The behaviour and activity budgets of two sympatric sloths; Bradypus variegatus and Choloepus hoffmanni.

It is usually beneficial for species to restrict activity to a particular phase of the 24-hour cycle as this enables the development of morphological and behavioural adaptations to enhance survival under specific biotic and abiotic conditions. Sloth activity patterns are thought to be strongly related to the environmental conditions due to the metabolic consequences of having a low and highly variable core body temperature. Understanding the drivers of sloth activity and their ability to withstand environmental fluctuations is of growing importance for the development of effective conservation measures, particularly when we consider the vulnerability of tropical ecosystems to climate change and the escalating impacts of anthropogenic activities in South and Central America. Unfortunately, the cryptic nature of sloths makes long term observational research difficult and so there is very little existing literature examining the behavioural ecology of wild sloths. Here, we used micro data loggers to continuously record, for the first time, the behaviour of both Bradypus and Choloepus sloths over periods of days to weeks. We investigate how fluctuations in the environmental conditions affect the activity of sloths inhabiting a lowland rainforest on the Caribbean coast of Costa Rica and examined how this might relate to their low power lifestyle. Both Bradypus and Choloepus sloths were found to be cathemeral in their activity, with high levels of between-individual and within-individual variation in the amounts of time spent active, and in the temporal distribution of activity over the 24-hour cycle. Daily temperature did not affect activity, although Bradypus sloths were found to show increased nocturnal activity on colder nights, and on nights following colder days. Our results demonstrate a distinct lack of synchronicity within the same population, and we suggest that this pattern provides sloths with the flexibility to exploit favourable environmental conditions whilst reducing the threat of predation.

Pump the brakes! The hindlimbs of three-toed sloths decelerate and support suspensory locomotion.

Modern tree sloths are one of few mammalian taxa for which quadrupedal suspension is obligatory. Sloth limb musculature is specialized for slow velocity, large force contractions that stabilize their body below branches and conserve energy during locomotion. However, it is unknown whether two- and three-toed sloths converge in their use of limb kinetics and if these patterns are comparable to how primates perform arboreal suspensory locomotion. This study addressed this need by collecting limb loading data in three-toed sloths (Bradypus variegatus; N=5) during suspensory walking. Sloths performed locomotor trials at their preferred speed on an instrumented beam apparatus with a force platform as the central supporting segment. Peak forces and impulses of the forelimb and hindlimb were recorded and analyzed in three dimensions. The hindlimbs of B. variegatus apply large braking forces greater in magnitude than peak forces generated by the forelimbs in propulsion, a pattern consistent with that observed in two-toed sloths. However, B. variegatus exhibits hindlimb-biased body weight support in vertical peak forces and impulse, with appreciable laterally directed forces in each limb pair, both of which vary from limb loading distributions in two-toed sloths. Moreover, body weight distribution between limb pairs is opposite to that employed by primates during quadrupedal suspension. Thus, there appear to be multiple strategies for achieving suspensory locomotion in arboreal mammals. These differences may be attributable to anatomical variation or phylogenetic position, but as of yet an explanation remains unknown. Future EMG analyses are expected to provide insight into how specific hindlimb muscle groups contribute to braking forces and stabilizing the center of mass of sloths during suspension.

Three toes and three modes: Dynamics of terrestrial, suspensory, and vertical locomotion in brown-throated three-toed sloths (Bradypodidae, Xenarthra).

