The acute phase response in bats (Carollia perspicillata) varies with time and dose of the immune challenge

Animals are constantly exposed to a myriad of pathogens, and their immune system plays a pivotal role in mitigating the potential deleterious effects of such exposure (Acevedo-Whitehouse and Duffus, 2009). From a methodological perspective, ecoimmunological studies ultimately have attempted to use some metrics that portray immunocompetence; that is, the ability of the immune system to fight pathogens (Demas et al., 2011; Schoenle et al., 2018). The acute phase response (APR) is a core component of the innate immune response, and its quantification is widely used to assess the immunocompetence of different species of animals (Merlo et al., 2016; Ramirez-Otarola et al., 2018; Roy et al., 2016). The APR is the first line of induced defense used by all animals in response to infections, and the most common technique employed to activate it in experimental studies is the central or peripherical administration of lipopolysaccharide (LPS) (Cray et al., 2009; Rudaya et al., 2005; Sampath, 2018). LPS is an immunogenic component of the outer membrane of gram-negative bacteria that causes the release of proinflammatory cytokines, which in turn trigger a suite of behavioral (decreased activity, anorexia and adipsia) and physiological responses [increase in body temperature, metabolic costs and total white blood cell (WBC) count, and activation of the hypothalamic–pituitary–adrenal (HPA) axis; Lochmiller and Deerenberg, 2000; Sampath, 2018; Lopes et al., 2021]. The APR accelerates pathogen elimination and enhances the activation of the adaptive immune system, thus conferring an immediate benefit in controlling infection (Cray et al., 2009; Cray, 2012). However, the APR is thought to have a high cost/benefit ratio because its protective value is lower when compared with adaptive constitutive responses, such as those mediated by leukocytes, and the energetic cost of its activation is the highest of all immune system defenses (Hasselquist and Nilsson, 2012; Martin et al., 2008).

The magnitude of behavioral and physiological components of the LPS-induced APR has been measured in several vertebrate groups. Although a decrease in food intake and body mass seems to be a universal response observed after LPS inoculation in all taxa, there was a great variability in the magnitude of these responses, both within and between species (Burness et al., 2010; Coon et al., 2011; Gayle et al., 1998; Haba et al., 2012; Llewellyn et al., 2011; Mathias et al., 2000; Skold-Chiriac et al., 2015; Volkoff and Peter, 2004; Wang et al., 2018; Webel et al., 1998). Such variability can also be noticed from results of several studies that reported changes in body temperature, metabolic rate, total WBC count or levels of circulating glucocorticoid hormones after LPS inoculation (Johnson et al., 1993; Kozak et al., 1994; Kimura et al., 1994; Mathias et al., 2000; Koutsos and Klasing, 2001; Wang et al., 2003; Jacobsen et al., 2005; Grion et al., 2007; de Boever et al., 2009; Pérez-Nievas et al., 2010; Burness et al., 2010; MacDonald et al., 2012; Meitern et al., 2013; King and Swanson, 2013; Voigt and Kingston, 2016; Yamashita et al., 2017; Bowers et al., 2017). However, unlike changes in food intake and body mass, the universality of these changes is disputable, as some studies did not report changes in body temperature, metabolic costs, HPA axis activation or total WBC count (Amaral-Silva et al., 2021; Cabrera-Martinez et al., 2019; Copeland et al., 2005; Guerrero-Chacón et al., 2018; Lind et al., 2020; Melhado et al., 2020; Stockmaier et al., 2015; Weber et al., 2005). Some of these differences certainly reflect species-specific differences and they are contingent on the specific context of the study. However, some of these variations, especially at the intraspecific level, might also reflect differences in the time (active and rest periods) and dose of LPS injections.

