Combined effects of ambient temperature and food availability on induced innate immune response of a fruit-eating bat (Carollia perspicillata).

Resilience of mammals to anthropogenic climate and land-use changes is associated with the maintenance of adequate responses of several fitness-related traits such as those related to immune functions. Isolated and combined effects of decreased food availability and increased ambient temperature can lead to immunosuppression and greater susceptibility to disease. Our study tested the general hypothesis that decreased food availability, increased ambient temperature and the combined effect of both factors would affect selected physiological and behavioral components associated with the innate immune system of fruit-eating bats (Carollia perspicillata). Physiological (fever, leukocytosis and neutrophil/lymphocyte ratio) and behavioral (food intake) components of the acute phase response, as well as bacterial killing ability of the plasma were assessed after immune challenge with lipopolysaccharide (LPS: 10 mg/kg) in experimental groups kept at different short-term conditions of food availability (ad libitum diet or 50% food-deprived) and ambient temperature (27 and 33°C). Our results indicate that magnitude of increase in body temperature was not affected by food availability, ambient temperature or the interaction of both factors, but the time to reach the highest increase took longer in LPS-injected bats that were kept under food restriction. The magnitude of increased neutrophil/lymphocyte ratio was affected by the interaction between food availability and ambient temperature, but food intake, total white blood cell count and bacterial killing ability were not affected by any factor or interaction. Overall, our results suggest that bacterial killing ability and most components of acute phase response examined are not affected by short-term changes in food availability and ambient temperature within the range evaluated in this study, and that the increase of the neutrophil/lymphocyte ratio when bats are exposed to low food availability and high ambient temperature might represent an enhancement of cellular response to deal with infection.

Non-destructive methods to assess pesticide exposure in free-living bats.

Bat populations are dwindling worldwide due to anthropogenic activities like agriculture, however the role that pesticide exposure plays on these declines is unclear. To address these research gaps, we first need to develop reliable methods to detect and monitor exposure to environmental pollutants and its effects on free-living bats. The use of biomarkers is a sensitive and informative tool to study sublethal effects in wildlife, however it requires laboratory validation and integrative approaches to be applicable to free-living species. In this study, we propose a set of non-destructive biomarkers to evaluate pesticide exposure in free-ranging bats and validated their suitability with dose-exposure experiments in captivity. We selected three biomarkers that have been widely used in vertebrate ecotoxicology and that combined represent sensitive, specific, and ecologically relevant responses to pollutants: DNA damage, AChE activity, and leukocyte profiles. We used two insectivorous bat species as model species Eptesicus fuscus (laboratory) and Pteronotus mexicanus (field). We found that micronuclei frequency (genotoxicity) and AChE activity (exposure and neurotoxicity) were robust indicators of toxicant exposure. The validity of this set of endpoints was supported by their consistent performance in laboratory and field experiments as well as by the significant correlation among them. Leukocyte profile (systemic stress) results were not consistent between laboratory and field studies, suggesting further evaluation of its suitability is needed. Integrative approaches, like the one we used here, maximize the insights about toxicant effects by combining the information of single biomarkers into more meaningful inferences, which can be applied to environmental risk assessments in wildlife. Furthermore, the use of non-destructive, cost-effective biomarkers is imperative when assessing toxicant exposure and effects in vulnerable wildlife and it should be a priority in the field of wildlife toxicology.

Sick bats stay home alone: fruit bats practice social distancing when faced with an immunological challenge.

Along with its many advantages, social roosting imposes a major risk of pathogen transmission. How social animals reduce this risk is poorly documented. We used lipopolysaccharide challenge to imitate bacterial infection in both a captive and a free-living colony of an extremely social, long-lived mammal-the Egyptian fruit bat. We monitored behavioral and physiological responses using an arsenal of methods, including onboard GPS to track foraging, acceleration sensors to monitor movement, infrared video to record social behavior, and blood samples to measure immune markers. Sick-like (immune-challenged) bats exhibited an increased immune response, as well as classic illness symptoms, including fever, weight loss, anorexia, and lethargy. Notably, the bats also exhibited behaviors that would reduce pathogen transfer. They perched alone and appeared to voluntarily isolate themselves from the group by leaving the social cluster, which is extremely atypical for this species. The sick-like individuals in the open colony ceased foraging outdoors for at least two nights, thus reducing transmission to neighboring colonies. Together, these sickness behaviors demonstrate a strong, integrative immune response that promotes recovery of infected individuals while reducing pathogen transmission inside and outside the roost, including spillover events to other species, such as humans.