Living sloths exhibit numerous anatomical specializations towards inverted quadrupedalism, however, previous studies have noted a more varied locomotor repertoire than previously anticipated. In this study, we present spatiotemporal gait characteristics and triaxial kinetic data from the brown-throated three-toed sloth (Bradypus variegatus) across three locomotor modes: terrestrial quadrupedal "crawling", suspensory walking, and vertical climbing. Compared to quadrupedal crawling and suspensory walking, B. variegatus adopted longer contact times and stride durations, larger duty factors, and greater speed during vertical climbing. Net fore-aft impulses were significantly greater during vertical climbing in both limb pairs than in quadrupedal crawling and suspensory walking. Functionally, during quadrupedal crawling and vertical climbing, both limb pairs served propulsive roles, while differentiation between a propulsive forelimb and braking hindlimb was observed during suspension. Net tangential forces differentiated vertical climbing kinetics from the other modes of locomotion, with the introduction of bidirectional pulling and pushing forces in the forelimb and hindlimb, respectively. The net mediolateral impulses were similar in vertical climbing and quadrupedal crawling as both limb pairs directed forces in one direction, whereas during suspensory walking, the laterally dominant forelimb was opposed by the medially dominant hindlimb. In total, this study provides novel data on the diverse locomotor dynamics in a slow-moving arboreal tetrapod and posits new testable hypotheses about the neuroplasticity and ease of transitioning between locomotor behaviors. The strikingly similar kinetic profiles of quadrupedal crawling and suspensory walking compared to vertical climbing suggest shared neuromuscular and mechanical demands between these mirrored locomotor modes.

Differences in fungal communities in the fur of two- and three-toed sloths revealed by ITS metabarcoding.

Sloths have dense fur on which insects, algae, bacteria and fungi coexist. Previous studies using cultivation-dependent methods and 18S rRNA sequencing revealed that the fungal communities in their furs comprise members of the phyla Ascomycota and Basidiomycota. In this note, we increase the resolution and knowledge of the mycobiome inhabiting the fur of the two- (Choloepus hoffmanni) and three-toed (Bradypus variegatus) sloths. Targeted amplicon metagenomic analysis of ITS2 nrDNA sequences obtained from 10 individuals of each species inhabiting the same site revealed significant differences in the structure of their fungal communities and also in the alpha-diversity estimators. The results suggest a specialization by host species and that the host effect is stronger than that of sex, age and animal weight. Capnodiales were the dominant order in sloths' fur and Cladosporium and Neodevriesia were the most abundant genera in Bradypus and Choloepus, respectively. The fungal communities suggest that the green algae that inhabit the fur of sloths possibly live lichenized with Ascomycota fungal species. The data shown in this note offer a more detailed view of the fungal content in the fur of these extraordinary animals and could help explain other mutualistic relationships in this complex ecosystem.

Antibiotic-producing Micrococcales govern the microbiome that inhabits the fur of two- and three-toed sloths.

Sloths have a dense coat on which insects, algae and fungi coexist in a symbiotic relationship. This complex ecosystem requires different levels of controls; however, most of these mechanisms remain unknown. We investigated the bacterial communities inhabiting the hair of two- (Choloepus Hoffmanni) and three-toed (Bradypus variegatus) sloths and evaluated their potential for producing antibiotic molecules capable of exerting control over the hair microbiota. The analysis of 16S rRNA amplicon sequence variants revealed that the communities in both host species are dominated by Actinobacteriota and Firmicutes. The most abundant genera were Brevibacterium, Kocuria/Rothia, Staphylococcus, Rubrobacter, Nesterenkonia and Janibacter. Furthermore, we isolated nine strains of Brevibacterium and Rothia capable of producing substances that inhibited the growth of common mammalian pathogens. The analysis of the biosynthetic gene clusters of these nine isolates suggests that the pathogen-inhibitory activity could be mediated by the presence of siderophores, terpenes, beta-lactones, Type III polyketide synthases, ribosomally synthesized and post-translationally modified peptides, non-alpha poly-amino acids like e-Polylysine, ectoine or non-ribosomal peptides. Our data suggest that Micrococcales that inhabit sloth hair could have a role in controlling microbial populations in that habitat, improving our understanding of this highly complex ecosystem.

Myology of the pelvic limb of the brown-throated three-toed sloth (Bradypus variegatus).