Although several studies analyzed the direction and magnitude of the LPS-induced APR in diverse vertebrate taxa, relatively few studies attempted to understand the direction of changes in APR components as a function of the period when the immune system was challenged, and the dose used to activate this response (Tables 1 and 2). Studies with rodents, birds and amphibians show that the period when LPS is administered has different effects on components of the LPS-induced APR (Table 1). In general, when animals are immune challenged during the active period, they respond with a greater decrease of food intake and body mass, increased leukocyte recruitment and higher or similar levels of corticosterone. In contrast, hypothermia and/or fever occurs during both active and rest periods (Table 1). Furthermore, studies with several groups of vertebrates show that the highest LPS doses result in a greater decrease of food intake and of body mass, higher levels of corticosterone and leukocytes, and a longer and stronger fever response in some cases preceded by hypothermia, or a long-lasting hypothermic response (Table 2). Fever and hypothermia have been suggested to be two alternative strategies to mitigate infection in birds and mammals. Hypothermia would be favored when animals face a highly demanding trade-off between the benefits and costs of mounting a fever response (Amaral-Silva et al., 2021; Ganeshan et al., 2019; Romanovsky and Székely, 1998; Steiner and Romanovsky, 2019). In general, it seems clear that the period of injection (active or rest period) and LPS dosage determines the direction and/or magnitude of APR component responses. However, there are exceptions to this trend, and, to the best of our knowledge, no study has attempted to measure the synergistic effects of dose and period of injection. Encounters with bacteria are likely to be maximal during the active phase, when animals are foraging and exploring the environment (Scheiermann et al., 2012; Scheiermann et al., 2013). Therefore, the activation of the APR must vary simultaneously with the period of infection and pathogenic load.

Analysis of bats’ immune response repertoire is fundamental to understand why this order hosts a wide variety of pathogens without showing clear signs of developing disease in most cases (Kacprzyk et al., 2017; Moratelli and Calisher, 2015; O'Shea et al., 2014). During their active period, bats increase their exposure to pathogens while foraging (Kuzmin et al., 2011; Patz et al., 2008; Wong et al., 2007), whereas aggregation behavior within their shelters and grooming during the rest period increases the potential for transmission of infectious agents (Mühldorfer, 2013; White and Razgour, 2020). Studies that have analyzed the APR in bats examined only certain components of it during specific periods and using a single LPS dose (Table 3). In these studies, LPS triggered fever when administered during the rest period, but no change in body temperature was observed when it was administered during the active period. Some studies report leukocytosis after LPS injection during the rest or active periods, while others show no evidence of leukocytosis during the active or rest periods. The neutrophil/lymphocyte (N/L) ratio increased after LPS injection during the active period in feeding bats, whereas during the rest period, using a similar LPS dose, the N/L ratio increased only in food-deprived bats. Although some studies reported body mass loss 24 h after LPS injection and assumed that it was partly due to anorexia, not all examined the effect of LPS on food intake rate, and just one evaluated this response during the active period. Furthermore, not all studies reported body mass changes after LPS injection. A common factor in bat studies is that they all immuno-challenged the animals with doses of LPS higher than or equal (1–5 mg kg⁻¹) to those used in most vertebrate studies (Tables 1 and 2), and even then, in some studies no change was observed in thermoregulatory response, total WBC count and N/L ratio (Table 3), reinforcing that bats may not show clear signs of disease even at higher LPS doses (a proxy of pathogen load).

In this study, we tested the interplay between LPS dose and time of injection (active and rest periods) on selected physiological (body temperature change, body mass change, total WBC count and N/L ratio) and behavioral (food intake) components of the APR using a Neotropical fruit-eating bat (Carollia perspicillata) as a model organism. We predicted that LPS would trigger a dose-dependent and/or period-dependent response on bat APR components. We expected that the highest LPS doses would result in a greater decrease of food intake and body mass loss, leukocytosis and an increase in the N/L ratio, and that these responses would be more pronounced when LPS was injected at the beginning of the active period. We used the N/L ratio as a proxy for HPA axis activation, as HPA axis activation results in the release of glucocorticoids, which in turn can lead to immune cell redistribution (Bornstein et al., 2006; Cain and Cidlowski, 2017; Davis et al., 2008). We further expected that LPS would trigger a dose-dependent change in body temperature during the rest and active periods, with the highest LPS doses resulting in an increase on magnitude and/or duration of this response. However, given the absence of a thermoregulatory response in bats during the active period (Table 3), and the variety of thermoregulatory responses observed in small rodents and birds after immune challenge during active and rest periods (Table 1), we made no predictions about the direction of body temperature change (e.g. fever or hypothermia).

Non-reproductive adults of Carollia perspicillata (Linnaeus 1758) were captured between June 2018 and July 2019 in three municipalities at São Paulo State, Southeastern Brazil: Edmundo Navarro de Andrade State Forest, located in Rio Claro (22°25′54.2″S 47°32′11.1″W), forest remnants of the Federal University of São Carlos−UFSCAR, located in São Carlos (23°21′32.5″S 46°15′15.1″W), and RPPN Rio dos Pilões located in Santa Isabel (21°59′6.2″S 47°52′55.3″W). Bats were captured with mist nets and transported to an outdoor cage (3×3×3 m), exposed to natural conditions of photoperiod and temperature (mean±s.d. 22±1°C; CEAPLA/IGCE/UNESP) at Universidade Estadual Paulista at Rio Claro. The bats were fed papaya and bananas for 5 days before the immune challenge. Permits to capture and house bats were issued by the Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio, process number 66452-1). Ethical permits for this study were issued by the animal ethics committee of the Universidade Estadual Paulista at Rio Claro (Authorization no. 3381).