DNA methylation predicts age and provides insight into exceptional longevity of bats.

Exceptionally long-lived species, including many bats, rarely show overt signs of aging, making it difficult to determine why species differ in lifespan. Here, we use DNA methylation (DNAm) profiles from 712 known-age bats, representing 26 species, to identify epigenetic changes associated with age and longevity. We demonstrate that DNAm accurately predicts chronological age. Across species, longevity is negatively associated with the rate of DNAm change at age-associated sites. Furthermore, analysis of several bat genomes reveals that hypermethylated age- and longevity-associated sites are disproportionately located in promoter regions of key transcription factors (TF) and enriched for histone and chromatin features associated with transcriptional regulation. Predicted TF binding site motifs and enrichment analyses indicate that age-related methylation change is influenced by developmental processes, while longevity-related DNAm change is associated with innate immunity or tumorigenesis genes, suggesting that bat longevity results from augmented immune response and cancer suppression.

Synergetic effects of immune challenge and stress depress cortisol, inflammatory response and antioxidant activity in fish-eating Myotis.

One of the most common tools in conservation physiology is the assessment of environmental stress via glucocorticoid measurement. However, little is known of its relationship with other stress-related biomarkers, and how the incidence of an immune challenge during long-term stress could affect an individual's overall stress response. We investigated here the relationship between basal and post-acute stress fecal cortisol metabolite (FC) with different antioxidant enzymes, oxidative damage and immune parameters in the fish-eating bat, Myotis vivesi We found that in both basal and post-stress conditions, FC was highly related with a number of antioxidant enzymes and immune parameters, but not to oxidative damage. We also assessed changes of FC through the seasons. Basal FC samples and stress reactivity after short-duration stress displayed similar levels during summer, autumn and early winter, but lower concentrations in late winter. Stress reactivity after long-duration stress was greater in summer and early winter. Finally, we tested the effect of a simultaneous exposure to a long, strong stress stimulus with an immune response stimulation by administrating adrenocorticotropic hormone (ACTH) and phytohemagglutinin (PHA) after 42 h. Results showed that when both stimuli were administrated, FC concentrations, inflammation and some antioxidant activity were lowered in comparison with the control and individual administration of the challenges. Our findings support the idea that animals maintain constant basal glucocorticoid levels when living in challenging environments, but response to acute stress differs seasonally and immune defense mechanisms and stress responses might be compromised when confronted with multiple challenges.

Bats respond to simulated bacterial infection during the active phase by reducing food intake.

Sickness triggers a series of behavioral and physiological processes collectively known as acute phase response (APR). Bats are known as reservoirs of a broad variety of pathogens and the physiological changes resulting from APR activation have been tested predominantly during the resting phase (daytime) in several species exposed to lipopolysaccharide (LPS). In contrast, behavioral consequences of sickness for bats and other wild mammals have received less attention. We examined the physiological and behavioral consequences of APR activation in a fruit-eating bat (Carollia perspicillata) challenged with LPS during the active phase (nighttime). We measured changes in food intake, body mass, body temperature, total white blood cell counts, and the neutrophil/lymphocyte ratio (N/L). No fever and leukocytosis were observed in bats injected with LPS, but food intake decreased, bats lost body mass and their N/L ratio increased. The effect of LPS on daily energy balance is remarkable and, along with the increase in N/L ratio, it is assumed to be beneficial to fight disease. On the basis of our findings and those with other bats, it is probable that the physiological and behavioral components of the immune response to LPS follow circadian rhythms, but a formal test of this hypothesis is warranted.