Tree sloths rely on their limb flexors for bodyweight support and joint stability during suspensory locomotion and posture. This study aims to describe the myology of three-toed sloths and identify limb muscle traits that indicate modification for suspensorial habit. The pelvic limbs of the brown-throated three-toed sloth (Bradypus variegatus) were dissected, muscle belly mass was recorded, and the structural arrangements of the muscles were documented and compared with the available myological accounts for sloths. Overall, the limb musculature is simplified by containing muscles with generally long and parallel fascicles. A number of specific and informative muscle traits are additionally observed in the pelvic limb of B. variegatus: well-developed hip flexors and hip extensors each displaying several fused bellies; massive knee flexors; two heads of the m. adductor longus and m. gracilis; robust digital flexors and flexor tendons; m. tibialis cranialis muscle complex originating from the tibia and fibula and containing a modified m. extensor digitorum I longus; appreciable muscle mass devoted to ankle flexion and hindfoot supination; only m. extensor digitorum brevis acts to extend the digits. Collectively, the findings for tree sloths emphasize muscle mass and organization for suspensory support namely by the hip flexors, knee flexors, and limb adductors, for which the latter two groups may stabilize suspensory postures by exerting appreciable medially-directed force on the substrate. Specializations in the distal limb are also apparent for sustained purchase of the substrate by forceful digital flexion coupled with strong ankle flexion and supination of the hind feet, which is permitted by the reorganization of several digital extensors. Moreover, the reduction or loss of other digital flexor and ab-adductor muscles marks a dramatic simplification of the intrinsic foot musculature in B. variegatus, the extent to which varies across extant species of two- and three-toed tree sloths and likely is related to substrate preference/use.

Coming to grips with life upside down: how myosin fiber type and metabolic properties of sloth hindlimb muscles contribute to suspensory function.

Sloths exhibit almost obligatory suspensory locomotion and posture. These behaviors require both strength and fatigue resistance, although we previously found muscle fiber type characteristics in the forelimbs of sloths that belied these initial expectations. Based on locomotor roles of the forelimbs versus hindlimbs in propulsion and braking, respectively, sloth hindlimb musculature should be adapted for force production and energy savings by a near homogeneous expression of slow myosin heavy chain (MHC) fibers. This hypothesis was tested by determining MHC fiber type (%) distribution and energy metabolism in the hindlimbs of three-toed (B. variegatus, N = 5) and two-toed (C. hoffmanni, N = 3) sloths. A primary expression of the slow MHC-1 isoform was found in the hindlimbs of both species. Slow MHC fiber type (%) was significantly greater in the flexors of B. variegatus, whereas expression of fast MHC-2A fibers was significantly greater in the extensors of C. hoffmannni. MHC-1 fibers were largest in cross-sectional area (CSA) and comprised the greatest %CSA in each muscle sampled from both species. Enzyme assays showed elevated activity for anaerobic enzymes (CK and LDH) compared with low-to-moderate activity for aerobic enzymes (3-HAD and CS), and only CK activity was related to body size. These findings emphasize a joint stabilization role by the hindlimbs during suspension, especially in smaller three-toed sloths, and suggest that larger two-toed sloths could have muscles further modified for greater power output and/or prolonged arboreal maneuvering. Moreover, modifications to muscle metabolism rather than MHC expression may be more reflective of functional adaptation in sloth limbs.

Genetic divergence and evidence of human-mediated translocation of two-fingered sloths (Choloepus hoffmanni) in Costa Rica.

Sloths are notoriously slow and consequently have limited dispersal ability, which makes them particularly vulnerable to the effects of habitat fragmentation and degradation. Sloths in Costa Rica are considered of conservation concern due to habitat loss, livestock production and increasing urbanization. Reintroductions from rescue centres are commonplace across the country, yet their genetic diversity and population structure are unknown, and there is currently little consideration of the genetic background prior to intervention or releases. We used microsatellite analysis to undertake the first exploratory investigation into sloth population genetics in Costa Rica. Using data from 98 two-fingered sloths (Choloepus hoffmanni) from four different geographic regions, we determined the presence of four potential genetic groups, three of them with minimal population structuring despite the limited dispersal ability and presence of physical barriers. Sloths from the North appear to represent a highly distinct population that we propose may require management as a discrete unit for conservation. We stress the need for additional analyses to better understand the genetic structure and diversity of North andWest regions and suggest that rescue facilities in Costa Rica should consider the genetic background of rehabilitated sloths when planning future reintroductions. Our results also highlight the threat posed by physical isolation due to widespread urbanization and agriculture expansion for a species with a weak dispersal ability.

