Thesis (Ph.D.)--Auburn University, 2007. / Abstract. Vita. Includes bibliographic references (ℓ. 81-86)

Insectivorous bats alter relative use of metabolic substrates to match requirements of their activities, including energetically expensive flight. The “fasting while foraging” hypothesis states that the metabolic demands of flight often exceed energy intake while foraging, hence bats may metabolize fat stores (especially early in the night) to power flight with ketones, a byproduct of the normal oxidation of fatty acids. Previous studies in bats have found increases in the plasma ketone ß-hydroxybutyrate following food consumption paired with or without flight. However, no study has explored whether increases in plasma ß-hydroxybutyrate occur following flight without food consumption. We used metabolite analysis to examine changes in plasma ß-hydroxybutyrate as a function of flight duration in 2 groups (fall and spring) of captive big brown bats (Eptesicus fuscus). We fasted bats for 12 hours prior to flight (exercise treatment) or rest (control), and then collected interfemoral vein blood. Exercise activity was quantified as flight time. For the Fall group, we collected three rest samples and one flight sample. Results for the Fall group were variable; interpretation of data patterns for this group may be complicated by changes in metabolism that occur in the fall when bats physiologically prepare for hibernation. To control for seasonal effects, we tested a second group of bats in the spring, collecting two rest and three flight samples. We found a positive correlation between flight duration and levels of plasma ß-hydroxybutyrate in the Spring group, which supports the fasting while foraging hypothesis. / Thesis / Master of Science (MSc) / The “fasting while foraging” hypothesis states that the metabolic demands of flight can exceed energy intake from recently consumed prey items, so insectivorous bats may metabolize fat stores (especially early in the night) to power flight with ketones, a byproduct of the normal oxidation of fatty acids. Previous studies in bats have found increases in the plasma ketone ß-hydroxybutyrate following food consumption, but no study has explored whether increases in plasma ß-hydroxybutyrate occur following flight without food consumption. We collected and analyzed blood to examine changes in plasma ß-hydroxybutyrate following different flight durations in big brown bats. We explored both seasonal and captivity effects. To explore seasonal effects, we sampled blood from bats in the fall and the spring, times that are biologically significant to big brown bats. The spring is when bats move out of torpor, a form of hibernation, into an active state and the fall is when bats are preparing for entering into torpor. To explore captivity effects, we sampled blood from bats recently introduced to or established to captivity. Bats were fasted for 12 hours prior to flight (exercise treatment) or rest (control), and then blood was collected. We characterized exercise using flight time. We found that plasma ß-hydroxybutyrate increased after longer flight durations, which supports the fasting while foraging hypothesis.

Historically, physiologists have believed steroid hormones act exclusively within the individual producing them. However, studies in mice have shown that bioactive 17β-estradiol (E2) is excreted in male urine and absorbed by female conspecifics where it binds to estrogen receptors in reproductive other tissues. This can lead to pregnancy disruption and/or cause precocious puberty in female conspecifics. In bats the transfer of tritium-labelled estradiol (3H-E2) from male to females has been shown during the mating season. I investigated the influence of season on 3H-E2 transfer and showed that females housed with 3H-E2 injected males had significantly higher levels of radioactivity in reproductive, neural, and peripheral tissues during reproductively relevant timepoints compared to the non-reproductive season. Because urine has been hypothesized as the vector of steroid hormone transmission in mice, I examined the natural patterns of sex steroids in the urine of male and female big brown bats (Eptesicus fuscus) across the annual reproductive cycle. I found that creatinine-adjusted E2, which corrects for animal hydration and activity, was significantly higher in male than female urine, and in adult compared to yearling urine. Seasonal differences in urinary E2 levels were observed within and between sexes. Finally, I designed a protocol to investigate the attractant properties of body odour and urine between bat sexes during the mating season. Using a two-alternative Y-maze arena, I found that female bats first approach the test arm containing urine of a male conspecific before exploring the arm with female urine. Females also tended to spend more time in the test-arm containing male urine and being in the male test-arm at the conclusion of the 5 min trial. My data supports the hypothesis that E2 has the potential to act as reproductive pheromone with urine as a likely vector. Given the close proximity of individual bats within a maternity roost, steroid hormone transfer between conspecifics quite likely occurs in nature and could have profound influences on female reproductive behaviour (e.g. receptivity) and physiology (e.g. estrus cycling). My research provides new evidence for the potential pheromonal actions of E2 in bats that is consistent with how sex steroids act as pheromones in other mammals. / Dissertation / Doctor of Philosophy (PhD) / Historically, physiologists have believed steroid hormones act exclusively within the individual producing them. However, studies in mice have shown that bioactive 17β-estradiol (E2) is excreted in male urine and absorbed by female conspecifics where it binds to estrogen receptors in reproductive and other tissues. This exogenous E2 can result in changes to female reproductive physiology and behaviour. Our lab has previously observed E2 to transfer between male and female captive big brown bats during the mating season. Research from this thesis provides evidence that the transfer of E2 from male to female bats is highest during times of mating and ovulation/fertilization. I also demonstrate that E2 naturally occurs in the urine of both male and females with age, sex, and seasonal differences in concentration. Lastly, I show that female bats are attracted to the urine of males. My research provides new evidence for the potential pheromonal actions of E2 in bats that is consistent with how sex steroids act as pheromones in other mammals.