The immune challenges were conducted in a climatic chamber (3×2×3 m), with a controlled room temperature (27°C – thermoneutral zone of C. perspicillata; Cruz-Neto and Jones, 2006) and photoperiod (12 h:12 h). Lights were switched on at 06:00 h and switched off at 18:00 h. Bats were transferred to the climatic chamber 1 day before the immune challenge for acclimation, and they were kept in individual cages (1×1×0.5 m). After acclimation, 24 bats were assigned to the immune challenge at 18:00 h (night-time) and 24 bats were assigned to the immune challenge at 08:00 h (day-time) using three LPS doses and one control group. Six bats of each injection period group (night- and day-time injections) were randomly assigned to either the LPS groups or the control group. Bats were injected subcutaneously into the scapula with 5, 10 or 15 mg kg⁻¹ of LPS (LPS L2630; Sigma-Aldrich) diluted in 50 μl of phosphate-buffered saline (PBS; P4417, Sigma-Aldrich), while control groups were injected only with 50 μl of PBS. In this way, we conducted four experimental trials for day- and night-time injections, respectively. The experimental trials were conducted in the climatic chamber with 6 individuals at a time, and the bats were assigned to only one period and dose treatment. The range of LPS doses represents the maximum dose used in previous studies with bats (5 mg kg⁻¹ LPS; Stockmaier et al., 2015, 2018) and doses at least 2–3 times that value to maximize the detection of the effects APR activation, as proposed by previous studies of birds and small mammals that used higher LPS doses (King and Swanson, 2013; Koutsos and Klasing, 2001; Scheiermann et al., 2012).

Before and after injections, bats were fed ad libitum with mango nectar (>95% of the composition in the form of simple sugars; Serigy^®) supplemented with 4 mg l⁻¹ of hydrolyzed casein (Sigma-Aldrich). A known amount of nectar was placed in a feeding device at 18:00 h and was weighed at 08:00 h to measure food intake. The amount of food given exceeded daily consumption based on preliminary observations. We also placed a feeding device in the climatic chamber with a known amount of nectar to measure evaporative loss. We assessed food intake changes (ΔFI) in relative terms as: ΔFI=(FI after injections−FI before injections)/(FI before injections). Bats were weighed to the nearest 0.1 g (Ohaus Precision Balance) before and after injections. The body mass of bats injected during the active period was measured at 18:00 h and 08:00 h in the period before injection, immediately before the injection at 18:00 h, and then in the period after injection at 08:00 h and 18:00 h. The body mass of bats injected during the rest period was measured around 08:00 h and 18:00 h in the period before injection, immediately before the injection at 08:00 h, and then in the period after the injection at 18:00 h and 08:00 h. We assessed the body mass changes (ΔM_b) in relative terms as: ΔM_b=(mean M_b after injections−mean M_b before injections)/(mean M_b before injections) (see Fig. S1A,B).

We collected ∼10–15 μl of blood from the propatagial vein 24 h before and after injections at around 18:00 h (groups injected during the active period) or 08:00 h (groups injected during the rest period) and prepared two blood smears to examine WBC count and N/L ratio. Total and differential WBC count in blood smears is the most common method used in bat studies that evaluated the APR after LPS immune challenge (Cabrera-Martinez et al., 2019; Guerrero-Chacón et al., 2018; Melhado et al., 2020; Moreno et al., 2021; Paksuz et al., 2009; Schneeberger et al., 2013; Seltmann et al., 2022; Stockmaier et al., 2015; Voigt et al., 2020). We estimate the WBC count by counting the mean number of immune cells in 20 field views of each blood smear under a microscope at 400× magnification. In addition, we counted the mean number of neutrophils and lymphocytes at 1000× magnification in two replicate sets of 100 immune cells on each blood smear to calculate the N/L ratio. We assessed changes in WBC count (ΔWBC) and N/L ratio (ΔN/L) in relative terms as: ΔWBC=(WBC after injection−WBC before injection)/(WBC before injection); and ΔN/L=(N/L after injection−N/L before injection)/(N/L before injection).

