our study area, in northeastern Argentina, can be considered a southern marginal region for the presence of leishmaniases and includes the habitat of black and gold howler monkeys, Alouatta caraya. To evaluate if A. caraya serve as potential hosts in the Leishmania cycle, we used molecular methods to examine infection by Leishmania spp. in 109 howler monkeys of different ages captured between July and August 2010. External ear tissue samples were subjected to PCR amplification for the Leishmania ribosomal internal transcribed spacer (ITS-1) and a RFLP assay with the Hae III restriction enzyme, and finally confirmed by sequencing. Nine howler monkeys (8.3%) were infected with Le. braziliensis (2.8%), Le. amazonensis (2.8%) and/or Le. infantum (3.7%). The results also suggest a case of co-infection between Le. braziliensis and Le. amazonensis. Further, we report the first observation of Le. amazonensis in the northeastern region of Argentina. The detection of Leishmania spp. in free-ranging howler monkeys gives rise to questions about the actual prevalence of the parasite in the wild, as well as if the number of infected wild monkeys detected may present a risk of leishmaniasis emergence in surronding human populations. Anyway, the presence of Leishmania spp. in A. caraya suggests the possible importance of these monkeys in the sylvatic and periurban transmission.

Germination of native and exotic seeds dispersed by wild black-and-gold howler monkeys (Alouatta caraya): assessing deinhibition and scarification effects.

Fruit ingestion by frugivores and the subsequent passage of seeds through their digestive tract can influence seed germination patterns. The removal of germination inhibitors contained in the fruit pulp (deinhibition effect), and/or the abrasion of the seed coat (scarification effect), can modify the probability and speed of seed germination. We followed seven groups of black-and-gold howler monkeys (Alouatta caraya) in northeastern Argentina between November 2012 and January 2013 to study the effect of seed passage through the howler monkeys' gut on seed germination. For three native and one exotic species, we compared the proportion of germination and germination times among seeds in three different treatments: gut-passed seeds (GP), manually extracted seeds from fruits (ME), and seeds in intact fruits (IF). Paired comparisons between treatments allowed us to assess the overall effect of seed passage (total effect without distinguishing individual effects, GP vs. IF), deinhibition effect (ME vs. IF), and scarification effect (GP vs. ME). Our results suggest that passage through the howler monkeys' gut enhances seed germination by an increase in the proportion of germinated seeds and/or by a reduction in germination times. We found that deinhibition enhanced germination in three out of the four studied species, while scarification enhanced germination in one and decreased germination in another species. Our work highlights that gut passage may affect seed germination by different mechanisms, and all of these mechanisms should be considered in germination studies. It also emphasizes the importance of A. caraya as a seed disperser in northeastern Argentina, showing the high quality of the treatment provided to seeds.

Biodiversity of protists and nematodes in the wild nonhuman primate gut.

Documenting the natural diversity of eukaryotic organisms in the nonhuman primate (NHP) gut is important for understanding the evolution of the mammalian gut microbiome, its role in digestion, health and disease, and the consequences of anthropogenic change on primate biology and conservation. Despite the ecological significance of gut-associated eukaryotes, little is known about the factors that influence their assembly and diversity in mammals. In this study, we used an 18S rRNA gene fragment metabarcoding approach to assess the eukaryotic assemblage of 62 individuals representing 16 NHP species. We find that cercopithecoids, and especially the cercopithecines, have substantially higher alpha diversity than other NHP groups. Gut-associated protists and nematodes are widespread among NHPs, consistent with their ancient association with NHP hosts. However, we do not find a consistent signal of phylosymbiosis or host-species specificity. Rather, gut eukaryotes are only weakly structured by primate phylogeny with minimal signal from diet, in contrast to previous reports of NHP gut bacteria. The results of this study indicate that gut-associated eukaryotes offer different information than gut-associated bacteria and add to our understanding of the structure of the gut microbiome.

Evolutionary trends in host physiology outweigh dietary niche in structuring primate gut microbiomes.

Over the past decade several studies have reported that the gut microbiomes of mammals with similar dietary niches exhibit similar compositional and functional traits. However, these studies rely heavily on samples from captive individuals and often confound host phylogeny, gut morphology, and diet. To more explicitly test the influence of host dietary niche on the mammalian gut microbiome we use 16S rRNA gene amplicon sequencing and shotgun metagenomics to compare the gut microbiota of 18 species of wild non-human primates classified as either folivores or closely related non-folivores, evenly distributed throughout the primate order and representing a range of gut morphological specializations. While folivory results in some convergent microbial traits, collectively we show that the influence of host phylogeny on both gut microbial composition and function is much stronger than that of host dietary niche. This pattern does not result from differences in host geographic location or actual dietary intake at the time of sampling, but instead appears to result from differences in host physiology. These findings indicate that mammalian gut microbiome plasticity in response to dietary shifts over both the lifespan of an individual host and the evolutionary history of a given host species is constrained by host physiological evolution. Therefore, the gut microbiome cannot be considered separately from host physiology when describing host nutritional strategies and the emergence of host dietary niches.