Salt has contrasting effects on the digestive processing of dilute nectar by two Neotropical nectarivorous bats.

Nectarivorous vertebrates might include sugar-dilute nectar in their diet and they are expected to undergo compensatory feeding. However, physiological constraints might limit the intake of sugar-dilute nectar, affecting energy budgets. Among other physiological processes, the limiting role of osmoregulation is supported by enhanced intake rate of dilute sugar solutions by avian nectarivores when salt is added. We tested if the Greater Antillean Long-tongued bat (Monophyllus redmani) and the Brown flower bat (Erophylla sezekorni) compensated energy intake when fed dilute-sugar solutions (2.5 and 5% sucrose), and if salt content (11, 20 and 40 mM NaCl l-1) modulated the intake rate of these solutions. Both species were unable to compensate intake of solutions with varying sugar densities, and energy intake on the 2.5 and 5% diets was lower than on the most concentrated diets (10, 20 and 30% sucrose). Both species responded differently to the addition of salt. Salt addition did not affect the intake of 2.5% sugar solutions by the Greater Antillean Long-tongued bat, and it decreased the intake of 5% sugar solutions. In contrast, the Brown flower bat increased the intake of 2.5 and 5% sugar solutions when salt was added. Intake responses to varying sugar densities of our two focal species and that of other bat species previously studied indicate that they are not uniform and that they might be modulated by digestive and osmoregulatory physiological traits.

Low ambient temperature reduces the time for fuel switching in the ruby-throated hummingbird (Archilochus colubris).

Physiological adaptations that enhance flux through the sugar oxidation cascade permit hummingbirds to rapidly switch between burning lipids when fasted to burning ingested sugars when fed. Hummingbirds may be able to exert control over the timing and extent of use of ingested sugars by varying digestive rates when under pressure to accumulate energy stores or acquire energy in response to heightened energy demands. We hypothesized that hummingbirds would modulate the timing of a switch to reliance on ingested sugars differently when facing distinct energetic demands (cool versus warm ambient temperatures). The timing of the oxidation of a single nectar meal to fuel metabolism was assessed by open-flow respirometry, while the time to first excretion following the meal was used as a proxy for digestive throughput time. As predicted, birds showed a more rapid switch in respiratory exchange ratio (RER = rate of O2 consumption/CO2 production) and excreted earlier when held at cool temperatures compared to warm. In both cases, RER peaked barely above 1.0 indicating ingested sugar fueled ≈100% of resting metabolism. Our findings suggest that energetic demands modulate the rate of fuel switching through shifts of the sugar oxidation cascade. The speed of this shift may involve decreases in gut passage times which have previously been thought to be inflexible, or may be caused by changes in circulation as a result of low ambient temperature.

Acoustic evaluation of behavioral states predicted from GPS tracking: a case study of a marine fishing bat.

BACKGROUND: Multiple methods have been developed to infer behavioral states from animal movement data, but rarely has their accuracy been assessed from independent evidence, especially for location data sampled with high temporal resolution. Here we evaluate the performance of behavioral segmentation methods using acoustic recordings that monitor prey capture attempts. METHODS: We recorded GPS locations and ultrasonic audio during the foraging trips of 11 Mexican fish-eating bats, Myotis vivesi, using miniature bio-loggers. We then applied five different segmentation algorithms (k-means clustering, expectation-maximization and binary clustering, first-passage time, hidden Markov models, and correlated velocity change point analysis) to infer two behavioral states, foraging and commuting, from the GPS data. To evaluate the inference, we independently identified characteristic patterns of biosonar calls ("feeding buzzes") that occur during foraging in the audio recordings. We then compared segmentation methods on how well they correctly identified the two behaviors and if their estimates of foraging movement parameters matched those for locations with buzzes. RESULTS: While the five methods differed in the median percentage of buzzes occurring during predicted foraging events, or true positive rate (44-75%), a two-state hidden Markov model had the highest median balanced accuracy (67%). Hidden Markov models and first-passage time predicted foraging flight speeds and turn angles similar to those measured at locations with feeding buzzes and did not differ in the number or duration of predicted foraging events. CONCLUSION: The hidden Markov model method performed best at identifying fish-eating bat foraging segments; however, first-passage time was not significantly different and gave similar parameter estimates. This is the first attempt to evaluate segmentation methodologies in echolocating bats and provides an evaluation framework that can be used on other species.