We measured skin temperature as an approximation of body temperature (Williams et al., 2009) using Sub-Cue Temperature Transmitters (2.71±0.05 g; Canadian Analytical Technologies, Calgary, AB, Canada) attached to the skin of bats on the scapular region 24 h before injection. Skin temperature is considered a good estimator of body temperature in bats (Melhado et al., 2020; Otálora-Ardila et al., 2016). Body temperature was recorded every hour from 24 h before to 24 h after injection, starting at 18:00 h in groups injected during the active period, and at 06:00 h in groups injected during the rest period. We assessed the change in body temperature (ΔT_b) 12 h after injection in absolute terms by subtracting hourly skin temperature after injection from the respective hourly skin temperature before injection (see Fig. S2A,B). We used the values of temperature from the calibration curves provided by the manufacturer before checking their readings against values obtained simultaneously with a thermometer and with eight transmitters placed in the climate chamber at different temperatures. Values obtained with the calibration curve and direct readings differed by 0.22±0.11°C (mean±s.d.).

A two-way ANOVA was used to test for the interaction effects of dose (PBS, 5 mg kg⁻¹ LPS, 10 mg kg⁻¹ LPS and 15 mg kg⁻¹ LPS) and period of injection (rest and active periods) on food intake, body mass change, WBC and N/L ratio. When the two-way interaction term of these analyses was significant, we used Tukey HSD post hoc test for pairwise means comparisons. Main effects of two-way ANOVA were interpreted when they were significant in the absence of statistically significant interactions by a Tukey HSD post hoc test for pairwise means comparisons when applicable (Kiernan, 2014; Maxwell and Delaney, 2004). Three-way mixed ANOVA (two between-subject factors/one within-subject factor) were used to test for the interaction effects of dose (PBS, 5 mg kg⁻¹ LPS, 10 mg kg⁻¹ LPS and 15 mg kg⁻¹ LPS) and period of injection (rest or active) as between-subject factors, and time (ΔT_b for 12 h) as within-subject factors on ΔT_b. When the three-way interaction or two-way interaction was significant, we used a Bonferroni post hoc test for pairwise means comparisons. Main effects of three-way mixed ANOVA were interpreted when they were significant in the absence of statistically significant interactions by Bonferroni post hoc test for pairwise means comparisons when applicable (Kiernan, 2014; Maxwell and Delaney, 2004). All variables were checked for normality (Shapiro–Wilk test) and homogeneity (Levene's test), which validated the use of parametric tests. In cases where one of the assumptions was violated, data were transformed to meet the assumptions of normality and homogeneity Accordingly, ΔWBC was transformed to cubic root, and log transformation was applied on ΔN/L and ΔM_b. Studentized residuals tests were used to detect significant outliers (±3 s.d.) and there were removed from the analyses. ΔFI clearly showed one potential outlier in the group injected with PBS during the rest period. Sphericity was assessed with Mauchly's test, and the Greenhouse–Geisser correction was applied when necessary. Before injections, there was no difference in food intake, mean M_b, total WBC count, N/L ratio or mean T_b among localities where bats were collected (see Table S1). We did not include sex in the analyses performed because we had a small sample number of females (N=9), in relation to males (N=39), distributed among the experimental groups, and this did not allow us to include sex as a factor or covariate. All statistical analyses were performed in SPSS 26 for Windows (IBM Corp., Armonk, NY, USA), and a fiducial level of 0.05 was adopted to determine the significance of all comparations.

There were significant interaction effects of dose and period of injection on ΔFI (F_3,38=6.10, P=0.002, partial η²=0.325; see Table S2). FI decreased when bats were injected during the rest period. In this case, all injected groups showed a mean decrease of FI, but the magnitude of the decrease was greater in the groups injected with 5, 10 and 15 mg kg⁻¹ LPS doses than in those injected PBS (59%, 60% and 77%, respectively; P<0.050; Fig. 1; see Table S3). No significant difference was found in the mean decrease of FI among bats injected with different LPS doses (P>0.05). FI was also affected in bats injected during the active period. In this case, the decrease in mean FI was also greater for bats injected with 5, 10 and 15 mg kg⁻¹ LPS doses when compared with PBS-injected bats (67%, 93% and 92%, respectively; P<0.05; Fig. 1; see Table S3). No significant difference was observed in the decrease in mean FI between bats injected with 5, 10 or 15 mg kg⁻¹ LPS doses (P>0.050). The changes in FI were not significantly different between rest and active periods in bats injected with PBS, and those injected with 5 or 15 mg kg⁻¹ LPS (P>0.05; Fig. 1). Bats injected with 10 mg kg⁻¹ LPS during the active period showed a greater mean decrease in FI compared with those injected with the same dose during the rest period (P<0.050; Fig. 1).

