Fasting glucose levels

Neotropical bats exhibited fasting responses associated with diet, which is consistent with our predictions [26]. Similar patterns have been documented in Old World species [17,27], where bats consuming protein-rich insects or carbohydrate-rich fruits maintain constant glucose levels during fasting through metabolic reserves, whereas nectar-feeding bats that rely on continuous sugar intake showed an inability to regulate glucose after short-term fasting. Vampire bats (Desmodus rotundus) are particularly vulnerable to fasting stress [28], showing higher fasting blood glucose levels than frugivorous and nectarivorous species after fasting periods, potentially reflecting the use of glycogen. These bats maintain glucose levels compatible with most mammals (over 60 mg/dL) after a 12-hour fasting period [29]. However, after 24 hours of fasting, their glucose levels can drop significantly to around 30 mg/dL, indicating a potential loss of their ability to tolerate long-term fasting [28]. Nectarivorous bats also exhibit susceptibility to fasting [30], as evidenced by their low glucose levels after short-term fasting. This vulnerability reflects limited energetic reserves [30] since the energy extracted from sugar is primarily allocated to meet the high metabolic demands of flight [31] rather than stored for future use. Interestingly, frugivorous bats exhibited fasting glucose levels comparable to those of nectarivorous species after the same fasting period (~ 50 mg/dL or lower). These results contrast with captive Artibeus that maintained stable blood glucose levels (~ 100 mg/dL) after a 24-hour fasting period, declining to ~ 45 mg/dL only after a 72-hour and a six-day fasting period [32]. This discrepancy likely reflects methodological differences since, in the mentioned study, captive bats received ultra-rich sugar diets (bananas, watermelon, papayas, etc.) for one week before fasting trials, accumulating fat reserves, and experienced reduced metabolic demands due to a more sedentary lifestyle [32] compared to the recently captured free-flying bats tested in our study. Finally, insectivorous and carnivorous bats exhibited higher glucose levels after 12 hours of fasting, consistent with patterns observed in vertebrates consuming high-protein diets [33,34] and distinct from those of other dietary guilds. Therefore, these patterns suggest that dietary specialization drives divergent fasting strategies in Neotropical bats, with animalivorous protein consumers showing the greatest tolerance, frugivores intermediate capacity, and nectar specialists’ acute vulnerability, reflecting evolutionary trade-offs between metabolic efficiency for preferred resources and physiological buffering during scarcity.

In non-reproductive individuals, sex did not significantly affect fasting glucose levels, consistent with previous studies [28,35]. However, sex-specific differences may emerge during longer fasting periods: male Molossus molossus maintained stable glucose levels, whereas females showed declining levels after 48 hours of food deprivation [36]. This result suggests that sexual differences, with larger males of M. molossus exhibiting superior glucose regulation compared to smaller females, may be indicate metabolic differentiation and resource partitioning between sexes. This hypothesis warrants investigation in other sexually dimorphic bat species with documented dietary segregation, which could reveal whether physiological niche differentiation reinforces sexual divergence in body size and ecology. Body mass showed no clear association with fasting glucose levels across all species (Fig 1C). However, previous research reported that smaller frugivorous bats have higher glucose levels than larger species [17], a pattern we observed within frugivore-omnivore genera (such as Artibeus and Carollia, S2 Fig) but not across the Stenodermatinae subfamily (Fig 1A). In humans, diet, activity, and body mass interactively influence fasting glucose levels [37]; therefore, the inconsistent association between body size and glucose regulation may reflect complex interactions among these factors rather than the isolated influence of body mass alone. Besides, the effect of body size on fasting glucose could be further explored by adding bats with higher body masses to the datasets.

Variation in sugar assimilation

Omnivorous and frugivorous bats exhibited the highest sugar assimilation capacity during the night, whereas nectarivorous species showed similar assimilation rates between day and night. The rapid nocturnal glucose level elevation observed in Phyllostomus and Artibeus is consistent with their activity patterns [37] and reflects efficient carbohydrate assimilation for flying and foraging during nighttime periods [38]. In contrast, nectarivorous Glossophaga species maintain minimal energy reserves [30] and rely primarily on exogenous glucose intake from their diet, using circulating blood glucose as their immediate energy source to sustain activity regardless of the time of day [31,39]. Therefore, they may rely on exercise to trigger sugar assimilation during the night [16]; thus, we probably did not find differences here because we maintained bats at rest during our sampling.