Auditory neurons selectively respond to frequency and amplitude of sound. In the auditory midbrain, duration-tuned neurons (DTNs) are subsets of neurons that selectively respond to the duration of sound. DTNs may help further understand the neural mechanism underlying temporal processing in the central nervous system. Temporal processing has been shown to play important roles in speech, discriminating species-specific signals, and echolocation. The goal of this thesis is to explore the role of DTNs through single-unit electrophysiological recordings in the auditory midbrain of the big brown bat (Eptesicus fuscus). Monotic and dichotic paired-tone stimulation was used to evoke excitatory and inhibitory responses from DTNs. Two stimuli consisted of best duration (BD) excitatory and non-excitatory (NE) tones. In the monotic condition, both tones were presented to the contralateral ear, and when they were close in time, the NE tone always suppressed spikes evoked by the BD tone. In the dichotic condition, the BD tone was presented to the contralateral ear. The NE tone was presented to the ipsilateral ear and suppressed BD tone evoked spiking in ~50% of cells. Properties of the ipsilaterally-evoked inhibition were investigated by varying the frequency of the NE tone from the best excitatory frequency (BEF), throughout a cell’s excitatory bandwidth (eBW). We measured the inhibitory frequency response area, best inhibitory frequency (BIF), and inhibitory bandwidth (iBW) of each cell. We found inhibition became weaker as the frequency of the NE tone moved further from the middle of the eBW. We found that a DTN’s BEF and BIF closely matched, but the eBW was broader than the iBW and overlapped the iBW measured from the same cell. This suggests temporal selectivity of midbrain DTNs are created by monaural inputs, with binaural inputs playing a lesser role in shaping duration selectivity. / Thesis / Master of Science (MSc)

Many neurons in the auditory midbrain are tuned to binaural cues. Two prominent binaural cues are the interaural intensity difference (IID) and the interaural time difference (ITD). The ITD cue can further be classified as either an ongoing ITD, which compares the phase difference in the waveform of low frequency stimuli present at either ear, or an onset ITD, which compares the onset time of arrival of two stimuli at either ear. Little research has been done on the sensitivity of single neurons to onset ITDs in the auditory system, particularly in bats. The current study examines the response properties of neurons in the inferior colliculus (IC) of the big brown bat, Eptesicus fuscus, to onset ITDs in response to high frequency pure tones. Measures of neurons’ dynamic response—the segment of the ITD function exhibiting the highest rate of change in activity—revealed an average change of 36% of its maximum response within the estimated behaviorally relevant range of ITDs. Time-intensity trading describes the ability of the brain to compensate the binaural time cue (ITD) cue for the binaural intensity cue (IID) and can be measured as the horizontal shift of an ITD function at various IIDs. Across all IC neurons, an average time-intensity trading ratio of 30 μs/dB was calculated to measure the sensitivity of IC neurons’ ITD response to changing IIDs. Minimum and maximum ITD responses were found to be clustered within a narrow range of ITDs. The average peak ITD response occurred at 268 μs and is consistent with findings in other mammals. All results in ITD tuning, time-intensity trading, and response maximum were invariant to stimulus frequency, confirming that IC neurons responded to onset ITDs and not ongoing ITDs. These results suggest the potential for high frequency onset cues to assist in the azimuthal localization of sound in echolocating bats. / Thesis / Master of Science (MSc)

No description available.