Cross-order host switches of hepatitis C-related viruses illustrated by a novel hepacivirus from sloths.

The genealogy of the hepatitis C virus (HCV) and the genus Hepacivirus remains elusive despite numerous recently discovered animal hepaciviruses (HVs). Viruses from evolutionarily ancient mammals might elucidate the HV macro-evolutionary patterns. Here, we investigated sixty-seven two-toed and nine three-toed sloths from Costa Rica for HVs using molecular and serological tools. A novel sloth HV was detected by reverse transcription polymerase chain reaction (RT-PCR) in three-toed sloths (2/9, 22.2%; 95% confidence interval (CI), 5.3-55.7). Genomic characterization revealed typical HV features including overall polyprotein gene structure, a type 4 internal ribosomal entry site in the viral 5'-genome terminus, an A-U-rich region and X-tail structure in the viral 3'-genome terminus. Different from other animal HVs, HV seropositivity in two-toed sloths was low at 4.5 per cent (3/67; CI, 1.0-12.9), whereas the RT-PCR-positive three-toed sloths were seronegative. Limited cross-reactivity of the serological assay implied exposure of seropositive two-toed sloths to HVs of unknown origin and recent infections in RT-PCR-positive animals preceding seroconversion. Recent infections were consistent with only 9 nucleotide exchanges between the two sloth HVs, located predominantly within the E1/E2 encoding regions. Translated sequence distances of NS3 and NS5 proteins and host comparisons suggested that the sloth HV represents a novel HV species. Event- and sequence distance-based reconciliations of phylogenies of HVs and of their hosts revealed complex macro-evolutionary patterns, including both long-term evolutionary associations and host switches, most strikingly from rodents into sloths. Ancestral state reconstructions corroborated rodents as predominant sources of HV host switches during the genealogy of extant HVs. Sequence distance comparisons, partial conservation of critical amino acid residues associated with HV entry and selection pressure signatures of host genes encoding entry and antiviral protein orthologs were consistent with HV host switches between genetically divergent mammals, including the projected host switch from rodents into sloths. Structural comparison of HCV and sloth HV E2 proteins suggested conserved modes of hepaciviral entry. Our data corroborate complex macro-evolutionary patterns shaping the genus Hepacivirus, highlight that host switches are possible across highly diverse host taxa, and elucidate a prominent role of rodent hosts during the Hepacivirus genealogy.

Keep calm and hang on: EMG activation in the forelimb musculature of three-toed sloths (Bradypus variegatus).

Sloths exhibit below branch locomotion whereby their limbs are loaded in tension to support the body weight. Suspensory behaviors require both strength and fatigue resistance from the limb flexors; however, skeletal muscle mass of sloths is reduced compared with other arboreal mammals. Although suspensory locomotion demands that muscles are active to counteract the pull of gravity, it is possible that sloths minimize muscle activation and/or selectively recruit slow motor units to maintain support, thus indicating neuromuscular specializations to conserve energy. Electromyography (EMG) was evaluated in a sample of three-toed sloths (Bradypus variegatus; N=6) to test this hypothesis. EMG was recorded at 2000 Hz via fine-wire electrodes implanted into two suites of four muscles in the left forelimb while sloths performed suspensory hanging (SH), suspensory walking (SW) and vertical climbing (VC). All muscles were minimally active for SH. During SW and VC, sloths moved slowly (duty factor: 0.83) and activation patterns were consistent between behaviors; the flexors were activated early and for a large percentage of limb contact, whereas the extensors were activated for shorter burst durations on average and showed biphasic (contact and swing) activity. Muscle activities were maximal for the elbow flexors and lowest for the carpal/digital flexors, and overall activity was significantly greater for SW and VC compared with SH. Wavelet analysis indicated high mean EMG frequencies from the myoelectric intensity spectra coupled with low burst intensities for SH, although the opposite pattern occurred for SW and VC, with the shoulder flexors and elbow flexor, m. brachioradialis, having extremely low mean EMG frequencies that are consistent with recruitment of slow fibers. Collectively, these findings support the hypothesis and suggest that sloths may selectively recruit smaller, fast motor units for suspensory postures but have the ability to offset the cost of force production by recruitment of large, slow motor units during locomotion.