Sucrose assimilation capacity differed significantly between bats with high (frugivorous) and low-sugar diets (insectivorous) at 10 and 60 minutes post-ingestion (Fig 2C), reflecting diet-specific physiological and anatomical adaptations. Frugivorous species possess: (i) elongated duodenum providing greater surface area for paracellular glucose diffusion, (ii) intestinal glucose transporters enhancing transcellular glucose absorption, and (iii) elevated sucrase expression enabling rapid disaccharide hydrolysis [9,10,14].

The temporal dynamics of sugar assimilation in the frugivorous bat A. lituratus revealed no detectable glucose absorption peak within 5 minutes following sucrose ingestion, suggesting that the initial digestive and absorption process required for disaccharide hydrolysis and glucose assimilation may take at least 10 minutes [15]. For sucrose, peak absorption occurred at 10 minutes in A. lituratus, followed by declining levels at 20 minutes as glucose was transported from the blood to tissues [40], and returned to fasting levels by 60 minutes. Notably, insectivorous bats showed peak glucose levels at 60 minutes, when frugivores had already returned to baseline. These contrasting temporal assimilation profiles, with 10 and 60 minutes emerging as critical assessment points, have been documented across several bat species and sugar types [9].

To compare the assimilation of the three sugars (sucrose, glucose, and trehalose) among bats with different diets, we fed each individual with all three sugars separately. Results aligned with previous reports of assimilation capacity and enzymatic activity [9,10], though omnivorous bats showed greater variability than previously documented [9]. Frugivorous bats demonstrated high glucose and sucrose assimilation but slow trehalose processing, consistent with their sugary-rich fruit-based diets. Limited trehalose assimilation was expected, as the treh gene encoding trehalase is pseudogenized in non-insectivorous bats [9,41], and trehalase activity decreased during evolutionary transition from insectivory to other diets [10]. Conversely, the insectivorous Molossus pretiosus exhibited minimal sugar assimilation across all three substrates, with no significant changes in blood glucose following ingestion. Molossus species feed primarily on coleopterans [41,42], which are rich in proteins and lipids rather than carbohydrates [43,44], suggesting that their digestive physiology is specialized for protein and lipid metabolism rather than sugar assimilation [10]. In contrast, omnivorous bats exhibited substantial intra- and interspecific variation in sugar assimilation. This heterogeneity likely reflects: i) variations among species in the degree of omnivory as a result of intermediate physiological and metabolic adaptations between faunivorous and herbivorous dietary extremes [45], and ii) high physiological plasticity in omnivorous species, which enables flexible metabolic responses to variation in diet composition and resource availability, even at the individual level [45–47].

Artibeus and Carollia showed similar glucose and sucrose assimilation patterns (Fig 4A), despite distinct fruit preferences: species of Artibeus primarily consume figs and Cecropia, while Carollia preferentially feed on fruits of Piper plants [48]. Although these fruits differ in nutritional composition [49,50], all are carbohydrate-rich, producing a Fast assimilation pattern [9] with high glucose digestive efficiency [50] and sugar-rich diet adaptations. Trehalose assimilation, however, revealed notable metabolic differences in the assimilation between genera. Species of Carollia exhibited higher trehalose assimilation than species of Artibeus, consistent with previous findings on these genera [9]. This ability to digest trehalose, a sugar commonly found in insects, is likely related to the significant consumption of insects in the diet of Carollia species [51]. While Carollia species can assimilate all three sugar types, Artibeus shows specialization primarily for fruit-derived sugars, suggesting Carollia maintains broader metabolic flexibility for the assimilation of diverse carbohydrates.

Male and non-reproductive female A. lituratus individuals showed similar patterns of sugar assimilation (Fig 4B). However, reproductive status may reveal sex-specific metabolic differences, as reproduction imposes substantial energetic demands on individuals of this genus [52], potentially creating temporally divergent metabolic requirements between males and females that warrant further investigation. Besides, future studies should examine whether sex-specific sugar assimilation emerges in species exhibiting sexual size dimorphism or sex-based dietary partitioning, which does not apply to species of Artibeus [53].