There was only a significant main effect of dose on mean ΔM_b (F_3.40=11.9, P<0.001, partial η²=0.472; Fig. 2; see Table S2). All injected groups showed a decrease of mean M_b, but the magnitude of the decrease was greater in bats injected with 10 and 15 mg kg⁻¹ LPS (5.6% and 5.9%, respectively; P<0.050; Fig. 2; see Table S3) than in those injected with PBS (1.4%). Bats injected with 5 mg kg⁻¹ LPS showed a greater decrease of mean M_b (3.4%) compared with those injected with PBS, but this decrease was not significant (P=0.096; Fig. 2). Bats injected with 15 mg kg⁻¹ LPS showed a greater decrease of mean M_b compared with those injected 5 mg kg⁻¹ LPS (P<0.050; Fig. 2).

Neither the effects of the dose×period of injection interaction (F_3.40=0.48, P=0.701, partial η²=0.034) nor the main effects of dose (F_3.40=0.880, P=0.460, partial η²=0.062) and period of injection (F_1.40=0.07, P=0.799, partial η²=0.002) on ΔWBC were significant (Fig. 3A). In contrast, there was a significant interaction effect of dose×period of injection on ΔN/L (F_3,40=3.11, P=0.037, partial η²=0.189; see Table S2). ΔN/L varied among doses when bats were injected during the rest period: the N/L ratio increased at a greater rate for bats injected with 10 and 15 mg kg⁻¹ LPS when compared with PBS-injected bats (189% and 91%, respectively; P<0.050), and the magnitude of this increase was greater for bats injected with 10 mg kg⁻¹ LPS than for those injected with 5 mg kg⁻¹ LPS (P<0.050; Fig. 3B; see Table S3). No significant difference was observed in ΔN/L between bats injected with 5, 10 or 15 mg kg⁻¹ LPS (P>0.050). For bats injected during the active period, injection dose also affected ΔN/L. In this case, ΔN/L was greater for bats injected with 5, 10 and 15 mg kg⁻¹ LPS when compared with PBS-injected bats (157%, 328% and 311%, respectively; P<0.050), and the magnitude of this increase was greater for bats injected with 10 and 15 mg kg⁻¹ LPS than with 5 mg kg⁻¹ LPS (P<0.050; Fig. 3B; see Table S3). However, no significant difference was observed in ΔN/L between bats injected with 10 and 15 mg kg⁻¹ LPS (P>0.050). The N/L ratio changes were not significantly different between PBS-injected bats during the rest and active periods (P>0.691). The magnitude of the increase in N/L ratio was more pronounced for bats injected with 5, 10 and 15 mg kg⁻¹ LPS during the active period than in those injected with the same doses during the rest period (P<0.05; Fig. 3B; see Table S3).

There was a significant main effect of time (F_4.26,170.69=1.26, P<0.001, partial η²=0.864), dose (F_3.40 =3.62, P=0.021, partial η²=0.213) and the dose×time interaction (F_12.80,170.69=1.81, P=0.045, partial η²=0.120) on hourly ΔT_b. However, we found no main effect of period of injection or any interaction effect with period of injection (see Table S2), indicating that this change over time depends on the dose, but not on the period of injection. Hourly ΔT_b of bats injected with PBS did not change with time after injection (P>0.404; Fig. 4A). Two hours after injection there was a significant increase in T_b of bats injected with 5 mg kg⁻¹ LPS compared with the first hour (P<0.001; Fig. 4A), whereas bats injected 10 and 15 mg kg⁻¹ LPS increased their T_b 3 h after injection compared with that during the first hour (P<0.001; Fig. 4A). ΔT_b of bats injected with PBS, or 5, 10 and 15 mg kg⁻¹ LPS was not significantly different 1 h after injection (P>0.993). Between 2 and 5 h after immune challenge, bats injected with 5, 10 and 15 mg kg⁻¹ LPS showed a significant increase in T_b compared with those injected with PBS (0.6–0.9°C; P<0.05; Fig. 4A; see Table S4). No difference was found in ΔT_b among bats that received LPS doses at any time point (P>0.05). After the maximum T_b response, bats of all LPS-injected groups steadily decreased their T_b, converging on the T_b of the PBS group (P>0.050; Fig. 4A). Eight hours after injection with 15 mg kg⁻¹ LPS, bats showed a new significative increase in T_b compared with PBS group (0.5°C; P<0.05; Fig. 4A; see Table S4), but this increase was not significantly different from the increase observed between 3 and 5 h after injection (P<0.05; Fig. 4A).