Sloths host Anhanga virus-related phleboviruses across large distances in time and space.

Sloths are genetically and physiologically divergent mammals. Phleboviruses are major arthropod-borne viruses (arboviruses) causing disease in humans and other animals globally. Sloths host arboviruses, but virus detections are scarce. A phlebovirus termed Anhanga virus (ANHV) was isolated from a Brazilian Linnaeus's two-toed sloth (Choloepus didactylus) in 1962. Here, we investigated the presence of phleboviruses in sera sampled in 2014 from 74 Hoffmann's two-toed (Choloepus hoffmanni, n = 65) and three-toed (Bradypus variegatus, n = 9) sloths in Costa Rica by broadly reactive RT-PCR. A clinically healthy adult Hoffmann's two-toed sloth was infected with a phlebovirus. Viral load in this animal was high at 8.5 × 107  RNA copies/ml. The full coding sequence of the virus was determined by deep sequencing. Phylogenetic analyses and sequence distance comparisons revealed that the new sloth virus, likely representing a new phlebovirus species, provisionally named Penshurt virus (PEHV), was most closely related to ANHV, with amino acid identities of 93.1%, 84.6%, 94.7% and 89.0% in the translated L, M, N and NSs genes, respectively. Significantly more non-synonymous mutations relative to ANHV occurred in the M gene encoding the viral glycoproteins and in the NSs gene encoding a putative interferon antagonist compared to L and N genes. This was compatible with viral adaptation to different sloth species and with micro-evolutionary processes associated with immune evasion during the genealogy of sloth-associated phleboviruses. However, gene-wide mean dN/dS ratios were low at 0.02-0.15 and no sites showed significant evidence for positive selection, pointing to comparable selection pressures within sloth-associated viruses and genetically related phleboviruses infecting hosts other than sloths. The detection of a new phlebovirus closely-related to ANHV, in sloths from Costa Rica fifty years after and more than 3,000 km away from the isolation of ANHV confirmed the host associations of ANHV-related phleboviruses with the two extant species of two-toed sloths.

The metabolic response of the Bradypus sloth to temperature.

Poikilotherms and homeotherms have different, well-defined metabolic responses to ambient temperature (T a ), but both groups have high power costs at high temperatures. Sloths (Bradypus) are critically limited by rates of energy acquisition and it has previously been suggested that their unusual departure from homeothermy mitigates the associated costs. No studies, however, have examined how sloth body temperature and metabolic rate vary with T a . Here we measured the oxygen consumption (VO2) of eight brown-throated sloths (B. variegatus) at variable T a 's and found that VO2 indeed varied in an unusual manner with what appeared to be a reversal of the standard homeotherm pattern. Sloth VO2 increased with T a , peaking in a metabolic plateau (nominal 'thermally-active zone' (TAZ)) before decreasing again at higher T a values. We suggest that this pattern enables sloths to minimise energy expenditure over a wide range of conditions, which is likely to be crucial for survival in an animal that operates under severe energetic constraints. To our knowledge, this is the first evidence of a mammal provisionally invoking metabolic depression in response to increasing T a 's, without entering into a state of torpor, aestivation or hibernation.

Cheap labor: myosin fiber type expression and enzyme activity in the forelimb musculature of sloths (Pilosa: Xenarthra).