Social play among black and gold howler monkey (Alouatta caraya) immatures during intergroup encounters.

We studied intergroup social play (IGSP) among immatures in wild black and gold howler monkeys (Alouatta caraya) in northeastern Argentina. IGSP events are one form of affiliative interaction that can occur during intergroup encounters. The main goal of this study was to analyze IGSP in A. caraya immatures and assess how intrinsic (e.g., age and sex) and extrinsic (e.g., seasonality) factors can influence the development of this type of social behavior. We followed 12 groups between 2008 and 2015 and recorded 182 encounters and 61 events of IGSP. Considering the composition of play partners, most IGSP events occurred among juveniles of both sexes (33%), followed by juveniles that were only-male (31%), and finally between mixed-sex juveniles and infants (20%) interactions. Additionally, most IGSP events occurred mainly in summer (56%), followed by spring (29%), with fewer events occurring in autumn (15%) and no IGSP events recorded in winter. Our results suggest that IGSP constitutes a beneficial activity in wild A. caraya that promotes behavioral flexibility, where immatures acquire social skills, such as tolerance, by interacting with unknown individuals. Moreover, the higher participation of young males in IGSP is consistent with the fact that adult black and gold howler males tend to be more actively involved in group encounters than females, supporting the hypothesis that social play provides benefits in the development of motor and social skills. Finally, seasonality in the frequencies of IGSP might be related to availability of foods with high and easily mobilized energy content in summer and spring.

Aged Black-and-Gold Howler Monkey Female (Alouatta caraya): A Sign of Reproductive Senescence?

Reproductive senescence patterns have been scarcely studied in Neotropical primates. The few studies available on the hormonal profiles of aging female monkeys indicate that the decline of ovarian function in nonhuman primates may resemble the hormonal events associated with the perimenopause in women. In this study, we explore a reproductive hormone profile of an aged black-and-gold howler monkey female (Alouatta caraya) from a wild population in northeastern Argentina and compare this profile with that of a cycling female in the same population. As part of a larger study, we recorded sociosexual behaviors in adult and subadult females belonging to two groups, and we collected urine (n = 877) to determine the sex hormone profile of each female. These samples were analyzed using enzyme immunoassays for estrone conjugates and pregnanediol-3-glucuronide (PdG). We found differences in mean values of PdG between the younger (cycling) and the older female. These hormone values were lower in the older female, and she did not show any signs of cyclicity for either reproductive hormone. Our results show that the aging female in this wild population shows signs of ovarian senescence, indicated by low, acyclic levels of progesterone metabolites.

Low Levels of Fruit Nitrogen as Drivers for the Evolution of Madagascar's Primate Communities.

The uneven representation of frugivorous mammals and birds across tropical regions - high in the New World, low in Madagascar and intermediate in Africa and Asia - represents a long-standing enigma in ecology. Several hypotheses have been proposed to explain these differences but the ultimate drivers remain unclear. Here, we tested the hypothesis that fruits in Madagascar contain insufficient nitrogen to meet primate metabolic requirements, thus constraining the evolution of frugivory. We performed a global analysis of nitrogen in fruits consumed by primates, as collated from 79 studies. Our results showed that average frugivory among lemur communities was lower compared to New World and Asian-African primate communities. Fruits in Madagascar contain lower average nitrogen than those in the New World and Old World. Nitrogen content in the overall diets of primate species did not differ significantly between major taxonomic radiations. There is no relationship between fruit protein and the degree of frugivory among primates either globally or within regions, with the exception of Madagascar. This suggests that low protein availability in fruits influences current lemur communities to select for protein from other sources, whereas in the New World and Old World other factors are more significant in shaping primate communities.

The Effects of Captivity on the Mammalian Gut Microbiome.

Recent studies increasingly note the effect of captivity or the built environment on the microbiome of humans and other animals. As symbiotic microbes are essential to many aspects of biology (e.g., digestive and immune functions), it is important to understand how lifestyle differences can impact the microbiome, and, consequently, the health of hosts. Animals living in captivity experience a range of changes that may influence the gut bacteria, such as diet changes, treatments, and reduced contact with other individuals, species and variable environmental substrates that act as sources of bacterial diversity. Thus far, initial results from previous studies point to a pattern of decreased bacterial diversity in captive animals. However, these studies are relatively limited in the scope of species that have been examined. Here we present a dataset that includes paired wild and captive samples from mammalian taxa across six Orders to investigate generalizable patterns of the effects captivity on mammalian gut bacteria. In comparing the wild to the captive condition, our results indicate that alpha diversity of the gut bacteria remains consistent in some mammalian hosts (bovids, giraffes, anteaters, and aardvarks), declines in the captive condition in some hosts (canids, primates, and equids), and increases in the captive condition in one host taxon (rhinoceros). Differences in gut bacterial beta diversity between the captive and wild state were observed for most of the taxa surveyed, except the even-toed ungulates (bovids and giraffes). Additionally, beta diversity variation was also strongly influenced by host taxonomic group, diet type, and gut fermentation physiology. Bacterial taxa that demonstrated larger shifts in relative abundance between the captive and wild states included members of the Firmicutes and Bacteroidetes. Overall, the patterns that we observe will inform a range of disciplines from veterinary practice to captive breeding efforts for biological conservation. Furthermore, bacterial taxa that persist in the captive state provide unique insight into symbiotic relationships with the host.