To our knowledge this is the first study investigating the interplay between dose and time of injection (rest or active periods) on components of the APR in vertebrates. We expected that LPS would trigger a dose-dependent and/or time-dependent response in the bats’ APR components. Our results indicate, however, that some components of the APR evaluated in this study were differently affected by dose (M_b variation and skin temperature), time (food intake) or the interaction between dose and time of injection (N/L ratio).

A decrease in FI after LPS was reported for several species of birds, non-flying mammals and bats, including a previous study with C. perspicillata (Melhado et al., 2020; see Tables 1–3). We observed the same general trend in our study. Although C. perspicillata individuals injected with PBS also showed a decrease in FI (5–22% decrease), this was significantly lower than the decrease observed for individuals injected with LPS (59–93% decrease). However, the magnitude of this decrease was contingent on the time of injection and dose. Like other studies with birds and non-flying mammals (Tables 1 and 2), higher doses of LPS elicited a greater decrease in FI in individuals of C. perspicillata, but it seems that the strength of this effect was more robust when these individuals were injected during the active period. This effect is best noted when observing the effects of period of injection and dose on the energy budget of this species. Despite the decrease in FI observed in the PBS-injected groups, the energy intake (50.75 and 52.88 kJ day⁻¹ for rest and active periods, respectively) was similar to that required by C. perspicillata to meet daily energy expenditure (50 kJ day⁻¹ estimated by allometric equation; see Cabrera-Martinez et al., 2019). The effect of LPS on energy balance was considerable: mean FI when bats were injected during the rest period provided 24.56, 28.25 and 14.18 kJ (from the lowest to the highest dose), whereas, when injected during the active period, mean FI provided 23.47, 3.80 and 3.05 kJ (from the lowest to the highest dose). Even though bats were caged during the experiment and their energy needs might be lower than those of free-ranging individuals, LPS exposure had dramatic effects on their energy balance in both injection periods. However, injections of 10 and 15 mg kg⁻¹ LPS resulted in a more robust decrease in energy intake during the active period (92.81–94.23%) than during the rest period (43.84–70,83%). Overall, our results reinforce the idea that a decrease in FI is in fact a universal response associated with the APR. Rather than considering it as a passive consequence of sickness, the decrease in FI has been suggested to be part of an adaptive response to confront disease because it might reduce foraging-related energy expenses and predation risk (Johnson, 2002). Our results also indicate that the expression of the anorexic disease behavior is regulated by the strength of the infection during the active period, with a more robust expression at higher doses, indicating a synergistic effect between LPS dose and period of injection.