Sloths are canopy-dwelling inhabitants of American neotropical rainforests that exhibit suspensory behaviors. These abilities require both strength and muscular endurance to hang for extended periods of time; however, the skeletal muscle mass of sloths is reduced, thus requiring modifications to muscle architecture and leverage for large joint torque. We hypothesize that intrinsic muscle properties are also modified for fatigue resistance and predict a heterogeneous expression of slow/fast myosin heavy chain (MHC) fibers that utilize oxidative metabolic pathways for economic force production. MHC fiber type distribution and energy metabolism in the forelimb muscles of three-toed ( Bradypus variegatus, n = 5) and two-toed ( Choloepus hoffmanni, n = 4) sloths were evaluated using SDS-PAGE, immunohistochemistry, and enzyme activity assays. The results partially support our hypothesis by a primary expression of the slow MHC-1 isoform as well as moderate expression of fast MHC-2A fibers, whereas few hybrid MHC-1/2A fibers were found in both species. MHC-1 fibers were larger in cross-sectional area (CSA) than MHC-2A fibers and comprised the greatest percentage of CSA in each muscle sampled. Enzyme assays showed elevated activity for the anaerobic enzymes creatine kinase and lactate dehydrogenase compared with low activity for aerobic markers citrate synthase and 3-hydroxyacetyl CoA dehydrogenase. These findings suggest that sloth forelimb muscles may rely heavily on rapid ATP resynthesis pathways, and lactate accumulation may be beneficial. The intrinsic properties observed match well with suspensory requirements, and these modifications may have further evolved in unison with low metabolism and slow movement patterns as means to systemically conserve energy. NEW & NOTEWORTHY Myosin heavy chain (MHC) fiber type and fiber metabolic properties were evaluated to understand the ability of sloths to remain suspended for extended periods without muscle fatigue. Broad distributions of large, slow MHC-1 fibers as well as small, fast MHC-2A fibers are expressed in sloth forelimbs, but muscle metabolism is generally not correlated with myosin fiber type or body size. Sloth muscles rely on rapid, anaerobic pathways to resist fatigue and sustain force production.

Sloths like it hot: ambient temperature modulates food intake in the brown-throated sloth (Bradypus variegatus).

Sloths are considered to have one of the lowest mass-specific metabolic rates of any mammal and, in tandem with a slow digestive rate, have been theorized to have correspondingly low rates of ingestion. Here, we show in a study conducted over five months, that three captive Bradypus variegatus (Brown-throated sloths) had a remarkably low mean food intake of 17 g kg(-1)day(-1) (SD 4.2). Food consumption was significantly affected by ambient temperature, with increased intake at higher temperatures. We suggest that the known fluctuation of sloth core body temperature with ambient temperature affects the rate at which gut fauna process digesta, allowing for increased rates of fermentation at higher temperatures. Since Bradypus sloths maintain a constantly full stomach, faster rates of fermentation should enhance digestive throughput, increasing the capacity for higher levels of food intake, thereby allowing increased energy acquisition at higher ambient temperatures. This contrasts with other mammals, which tend to show increased levels of food intake in colder conditions, and points to the importance of temperature in regulating all aspects of energy use in sloths.

Mitigating the squash effect: sloths breathe easily upside down.

Sloths are mammals renowned for spending a large proportion of time hanging inverted. In this position, the weight of the abdominal contents is expected to act on the lungs and increase the energetic costs of inspiration. Here, we show that three-fingered sloths Bradypus variegatus possess unique fibrinous adhesions that anchor the abdominal organs, particularly the liver and glandular stomach, to the lower ribs. The key locations of these adhesions, close to the diaphragm, prevent the weight of the abdominal contents from acting on the lungs when the sloth is inverted. Using ventilation rate and body orientation data collected from captive and wild sloths, we use an energetics-based model to estimate that these small adhesions could reduce the energy expenditure of a sloth at any time it is fully inverted by almost 13%. Given body angle preferences for individual sloths in our study over time, this equates to mean energy saving of 0.8-1.5% across individuals (with individual values ranging between 0.01 and 8.6%) per day. Given the sloth's reduced metabolic rate compared with other mammals and extremely low energy diet, these seemingly innocuous adhesions are likely to be important in the animal's energy budget and survival.