Duration tuning in the mammalian inferior colliculus (IC) is created by the interaction of excitatory and inhibitory synaptic inputs. We used extracellular recording and paired-tone stimulation to measure the strength and time-course of the contralateral inhibition and offset-evoked excitation underlying duration-tuned neurons (DTNs) in the IC of the awake bat. The onset time of a short, best duration (BD), excitatory probe tone was varied relative to the onset of a longer-duration, non-excitatory (NE) suppressor tone. Spikes evoked by the roving BD tone were suppressed or facilitated when the stationary NE tone was varied in frequency or amplitude. When the NE tone frequency was presented away from the cell’s best excitatory frequency (BEF) or at lower SPLs, the onset of inhibition was relatively constant whereas the offset and duration of inhibition decreased. Excitatory and inhibitory frequency response areas were measured and best inhibitory frequencies matched best excitatory frequencies; however, inhibitory bandwidths were broader than excitatory bandwidths. Excitatory rate-level and inhibitory suppression-level functions were also measured and the dynamic ranges and inflection points were similar, which is hypothesized to play a role in the level tolerance of responses measured from DTNs. We compared the latency of offset-locked facilitation to the onset or offset of inhibition as a function of frequency and amplitude; we found that the facilitation was more related to the onset of inhibition. Moreover, facilitation typically preceded the offset of inhibition – suggesting that it is a separate excitatory input to DTNs and not a rebound from inhibition. We conclude that DTNs receive inputs that generate and preserve temporal selectivity. / Dissertation / Doctor of Philosophy (PhD)

