Introduction

Fever is a highly conserved innate immune response [1]. In many organisms it has been shown that fever temperatures serve to increase host fitness by decreasing the rate of pathogen replication [2], [3] and/or increasing the efficiency of the immune system [4], [5]. However, even relatively brief periods of hyperthermia can impose significant costs on the host [6], [7], [8]. In the case of terrestrial ectotherms, attaining and maintaining fever temperatures requires that significant effort be devoted to basking behaviors, potentially detracting from time normally allocated to other essential functions [9]. In order to limit these costs, theory predicts that organisms should invest in fever according to the level of immune challenge.

Although fever in ectotherms has been well documented, the majority of studies have focused on exploring either the mechanisms of fever [10], [11], [12] or the net benefits of fever by quantifying fitness correlates such as survival and fecundity in fevering and non-fevering hosts [13]. In contrast, few studies have provided observations detailing how freely thermoregulating ectotherms behaviorally manage fever throughout the course of infection.

We have previously shown that house flies generate a behavioral fever when infected with a lethal strain of the entomopathogenic fungus, Beauveria bassiana [6], [14]. In this earlier work, fevering did not clear infection but nominally benefited fly fitness by reducing pathogen virulence, extending fly survival and enabling infected females to lay more eggs over their lifetime. However, fevering flies incurred costs in the form of decreased egg viability [6]. In order to balance these costs and benefits, we hypothesize that infected flies should adjust their fever behavior in accordance with the level of infection. To test this hypothesis, we explored how fungal dose affects the thermoregulatory behavior of individual house flies and complemented this behavioral assay with direct measures of the impact of fever temperatures on fungal growth.

Materials and Methods

Results

The overall pattern of thermoregulation showed flies recruited to a warm part of the gradient at the beginning of the heating period and then gradually redistributed to cooler parts, or left the gradient completely, as the day progressed (Fig. 1a). This general pattern was common across treatments. However, infected flies were found to select warmer areas of the gradient than uninfected controls (F_{2, 57.75} = 21.45, P<0.001), with the maximum temperature chosen positively correlated with dose (p’s <0.05, Fig. 1b). In addition, all infected flies remained on the gradient surface for a greater proportion of time than control flies (F_{2, 64.64} = 26.89, P<0.001) and flies infected with a higher dose tended to spend more time on the gradient surface than flies infected with the low dose, (P = 0.065, Fig. 1c.).

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Figure 1. Thermoregulatory behavior of house flies infected with either a high dose (5×10⁸ conidia/ml) or low dose (1×10⁸ conidia/ml) of Beauveria bassiana, compared to uninfected flies.

A) Represents an example distribution of the hourly mean temperature of flies in each treatment group on the third day of infection, while B) represents the hourly mean temperatures of flies in each treatment group over the course of infection during the time when the gradient was heated (6 hrs a day) over the course of the study. Flies were at ambient temperature (26°C) when they were not sitting on the gradient or when the gradient was turned off. Error bars were not included panel A-B to afford clarity of the fly temperature trends. C) Mean maximum daily preferred gradient temperatures, D) mean daily proportion of time spent on the gradient surface over the course of infection. Bars represent +1 SEM. Groups with different letters in the legends of each graph indicate significant differences (Linear-Mixed Model Repeated Measures ANOVA, sig. P<0.05).

https://doi.org/10.1371/journal.pone.0062269.g001

Simulated fever treatments impacted fungal growth (Fig. 2), with colony development reduced in all temperature treatments relative to controls (F_{6, 27} = 74.29, P<0.0001). There were significant main effects of temperature (F_1,27 = 66.89, P<0.001) and time (F_2,27 = 57.18, P<0.001), as well as a significant interaction term (F_2,27 = 8.45, P = 0.002) indicating that growth was increasingly inhibited as temperature and exposure period increased simultaneously.

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Figure 2. Mean colony diameter of Beauveria bassiana grown on agar (SDA) under different temperature regimes over 11 days.

Different letters indicate significant differences between groups (Tukey post-hoc test with Bonferroni correction, P<0.05). Bars represent ±1 SEM.

https://doi.org/10.1371/journal.pone.0062269.g002

Discussion

Our aim in the current study was to characterize the pattern of behavioral fever in response to different intensities of fungal infection. Fever in houseflies has been shown to provide survival benefits upon fungal infection [6]. Here we demonstrate that part of this benefit likely comes from direct negative effects of high temperatures on fungal growth. The more extreme the ‘fever temperature’, the greater the impact on the fungus, suggesting that higher and more prolonged fevers would be beneficial. However, there are also costs to mounting a fever response [6]. Given both costs and benefits we expect flies to tailor investment in behavioral fever in line with the severity of the immune elicitors causing infection and/or the extent to which temperatures remain conducive to pathogen growth during periods when the houseflies cannot thermoregulate. Consistent with this hypothesis, flies showed temporal variation in fever, selecting the highest temperatures for a relatively short period early in the day and then gradually moving to cooler areas. They also exhibited different intensities of fever, selecting hotter fever temperatures (and possibly investing marginally more time in active thermoregulation) when infected with a higher fungal dose.

Dose-response in fever has been shown previously [10], [11], [16]. However in most of these studies, test organisms were challenged with killed pathogens or pathogen derived immune elicitors to initiate the fever response, thereby eliminating the natural dynamics of infection. In our system, we suggest the degree of fever is likely correlated with the dose of immune elicitors presented by the fungus and so changes with the dynamics of infection. With a higher fungal dose, the magnitude of fever is increased. Similarly, in the early morning when the fungus has likely been growing at its optimal temperature throughout the night cycle, immune elicitors are at high levels and the fever response most intense. As the exogenous immune elicitors are reduced or removed from the hemolymph by the immune response and thermal stress placed on the fungus by fevering flies, the ‘signal’ to fever is attenuated, resulting in the fly gradually moving to cooler areas of the gradient. Overnight, the fungus recovers as the fly cannot fever to suppress fungal growth, and the cycle begins again the next day.

The patterns of fever could also be determined by energetics. Temperatures higher than the normal thermal optimum are energetically costly; it has been demonstrated in both ectotherms and endotherms that increasing body temperatures by only 3–5°C can increase metabolic rate by up to 60% [8], [17]. Therefore, infected flies might only be able to sustain peak fever temperatures for a relatively short time (1–2 hours) at the beginning of the daily heating period (Fig. 1a).

A third possibility is that the thermoregulatory behavior is under some sort of circadian rhythm, resulting in selection of highest temperatures during the morning hours. Elements of innate immune function have been shown to be under rhythmic control in other Diptera, such as Drosophila [18] and Anopheline mosquitoes [19]. However, previous work across numerous study systems has yielded little evidence for rhythmic control of thermal preference [20]–[25]. Moreover, circadian control would not account for the differences in intensities of fever in the different fungal treatments.

The current study is one of the first to describe the daily dynamics of fever to a live pathogen and to demonstrate dose-dependence of this immune response in an insect. The molecular pathways responsible for regulating body temperature and fever are highly conserved across taxa [12], [26], [27], implying ancient origins. Endotherms are able to fine tune fever responses depending on the intensity of infection via physiological means [28]. Our results reveal that house flies can similarly tailor patterns of fever via behavioral means, putatively limiting fever costs.

Acknowledgments

This research was funded, in part, by an NSF-EID grant (EF-0914384), and a grant with the Pennsylvania Department of Health using Tobacco Settlement Funds. The Department specifically disclaims responsibility for any analyses, interpretations or conclusions.