Food restriction, but not seasonality, modulates the acute phase response of a Neotropical bat.

Season and food intake are known to affect immune response of vertebrates yet their effects on metabolic rate have been rarely explored. We tested the effect of season and acute food restriction and their interaction on the energetic cost of immune response activation of a tropical vertebrate, the Seba's short-tailed fruit bat (Carollia perspicillata). We specifically stimulated the acute phase response (APR) with bacterial lipopolysaccharide (LPS) to measure metabolic changes along with changes in body temperature (Tb), body mass (Mb), white blood cell counts and the Neutrophil/Lymphocyte ratio (N/L). We found no effect of season on the different factors associated to the activation of the APR. In contrast to our expectations, unfed bats reached similar Tb increments and RMR peak values and had higher RMR scope values and higher caloric costs than fed bats after LPS injection. However, food deprivation led to delayed metabolic response indicated by longer time required to reach peak RMR values in unfed bats. Both food-deprived and fed bats did not present leukocytosis after APR activation and their WBC counts were similar, but unfed bats had a significant increase of N/L. APR activation represented a small fraction of the bat daily energy requirements which might explain why unfed bats were not limited to mount a metabolic response. Our study adds to recent evidence showing that activating the innate immune system is not an energetically expensive process for plant-eating bats.

The energetic cost of mounting an immune response for Pallas's long-tongued bat (Glossophaga soricina).

The acute phase response (APR) is the first line of defense of the vertebrate immune system against pathogens. Mounting an immune response is believed to be energetically costly but direct measures of metabolic rate during immune challenges contradict this assumption. The energetic cost of APR for birds is higher than for rodents, suggesting that this response is less expensive for mammals. However, the particularly large increase in metabolic rate after APR activation for a piscivorous bat (Myotis vivesi) suggests that immune response might be unusually costly for bats. Here we quantified the energetic cost and body mass change associated with APR for the nectarivorous Pallas's long-tongued bat (Glossophaga soricina). Activation of the APR resulted in a short-term decrease in body mass and an increase in resting metabolic rate (RMR) with a total energy cost of only 2% of the total energy expenditure estimated for G. soricina. This increase in RMR was far from the large increase measured for piscivorous bats; rather, it was similar to the highest values reported for birds. Overall, our results suggest that the costs of APR for bats may vary interspecifically. Measurement of the energy cost of vertebrate immune response is limited to a few species and further work is warranted to evaluate its significance for an animal's energy budget.

Baseline and post-stress seasonal changes in immunocompetence and redox state maintenance in the fishing bat Myotis vivesi.

Little is known of how the stress response varies when animals confront seasonal life-history processes. Antioxidant defenses and damage caused by oxidative stress and their link with immunocompetence are powerful biomarkers to assess animal´s physiological stress response. The aim of this study was A) to determine redox state and variation in basal (pre-acute stress) immune function during summer, autumn and winter (spring was not assessed due to restrictions in collecting permit) in the fish-eating Myotis (Myotis vivesi; Chiroptera), and B) to determine the effect of acute stress on immunocompetence and redox state during each season. Acute stress was stimulated by restricting animal movement for 6 and 12 h. The magnitude of the cellular immune response was higher during winter whilst that of the humoral response was at its highest during summer. Humoral response increased after 6 h of movement restriction stress and returned to baseline levels after 12 h. Basal redox state was maintained throughout the year, with no significant changes in protein damage, and antioxidant activity was modulated mainly in relation to variation to environment cues, increasing during high temperatures and decreasing during windy nights. Antioxidant activity increased after the 6 h of stressful stimuli especially during summer and autumn, and to a lesser extent in early winter, but redox state did not vary. However, protein damage increased after 12 h of stress during summer. Prolonged stress when the bat is engaged in activities of high energy demand overcame its capacity to maintain homeostasis resulting in oxidative damage.