The omnivorous species C. perspicillata is a primary consumer of Piper fruits that showed higher sucrose assimilation during the rainy season than during the dry season (Fig 4C), evidenced by a higher glucose peak at 10 minutes post-ingestion, indicating enhanced sucrose hydrolysis and glucose absorption through the small intestine [15]. This seasonal metabolic change aligns with plant phenology in dry tropical forests, with most animal-dispersed plants fruiting during the rainy season [54], including synchronized fruiting peaks in Piper species [55]. Thus, elevated sucrose processing capacity in Carollia coincides temporally with peak availability of their primary food resource, suggesting metabolic upregulation in response to seasonal resource abundance. Conversely, C. perspicillata exhibited a slight increase in trehalose assimilation during the dry season, potentially reflecting a metabolic adjustment associated with a dietary shift toward greater consumption of nocturnal insects when fruit availability declines in the forest understory [56,57]. Thus, this pattern suggests a potential seasonal dietary flexibility [58–60], with C. perspicillata adjusting foraging effort to exploit the most available resources [61]. Given documented seasonal shifts in nectar-feeding bat behavior and dietary flexibility in related insectivorous species in seasonal ecosystems [62], further research should examine how bat carbohydrate metabolism associates with seasonal resource availability [63,64]. Such studies will enhance our understanding of physiological plasticity and species vulnerability to habitat degradation and climate change.

Metabolic differentiation between syntopic congeneric species

To test whether syntopic congeneric species exhibit metabolic differentiation, potentially associated with food resource partitioning, we compared sugar assimilation among three Carollia species: C. perspicillata, C. brevicauda, and C. castanea. Despite morphological and ecological similarity, these three species coexist through niche partitioning, including varying degrees of insectivory versus frugivory [51]. Our results reveal metabolic differentiation, suggesting resource partitioning. Among the species with similar body sizes, C. brevicauda showed high glucose but low trehalose assimilation, suggesting specialization on fruit-derived sugars. Conversely, C. perspicillata exhibited lower glucose but higher trehalose assimilation than C. brevicauda, suggesting greater reliance on insect-derived sugars. This physiological divergence requires further confirmation by increasing the sample size. However, it is potentially associated with food resource segregation, which reduces competition by reinforcing dietary preferences despite similar body size and external morphology, with metabolic capacity possibly tracking resource-use patterns. C. castanea, the smallest congener, showed high assimilation of all three sugars, suggesting broader metabolic flexibility. This may reflect a different niche partitioning mechanism: a small body size and morphological differentiation in cranial and dental traits enable access to distinct food items, reducing reliance on metabolic specialization for competitive exclusion. These patterns are consistent with the possibility that metabolic differentiation facilitates coexistence among morphologically similar congeners even in habitats with limited food resources, while morphologically divergent species may rely more strongly on metabolic flexibility. If confirmed across additional species and systems, this would suggest that physiological specialization in food processing emerges as an adaptive response when other mechanisms for niche partitioning are constrained.

Capturing bats

We caught bats using mist nets set up in dry and humid forest understories in Colombia’s Valle del Cauca and Chocó departments between 2019 and 2020. The nets were active from 18:30–21:00 h and were checked every 30 minutes. We ensured that all captured bats were fed before a 10–12-hour fast post-capture. For taxonomic identification, we used a taxonomic key for South American bats [65]. We recorded the sex of the individuals and assessed the biological age by using transillumination of their metacarpal-phalangeal joints [66]. In addition, we evaluated the reproductive status of the bats, including only non-reproductive adults (applicable to females) in our study to minimize physiological variation that can occur due to growth, or increased stress during pregnancy, and lactation [7,20,67].

Dietary classification

We classified the species based on their dietary preferences obtained by previous research [68], examinations of teeth and cranial morphology [68–71], tongue and gut anatomy [72]. These traits relate to how species acquire, process, and assimilate food. We considered six species as primarily insect consumers (Peropteryx kappleri, Saccopteryx bilineata, Molossus molossus, Molossus pretiosus, Myotis, Garnerycteris crenulatum), one species as a blood feeder (Desmodus rotundus), one as a carnivorous (Vampyrum spectrum), two as nectar consumers (Glossophaga soricina, Choeroniscus godmani), eleven as preferring fruits (Sturnira ludovici, S. luisi, S. parvidens, S. giannae, Platyrrhinus helleri, Uroderma bakeri, U. convexum, Dermanura sp., Artibeus aequatorialis, A. planirostris, A. lituratus), and six as omnivores (Phyllostomus discolor, P. hastatus, Carollia castanea, C. brevicauda, C. perspicillata), consuming primarily at least two different food types and showing trehalose assimilation [9].