Detection of the mosquito-borne flaviviruses, West Nile, Dengue, Saint Louis Encephalitis, Ilheus, Bussuquara, and Yellow Fever in free-ranging black howlers (Alouatta caraya) of Northeastern Argentina.

Several medically important mosquito-borne flaviviruses have been detected in Argentina in recent years: Dengue (DENV), St. Louis encephalitis (SLEV), West Nile (WNV) and Yellow Fever (YFV) viruses. Evidence of Bussuquara virus (BSQV) and Ilheus virus (ILHV) activity were found, but they have not been associated with human disease. Non-human primates can act as important hosts in the natural cycle of flaviviruses and serological studies can lead to improved understanding of virus circulation dynamics and host susceptibility. From July-August 2010, we conducted serological and molecular surveys in free-ranging black howlers (Alouatta caraya) captured in northeastern Argentina. We used 90% plaque-reduction neutralization tests (PRNT90) to analyze 108 serum samples for antibodies to WNV, SLEV, YFV, DENV (serotypes 1and 3), ILHV, and BSQV. Virus genome detection was performed using generic reverse transcription (RT)-nested PCR to identify flaviviruses in 51 antibody-negative animals. Seventy animals had antibodies for one or more flaviviruses for a total antibody prevalence of 64.8% (70/108). Monotypic (13/70, 19%) and heterotypic (27/70, 39%) patterns were differentiated. Specific neutralizing antibodies against WNV, SLEV, DENV-1, DENV-3, ILHV, and BSQV were found. Unexpectedly, the highest flavivirus antibody prevalence detected was to WNV with 9 (8.33%) monotypic responses. All samples tested by (RT)-nested PCR were negative for viral genome. This is the first detection of WNV-specific antibodies in black howlers from Argentina and the first report in free-ranging non-human primates from Latin-American countries. Given that no animals had specific neutralizing antibodies to YFV, our results suggest that the study population remains susceptible to YFV. Monitoring of these agents should be strengthened to detect the establishment of sylvatic cycles of flaviviruses in America and evaluate risks to wildlife and human health.

Validation of an enzyme immunoassay and comparison of fecal cortisol metabolite levels in black and gold howler monkeys (Alouatta caraya) inhabiting fragmented and continuous areas of the humid Chaco region, Argentina.

In the last years, the study of how environmental stimuli influence the physiology and specifically the endocrinology of an organism became increasingly important, relying mainly on the quantification of glucocorticoids to monitor animal welfare. Most studies investigating cortisol levels in primates were focused on the impact of social stressors; however, a major concern for the conservation of howler monkeys is the increased habitat fragmentation led by the advancement of the agricultural frontier. We compared fecal cortisol metabolite levels (FGCM) in howler monkeys (Alouatta caraya) living in fragmented and continuous forests of the Argentine humid Chaco region, throughout the warm season (spring-summer). Fecal samples (n = 114) were collected from adult individuals, and steroid extracts analyzed with an enzyme immunoassay also validated in this work. Parallel displacement curves were obtained between dilutions of pooled fecal extracts and the cortisol standard curve (r2 = 0.99; P = 0.23). Efficiency of the fecal extraction procedure was 79.4% ± 38%; recovery of exogenous hormone added to fecal extracts indicated a low interference of components in the feces with antibody binding. The exogenous administration of ACTH in captive-bred animals demonstrated a "cause-and-effect" relationship between the adrenal gland activation and increased FGCM levels. Contrary to our initial prediction, we were not able to demonstrate a significant difference in FGCM levels of caraya monkeys inhabiting the continuous versus fragmented habitats in our study site (83.2 ± 4.9 ng/g [n = 10 individuals] vs. 71.5 ± 4.9 ng/g [n = 7 individuals]; P = 0.29); this could be the result of low levels of disturbance imposed by a moderate and selective logging, which has proved to be beneficial for this species with high resilience by adjusting their diet to cope with feeding in degraded habitats but with new leaves and buds. Regardless of the habitat, cortisol metabolites were significantly higher in females than in males (86.4 ± 4.2 ng/g [n = 12 individuals] vs. 60.7 ± 5.0 ng/g [n = 5 individuals] respectively; P = 0.007). RESEARCH HIGHLIGHTS: Validation of an enzyme immunoassay and comparison of fecal cortisol metabolite levels in black and gold howler monkeys (Alouatta caraya) inhabiting fragmented and continuous areas of the humid Chaco region, Argentina. Contrary to our initial prediction, no significant differences in Alouatta caraya fecal cortisol metabolite levels were detected; cortisol metabolites were significantly higher in females. Probably, animals adjusted their diet to cope with feeding in degraded habitats, but with new leaves and buds.