Metabolic Cost of the Activation of Immune Response in the Fish-Eating Myotis (Myotis vivesi): The Effects of Inflammation and the Acute Phase Response.

Inflammation and activation of the acute phase response (APR) are energetically demanding processes that protect against pathogens. Phytohaemagglutinin (PHA) and lipopolysaccharide (LPS) are antigens commonly used to stimulate inflammation and the APR, respectively. We tested the hypothesis that the APR after an LPS challenge was energetically more costly than the inflammatory response after a PHA challenge in the fish-eating Myotis bat (Myotis vivesi). We measured resting metabolic rate (RMR) after bats were administered PHA and LPS. We also measured skin temperature (Tskin) after the LPS challenge and skin swelling after the PHA challenge. Injection of PHA elicited swelling that lasted for several days but changes in RMR and body mass were not significant. LPS injection produced a significant increase in Tskin and in RMR, and significant body mass loss. RMR after LPS injection increased by 140-185% and the total cost of the response was 6.50 kJ. Inflammation was an energetically low-cost process but the APR entailed a significant energetic investment. Examination of APR in other bats suggests that the way in which bats deal with infections might not be uniform.

The cost of digestion in the fish-eating myotis (Myotis vivesi).

Flying vertebrates, such as bats, face special challenges with regards to the throughput and digestion of food. On the one hand, as potentially energy-limited organisms, bats must ingest and assimilate energy efficiently in order to satisfy high resting and active metabolic demands. On the other hand, the assimilation of nutrients must be accomplished using a digestive tract that is, compared with that of similarly sized non-flying vertebrates, significantly shorter. Despite these competing demands, and the relative breadth of dietary diversity among bats, little work has been done describing the cost of digestion, termed 'specific dynamic action' (SDA). Here, we provide the first systematic assessment of the SDA response in a bat, the fish-eating myotis (Myotis vivesi). Given the shorter digestive tract and the relatively higher resting and active metabolic rates of bats in general, and based on anecdotal published evidence, we hypothesized that the SDA response in fish-eating myotis would be dependent on meal size and both significantly more brief and intense than in small, non-flying mammals. In agreement with our hypothesis, we found that the peak metabolic rate during digestion, relative to rest, was significantly higher in these bats compared with any other mammals or vertebrates, except for some infrequently eating reptiles and amphibians. Additionally, we found that the magnitude and duration of the SDA response were related to meal size. However, we found that the duration of the SDA response, while generally similar to reported gut transit times in other small bats, was not substantially shorter than in similarly sized non-flying mammals.

The sugar oxidation cascade: aerial refueling in hummingbirds and nectar bats.

Most hummingbirds and some species of nectar bats hover while feeding on floral nectar. While doing so, they achieve some of the highest mass-specific V(O(2)) values among vertebrates. This is made possible by enhanced functional capacities of various elements of the 'O(2) transport cascade', the pathway of O(2) from the external environment to muscle mitochondria. Fasted hummingbirds and nectar bats fly with respiratory quotients (RQs; V(CO(2))/V(O(2))) of ~0.7, indicating that fat fuels flight in the fasted state. During repeated hover-feeding on dietary sugar, RQ values progressively climb to ~1.0, indicating a shift from fat to carbohydrate oxidation. Stable carbon isotope experiments reveal that recently ingested sugar directly fuels ~80 and 95% of energy metabolism in hover-feeding nectar bats and hummingbirds, respectively. We name the pathway of carbon flux from flowers, through digestive and cardiovascular systems, muscle membranes and into mitochondria the 'sugar oxidation cascade'. O(2) and sugar oxidation cascades operate in parallel and converge in muscle mitochondria. Foraging behavior that favours the oxidation of dietary sugar avoids the inefficiency of synthesizing fat from sugar and breaking down fat to fuel foraging. Sugar oxidation yields a higher P/O ratio (ATP made per O atom consumed) than fat oxidation, thus requiring lower hovering V(O(2)) per unit mass. We propose that dietary sugar is a premium fuel for flight in nectarivorous, flying animals.