Blood glucose measurements

We assessed sugar assimilation by measuring the increase in blood glucose concentration after feeding each individual a specific type of sugar, in accordance with the objectives of our tests. We measured blood glucose levels using a single drop of blood collected from the forearm of each individual with a lancet and a glucometer (Glucoquick G30a, Diabetrics®). The individuals were fed with a 20% sugar solution (glucose, sucrose, or trehalose) after a fasting period of 10–12 hours to record their baseline glucose levels. Following this, we measured blood glucose levels at 5, 10, 20, 30, and 60 minutes after ingesting a single 20% glucose solution. The amount of sugar provided to bats (5.4 g/kg) was calculated based on their body weight according to [73]. We tested the assimilation of glucose and sucrose—a monosaccharide and a disaccharide commonly found in fruits and nectar [11]—and trehalose, a disaccharide found in insects’ hemolymph [12], while bats were at rest in soft cloth bags. This methodology was followed both during the dry and the rainy seasons for several individuals of Carollia perspicillata since dietary shifts related to seasonality [59] could impact sugar assimilation.

To standardize the optimal time for measuring glucose levels, we offered sucrose to bats with different diets and compared their blood glucose levels 10 and 30 minutes after ingestion, both day and night. We observed a similar assimilation pattern between day and night, so the remaining assimilation tests were conducted from sunrise to sunset. On the other hand, to identify the most informative time for assessing sugar assimilation, we measured glucose levels at 5, 10, 20, 30, and 60 minutes after the bats consumed a disaccharide (sucrose), which requires an additional enzymatic step for assimilation compared to monosaccharides [15]. Our findings indicated that measuring glucose levels 10 minutes after feeding bats with a sugar-rich diet (like frugivores) provided helpful information, reflecting an absorption peak. In contrast, we observed an absorption peak for insectivorous bats 60 minutes after sugar ingestion.

To ensure consistency, we first measured the assimilation of the three sugars (glucose, sucrose, and trehalose) in each individual at two-time points: 10 and 60 minutes. This approach allowed us to control for intraspecific variation. Each sugar was offered separately, with a 5-hour interval between offerings, sufficient for the blood glucose levels of both frugivorous and insectivorous bats to return to baseline (S3 Fig). Additionally, we measured the assimilation of just one sugar (glucose, sucrose, or trehalose) per individual of the most frequently captured species at various time points throughout the hour (0, 10, 30, and 60 minutes). This method enabled us to compare glucose levels while minimizing the risk of excessive blood sampling from any bat.

The data included in the analysis and figures of the paper can be found at https://github.com/andreaber1/sugar_metabolism_bats.

Statistical analysis

To assess the effects of sex, weight, and diet on fasting glucose levels in bats, we performed a Bayesian multi-level phylogenetic model using the brms R package [74]. This model used blood glucose concentrations measured after fasting and incorporated a phylogeny that included multiple families and 16 species [75]. To account for both multiple individuals within species and shared evolutionary history, we included a species-level random intercept with a phylogenetic covariance structure based on the species tree, assuming Brownian motion evolution. This approach models observations as nested within species and incorporates phylogenetically structured covariance among species, thereby controlling for non-independence arising from both within-species pseudoreplication and phylogenetic relatedness. Additionally, we evaluated the phylogenetic signal of fasting glucose levels with Pagel’s lambda [76]. Furthermore, to individually assess differences in sugar assimilation among frugivore bats based on intra- or inter-genera comparisons, time of day (day/night), sex, and season, we employed the Wilcoxon test. This test allowed us to compare blood glucose levels across different time points and treatment conditions. All the statistical analyses were performed using R 4.3.1 [77].

Ethical statement

This research was authorized by the Colombian National Environmental Authorities: Autoridad Nacional de Licencias Ambientales and Ministerio del Ambiente y Desarrollo Sostenible (Permit 1070). Anesthesia, analgesia, or euthanasia were not required or applied during the study.