Le syndrome du museau blanc (SMB), causé par le champignon Pseudogymnoascus destructans (Pd), a mis en péril les populations de chauves-souris hibernantes en Amérique du Nord. Certaines espèces sont hautement vulnérables à la maladie alors que d’autres espèces semblent être résistantes ou tolérantes à l’infection. Plusieurs facteurs physiologiques et environnementaux peuvent expliquer ces différences. Or avant 2015, peu d’études avaient porté sur le microbiome de la peau en relation avec cette maladie. La présente thèse vise à caractériser le microbiome cutané de chiroptères affectés par le SMB afin d’identifier les facteurs de vulnérabilité ou de résistance à la maladie. L’objectif principal est de déterminer comment le microbiome est affecté par la maladie ainsi que de déterminer si celui-ci à un rôle dans la protection face à l’infection fongique. Au Chapitre 1, nous avons tout d’abord exploré et comparé le microbiote cutané de petites chauves-souris brunes (Myotis lucifugus) non affectées par le SMB avec celui de chauves-souris survivantes au SMB pour tester l’hypothèse selon laquelle le microbiote cutané est modifié par la maladie. Nos résultats montrent que le site d’hibernation influence fortement la composition et la diversité du microbiote cutané. Les sites d’hibernations Pd positifs et négatifs diffèrent significativement en termes de diversité, ainsi qu’en termes de composition du microbiote. La diversité est réduite au sein du microbiote des chauves-souris survivantes au SMB et enrichi en taxons tels que Janthinobacterium, Micrococcaceae, Pseudomonas, Ralstonia et Rhodococcus. Certains de ces taxons sont reconnus pour leur potentiel antifongique et des souches spécifiques de Rhodococcus et de Pseudomonas peuvent inhiber la croissance de Pd. Nos résultats sont cohérents avec l’hypothèse selon laquelle l’infection par Pd modifie le microbiote cutané des chauves-souris survivantes et suggèrent que le microbiote peut jouer un rôle de protection face au SMB. Au Chapitre 2, nous avons étudié le microbiote d’une espèce résistante au champignon Pd en milieu contrôlé avant et après infection afin d’établir la réponse potentielle à la maladie. L’espèce étudiée est la grande chauve-souris brune (Eptesicus fuscus) dont le microbiote cutané pourrait jouer un rôle de protection contre l’infection. Nos résultats montrent que la diversité du microbiote de la grande chauve-souris brune inoculée avec Pd est plus variable dans le temps, tandis que la diversité du microbiote des chauves-souris du groupe contrôle demeure stable. Parmi les taxons les plus abondants, Pseudomonas et Rhodococcus, deux taxons connus pour leur potentiel antifongique contre Pd et d’autres champignons, sont restés stables durant l’expérience. Ainsi, bien que l’inoculation par le champignon Pd ait déstabilisé le microbiote cutané, les bactéries aux propriétés antifongiques n’ont pas été affectées. Cette étude est la première à démontrer le potentiel du microbiote cutané d’une espèce de chauves-souris pour la résistance au SMB. Au Chapitre 3, le microbiome cutané de la petite chauve-souris brune a été évalué en milieu naturel dans le contexte du SMB, à l’aide de la métagénomique, une approche haute résolution pour observer le potentiel fonctionnel du microbiome (métagénome fonctionnel). Nos résultats ont permis d’établir que le temps depuis l’infection a un effet significatif sur le métagénome fonctionnel. En effet, les chauves-souris dans la première année suivant l’infection ont un métagénome fonctionnel perturbé qui subit une perte de diversité fonctionnelle importante. Toutefois, le métagénome fonctionnel revient à une structure et composition similaire d’avant infection après 10 ans. Certaines fonctions détectées suite à l’infection sont associées à des gènes reliés au transport et à l’assimilation de métaux, des facteurs limitants pour la croissance du champignon. Ces gènes pourraient donc avoir un rôle à jouer dans la résistance ou la vulnérabilité à la maladie. Globalement, l’étude du métagénome chez la petite chauve-souris brune indique une vulnérabilité du métagénome fonctionnel au champignon, mais que celui-ci semble se rétablir après 10 ans. Une telle réponse pourrait avoir un impact sur la résilience de M. lucifugus. Cette thèse a permis d’acquérir des connaissances fondamentales sur le microbiome cutané des chauves-souris en hibernation pour mieux comprendre les communautés microbiennes de la peau dans le contexte du SMB. Le microbiome pourrait en effet jouer un rôle dans la vulnérabilité et la résistance des chauves-souris à la maladie, et il est essentiel d’adapter notre façon d’aborder la protection de ces espèces et de leur microbiome. Nous souhaitons que les travaux de cette thèse permettent de sensibiliser les acteurs de la conservation à l’existence et à l’importance potentielle du microbiome pour la santé de son hôte. Cette thèse fait également état de l’avancement des méthodes d’analyses qui permettront d’être de plus en plus précis et d’appliquer les connaissances du microbiome en biologie de la conservation. / White-nose syndrome (WNS) caused by the fungus Pseudogymnoascus destructans (Pd) has put hibernating bat populations at risk in North America. Some species are highly vulnerable to the disease while other species appear to be resistant or tolerant. Several physiological and environmental factors can explain these differences. However, before 2015, few studies have focused on the skin microbiome in relation to this disease. The present thesis aims to characterize the cutaneous microbiome of bats affected by WNS in order to identify the factors of vulnerability or resistance to the disease. The main objective is to determine how the microbiome can protect against the Pd fungus, or conversely how the microbiome is altered by the fungal infection. In Chapter 1, we first explored and compared the skin microbiota of little brown bats (Myotis lucifugus) unaffected by WNS with that of WNS survivors to test the hypothesis that the skin microbiota is modified by the disease. Our results show that the hibernation site strongly influences the composition and diversity of the skin microbiota. The Pd positive and negative sites differ significantly in terms of diversity, as well as in terms of the composition of the microbiota. Diversity is reduced within the microbiota of bats surviving WNS and enriched in taxa such as Janthinobacterium, Micrococcaceae, Pseudomonas, Ralstonia, and Rhodococcus. Some of these taxa are recognized for their antifungal potential and specific strains of Rhodococcus and Pseudomonas may inhibit the growth of Pd. Our results are consistent with the hypothesis that Pd infection modifies the skin microbiota of surviving bats and suggest that the microbiota may play a protective role against WNS. In Chapter 2, we studied in a controlled environment the microbiota of a species that exhibits evidence of resistance with mild WNS symptoms, before and after infection, to establish the potential response to the disease. The species studied is the big brown bat (Eptesicus fuscus), whose skin microbiota could play a protective role against infection. Our results show that the diversity of the microbiota of big brown bats inoculated with Pd is more variable over time, while the diversity of the microbiota of the control bats remains stable. Among the most abundant taxa, Pseudomonas and Rhodococcus, two taxa known for their antifungal potential against Pd and other fungi, remained stable during the experiment. Thus, although inoculation with the Pd fungus destabilized the skin microbiota, bacteria with antifungal properties were not affected. This study is the first to demonstrate the potential of the skin microbiota of a bat species for resistance to WNS. In Chapter 3, the skin microbiome of the little brown bat was evaluated in the natural environment in the context of WNS, using metagenomics, a higher-resolution approach to observe the functional potential of the microbiome (functional metagenome). Our results established that the time since infection has a significant effect on the functional metagenome. Indeed, bats in the first year after infection have a disrupted functional metagenome that undergoes a significant loss of functional diversity. However, the functional metagenome returns to a similar structure and composition to that observed before infection after 10 years. Certain functions detected following infection are associated with genes linked to the transport and assimilation of metals, known limiting factors for the growth of the fungus. These genes could therefore have a role to play in resistance or vulnerability to the disease. Overall, this metagenomics study indicates functional metagenome vulnerability to the fungus, although the original functional metagenome is reestablished after 10 years. Such diversified response could impact M. lucifugus resilence. This thesis provides fundamental knowledge on the skin microbiome of hibernating bats to better understand the microbial communities of the skin in the context of WNS. The microbiome could indeed play a role in the vulnerability and resistance of bats to disease and it is essential to adapt our way of approaching the protection of these species and their microbiomes. We hope that the results of this thesis will raise awareness among conservation stakeholders about the existence and potential importance of the microbiome for the health of its host. This thesis also reports on the advancement of analytical methods that will make it possible to be more and more precise and to apply knowledge of the microbiome in conservation biology.