A nectar-feeding mammal avoids body fluid disturbances by varying renal function.

To maintain water and electrolyte balance, nectar-feeding vertebrates oscillate between two extremes: avoiding overhydration when feeding and preventing dehydration during fasts. Several studies have examined how birds resolve this osmoregulatory dilemma, but no data are available for nectar-feeding mammals. In this article, we 1) estimated the ability of Pallas's long-tongued bats (Glossophaga soricina; Phyllostomidae) to dilute and concentrate urine and 2) examined how water intake affected the processes that these bats use to maintain water balance. Total urine osmolality in water- and salt-loaded bats ranged between 31 +/- 37 mosmol/kgH(2)O (n = 6) and 578 +/- 56 mosmol/kgH(2)O (n = 2), respectively. Fractional water absorption in the gastrointestinal tract was not affected by water intake rate. As a result, water flux, body water turnover, and renal water load all increased with increasing water intake. Despite these relationships, glomerular filtration rate (GFR) was not responsive to water loading. To eliminate excess water, Pallas's long-tongued bats increased water excretion rate by reducing fractional renal water reabsorption. We also found that rates of total evaporative water loss increased with increasing water intake. During their natural daytime fast, mean GFR in Pallas's long-tongued bats was 0.37 ml/h (n = 10). This is approximately 90% lower than the GFR we measured in fed bats. Our findings 1) suggest that Pallas's long-tongued bats do not have an exceptional urine-diluting or -concentrating ability and 2) demonstrate that the bats eliminate excess ingested water by reducing renal water reabsorption and limit urinary water loss during fasting periods by reducing GFR.

Dietary sugar as a direct fuel for flight in the nectarivorous bat Glossophaga soricina.

It is thought that the capacity of mammals to directly supply the energetic needs of exercising muscles using recently ingested fuels is limited. Humans, for example, can only fuel about 30%, at most, of exercise metabolism with dietary sugar. Using indirect calorimetry, i.e. measurement of rates of O(2) consumption and CO(2) production, in combination with carbon stable isotope techniques, we found that nectarivorous bats Glossophaga soricina use recently ingested sugars to provide approximately 78% of the fuel required for oxidative metabolism during their energetically expensive hovering flight. Among vertebrate animals, only hummingbirds exceed the capacity of these nectarivorous bats to fuel exercise with dietary sucrose. Similar experiments performed on Anna's (Calypte anna) and rufous (Selasphorus rufus) hummingbirds show that they use recently ingested sugars to support approximately 95% of hovering metabolism. These results support the suggestion that convergent evolution of physiological and biochemical traits has occurred among hovering nectarivorous animals, rendering them capable of a process analogous to aerial refueling in aircraft.

Sucrose hydrolysis does not limit food intake by Pallas's long-tongued bats.

Nectarivorous bats include very dilute nectar in their natural diet, and recent work with Pallas's long-tongued bat Glossophaga soricina showed that sugar (energy) intake rate decreased at dilute sucrose solutions. However, chiropterophillous nectar is composed mainly of the hexoses glucose and fructose. Because bats fed hexose nectar would save the delay of hydrolyzing sucrose, we hypothesized that sugar intake rate should be higher on this diet than on sucrose nectar. We compared intake response in Pallas's long-tongued bats offered 1 : 1 glucose-fructose (hexose) and sucrose diets at 5%, 10%, 20%, 30%, and 40% (mass/volume) sugar solutions. We also tested the hypothesis that sucrose hydrolysis limits food intake in bats. Intake response was the same in bats fed both types of diet: sugar intake rate was lower in dilute solutions and then increased with sugar concentration. Similar intake responses in both diets indicate that sucrose hydrolysis alone does not limit food intake and support the idea that the burden of processing excess water in dilute solutions plays a major role.