A decrease in M_b after LPS injection is a common trend reported in several studies with birds and mammals, including bats (see Tables 1–3). Our study corroborates this trend, and our findings were, in general, similar to those hitherto reported for C. perspicillata (Cabrera-Martinez et al., 2019; Melhado et al., 2020; Schneeberger et al., 2013). Our study is also in accordance with those in birds and non-flying mammals, which showed that the magnitude of M_b loss is dose dependent (see Table 3). M_b loss over a 24 h period after LPS injection might reflect the mobilization of nutrient stores to cover the energetic costs associated with mounting an immune response and/or the decrease in FI (Owen-Ashley and Wingfield, 2007). If magnitude of M_b loss is somehow correlated with increased APR costs and/or decreased FI, we might expect that M_b loss would increase with higher LPS doses. Interestingly, although we used a higher LPS dose than most other studies, and observed a substantial decrease in energy intake at higher doses, our results (a decrease of 3–6%) were within the range of changes reported for bats (6–8% decrease after injection of 1–5 mg kg⁻¹ LPS; Schneeberger et al., 2013; Stockmaier et al., 2015, 2018; Guerrero-Chacón et al., 2018), birds (2–6% decrease after injection of 1 mg kg⁻¹ LPS; Owen-Ashley et al., 2008; Burness et al., 2010) and small rodents (4.8–9.9% decrease after injection of 0.05–3.5 mg kg⁻¹ LPS; Kozak et al., 1994; MacDonald et al., 2011, 2012). Increased metabolic cost associated with APR activation during rest was previously described for C. perspicillata, and the energy invested in mounting an innate immune response represents 0.3% of daily energy expenditure in this species (Cabrera-Martínez et al., 2019). Negligible metabolic cost of APR was also reported for the plant-eating bats Glossophaga soricine (2% of daily energy budget; Cabrera-Martínez et al., 2018) and Artibeus lituratus (<1% of daily energy budget; Guerrero-Chacón et al., 2018) immuno-challenged during rest, whereas significant costs (9.8–14% of daily energy budget) were reported for the fish-eating bat Myotis vivesi (Otálora-Ardila et al., 2016; 2017). Although we have not evaluated the metabolic costs of APR, it is possible that even high doses of LPS do not elicit a substantial increase in the metabolic costs of plant-eating bats, and then, even with a substantial reduction in FI, there may be no increase in energy mobilization beyond the minimum necessary. Future studies that analyze the metabolic cost and mobilization of triglycerides considering the magnitude of the LPS dose may better clarify the direction and magnitude of energy mobilization. Furthermore, is worth noting that we found no effect of period of injection on the magnitude of M_b loss after LPS injection, and that specific effects of dose on FI and M_b were not the same. A more robust decrease in M_b was not observed at higher LPS doses during the active period. The only study that compared the effects of time of LPS injection on M_b loss was carried out in birds, used a similar dose (0.10 mg kg⁻¹ LPS) and found that only injection during the active period resulted in body mass loss (Skold-Chiriac et al., 2015).

Leukocytosis, an increase in circulation levels of WBCs after LPS administration is considered a central part of the APR (Cray, 2012). Regardless of the injection time or LPS dose, there was no significative increase in the total WBC count 24 h after LPS injection. Previous studies on C. perspicillata showed evidence of leukocytosis when LPS was injected during the rest period (2 mg kg⁻¹ LPS; Schneeberger et al., 2013) and no evidence of leukocytosis when LPS was injected during the active and rest periods (3 mg kg⁻¹ LPS; Cabrera-Martinez et al., 2019; Melhado et al., 2020). In these previous studies, C. perspicillata were injected subcutaneously with a similar but lower LPS dose compared with those used in our study, but Schneeberger et al. (2013) and Melhado et al. (2020) counted WBCs 24 h after injection whereas Cabrera-Martinez et al. (2019) did so after a shorter period (9 h). A common feature of the four studies, including the present study, is the existence of individual variation in bat response with some individuals increasing, decreasing or maintaining the number of WBCs after LPS injection. Leukocytosis is initially mediated through the production of cortisol by the removal of marginal pool cells from the tissues and placement in the blood in what is often called an immediate stress response (Cray, 2012). Neutrophils constitute the first line of defense against bacterial infection and are the primary phagocytic cells that rapidly proliferate in response to infection and inflammation (Akira et al., 2016; Davis et al., 2008; Nathan, 2006; Weise et al., 2017). LPS activates the HPA axis, culminating in a quick glucocorticoid (GC) release, which increases the migration of bone marrow-derived neutrophils to the bloodstream, where they are essential in the fight against infection (Bornstein et al., 2006; Cain and Cidlowski, 2017; Shiraishi et al., 1996; Turmelle et al., 2010). GCs also redirect the traffic of circulating lymphocytes from the bloodstream to lymph nodes, spleen, bone marrow and skin, improving the innate or adaptive immune response (role of NK cells, memory T cells or B cell function; Dhabhar, 2002; Taves and Ashwell, 2021). True leukocytosis follows some days after the effects of the proinflammatory cytokine response and its positive effect on the bone marrow, and often involves higher levels of both lymphocytes and neutrophils (Cray, 2012). Similar to previous studies with bats, LPS triggered an increase in N/L ratio when injected during the rest and active periods (Weise et al., 2017; Stockmaier et al., 2018; Melhado et al., 2020), but the magnitude of this increase was dose and time dependent, with a more evident dose dependence and a more robust increase in bats injected with higher LPS doses during the active period. The dose-dependent increase in the N/L ratio may be a result of a dose-dependent increase in GCs in the bloodstream, which consequently redirect neutrophil and lymphocyte traffic (Dhabhar, 2002), whereas, a robust increase in N/L ratio during the active period may be a result of a higher prevalence of GCs in the bloodstream during the active period (Gong et al., 2015; Markowska et al., 2017; Scheiermann et al., 2013), improving immunological function against pathogens, which are likely to be maximal during the active phase (Scheiermann et al., 2013).

Regardless of the time of injection (rest or active period), our results indicate a dose-dependent effect on the duration, but not in the magnitude, of the increase in skin temperature. T_b peaked between 2 and 4 h after injection, similar to previous studies with bats (Cabrera-Martinez et al., 2019; Otálora-Ardila et al., 2016), small rodents (MacDonald et al., 2012; Morrow and Opp, 2005; Romanovsky et al., 1996a; Rudaya et al., 2005) and birds (Maloney and Gray, 1998; Nomoto, 1996; Skold-Chiriac et al., 2015). Additionally, bats injected with the highest dose of LPS maintained a significant increase in skin temperature for a longer time (3–5 h), with a second period of increase in skin temperature at 8 h (biphasic fever), indicating that this dose activates fever longer than lower doses, as observed in previous studies with small rodents (Romanovsky et al., 1996a; Rudaya et al., 2005). However, it is important to note that the peak of the temperature increase after LPS injection (0.5–0.9°C) as well as the duration of the febrile response after the highest LPS dose was not higher or longer lasting than observed in previous studies with bats, small rodents and birds that administered LPS doses at least 3 times lower (bats: up to 3°C, 7 h after 1.75–2.80 mg kg⁻¹ LPS; Otálora-Ardila et al., 2017, Guerrero-Chacón et al., 2018; Cabrera-Martinez et al., 2019; small rodents: up to 3°C, 2–11 h after 0.001–1 mg kg⁻¹ LPS; Romanovsky et al., 1996a; Morrow and Opp, 2005; Rudaya et al., 2005; MacDonald et al., 2012; birds: up to 1.2°C, 5–20 h after 0.001–1 mg kg⁻¹ LPS; Nomoto, 1996; Maloney and Gray, 1998; Adelman et al., 2010; Coon et al., 2011). Based on the limited work available on bats treated with LPS, we speculate that fever is a predominant response, and that bats do not face a highly demanding trade-off between the benefits and costs of mounting a fever response even at higher LPS doses. Higher LPS doses are able to trigger a long-lasting febrile response in bats, but the magnitude and duration of this response must vary depending on the species, independently of LPS dosage. The functional basis of this pattern is not known, but it is possible that, similar to small rodents and birds, the LPS receptor proteins on the surface of cells (Toll-like receptors; TLR4) are under varying selection, modifying their ability to recognize and respond to LPS (Fornůsková et al., 2013; Vinkler et al., 2014). In fact, recent studies with bats have shown that TLR3, 7, 8 and 9 (viral receptor proteins) sequences in eight bats from three different families (Pteropodidae, Vespertilionidae and Phyllostomidae) are evolving under purifying selection, and multiple mutations in the ligand-binding domain of these receptors have been reported (Banerjee et al., 2017; Escalera-Zamudio et al., 2015; Jiang et al., 2017; Romanovsky et al., 1996b). Subsequent haplotypes of TLRs and their specific ligands can result in altered thermoregulatory responses and contribute to the adaptation of the pathogen–host interaction (Fornůsková et al., 2013; Jiang et al., 2017).

Our study offers important insights into the dependence on time and LPS dosage of the APR of bats, and how they deal with the magnitude of infections at different times of day. In general, the results showed that LPS-evoked changes in skin temperature, FI and N/L depend on the LPS dose and/or time when LPS is administered (rest or active period), whereas changes in M_b and WBC must be contingent upon factors other than dose and period of injection. Allied to the fact that the direction of FI and N/L ratio changes is not determined by exactly the same factors or interaction between factors, and that the thermoregulatory response of bats appears to be less sensitive to immune challenge with higher LPS doses, our results suggest that studies that explore the components of the APR should consider the LPS dose and time of day in which the response is induced. Additionally, further studies should also evaluate metabolic cost and mobilization of triglycerides resulting from LPS immune challenge when testing the effects of dose and period of injection.

We thank Augusto G. Paulino, Gabriel Melhado, Lucia V. Cabrera-Martínez, Pedro Henrique Miguel, Poliana Arantes, Victor H. Bruno, Murilo Negreiros and Ayrton Nascimento for helping us in the field and/or in the performance of experimental tests. All experimental tests were carried out within the facilities of the Laboratório de Fisiologia Animal (LaFA), located at Departamento de Biodiversidade, Instituto de Biociências, Universidade Estadual Paulista Júlio de Mesquita Filho, Rio Claro, São Paulo, Brazil.