Physiological Ecology of Bat Migration

Migration is perhaps the most poorly understood aspect of bat biology and the underlying physiological basis is virtually unstudied. Although distantly related, bats and birds are both endothermic flying vertebrates and bird migration physiology has been studied for decades. Therefore, I used migratory birds as a model system to make predictions regarding the physiological ecology of bat migration. First, I compared brain size of migratory and sedentary bat species. Migratory species have smaller brains which suggests the costs of carrying and maintaining a large brain are incompatible with the demands of migration. Next, I studied silver-haired bats (Lasionycteris noctivagans) during migratory stopover. Bats arrived at the site with fat stores comparable to migratory birds, rarely foraged, and had short stopover durations. I proposed that bats use daily torpor to minimize energy expenditure during non-flight periods, thus sparing fuel stores for migratory flight. Finally, I compared body composition and flight muscle physiology in migrating and non-migrating hoary bats (Lasiurus cinereus). Changes in digestive and exercise organ sizes, the composition of adipose stores, and increased catabolic enzyme activities all reflected the increased energetic demands of migration. Sex-specific changes in muscle membrane fatty acid composition and the expression of fatty acid transport proteins suggest pregnant females are subject to different pressures than males. The energetic demands of bat migration lead to many physiological changes as observed in migratory birds. However, several factors specific to bats (especially heterothermy and the timing of reproduction) result in bat migration as a distinct phenomenon compared to birds

THE EFFECTS OF DIETARY FATTY ACIDS ON AVIAN MIGRATORY PERFORMANCE

Migratory birds use fat nearly exclusively to fuel their long distance movements. Little is known about how different types of fat affect their migratory performance, although dietary fats have been hypothesized to affect exercise through their roles as both oxidative substrates and membrane components. This thesis investigated the effects of fatty acids on avian flight performance and evaluated these two hypothesized mechanisms. I first investigated the selective mobilization of fatty acids from avian adipocytes. Unsaturated fatty acids and short chain fatty acids were most readily mobilized from avian adipocytes, indicating they may be important in increasing maximal metabolic rates. Further, this pattern did not change with migratory condition. I also investigated the effect of migratory condition and exercise on muscle phospholipid composition. Migratory disposition itself did not induce any endogenous changes to muscle phospholipid composition in preparation for migration, although exercise did have an effect on muscle phospholipids. Next, I demonstrated that birds fed a high co6 fatty acid diet achieved higher peak metabolic rates than those fed a high co3 diet. Further, I used a dietary manipulation to independently alter adipose triacylglycerol stores and muscle phospholipid composition. This experiment demonstrated that phospholipid composition does not drive exercise performance differences, but that triacylglycerol composition might be responsible for increased peak metabolic rate in birds fed the high ©6 diet. I also investigated the selectivity of the mitochondrial membrane fatty acid transporter carnitine palmitoyl transferase, and demonstrated that its activity was highest with shorter and polyunsaturated fatty acyl CoA substrates. Overall, maximal exercise in performance in birds may be affected by stored triacylglycerol composition due to selectivity of the fatty acid transport system, but does not appear to be affected by muscle phospholipid composition. Finally, I developed a theoretical framework for the study of avian fat composition that focuses on the tradeoffs between energy storage and transport of fatty acids

Genomic adaptations to a high sugar diet in birds

While excessive consumption of glucose- and fructose-sweetened soft drinks is a major risk factor for type 2 diabetes and metabolic syndrome in humans, several nectarivorous bird lin- eages have adapted their metabolism to rely mostly on simple sugars obtained from flower nectar. These lineages are spread around the world and include hummingbirds (Americas), honeyeaters and lorikeets (Australasia), and sunbirds (Africa and Asia). All these nectarivores have evolved distinct phenotypic traits allowing them to rely mostly on nectar as a source of nutrients. However, the genomic underpinnings of these natural adaptations to nectarivory are largely unknown. In order to identify genomic changes underlying metabolic adaptations of nectarivorous birds, we produced new genomic and transcriptomic data, and combining them with publically avail- able data, we ran a number of comprehensive comparative screens. To confirm our theoretical findings, we complemented them with experimental validation. The genome-wide screen for hummingbird-specific gene losses identified the loss of FBP2, a gene encoding a key gluconeogenic enzyme that is normally active in muscles of all tetrapods. Loss of FBP2 occurred around a time where energy-demanding hovering flight is thought to have evolved in hummingbirds. We hypothesized that FBP2 loss could have contributed to the evolution of their metabolic adaptations in muscles. To test this, we downregulated the gene in a bird muscle cell line. Even a partial knockout of FBP2 significantly upregulated glycolysis and mitochondrial respiration in cells. In our experiments, we also show that the latter is likely happening due to the increased number of mitochondria. Together, these results suggest that FBP2 loss contributed to metabolic adaptations that likely enhanced hummingbirds’ ability to immediately process newly ingested sugars thus providing energy for the hovering flight. To study the convergence of adaptations to nectarivory, we ran a number of screens search- ing for convergent and lineage-specific genomic changes. A screen for rapid adaptive evolution identified that a rate-limiting glycolytic enzyme (hexokinase 3) evolved under strong positive se- lection in the stem honeyeaters, potentially underlying similar metabolic changes to what FBP2 loss has introduced in hummingbirds. These results provide a deep insight into the genomic basis of adaptations to high-sugar ‘soft drink-like’ diets in birds. These findings have the potential not only to answer important evolu- tionary questions but also to teach us lessons concerning type 2 diabetes, metabolic syndrome, and obesity in humans.:Zusammenfassung 4 Abstract 6 Chapter I. Introduction 8 Diet diversity in birds 8 Nectarivory and its challenges 8 Adaptations to nectarivory 11 Hummingbirds 13 Other nectar-feeding birds 14 Genomic changes underlying phenotypic variation 15 Gene copy-number variation 16 Gene duplication mechanisms and its evolutionary fates 17 Gene duplications contribute to adaptive evolution 17 Detection of gene duplications 19 Gene loss 19 The use-it-or-lose-it hypothesis 20 The less-is-more hypothesis 20 Detection of gene losses 20 Selection pressure 21 Positive selection can drive adaptive evolution 21 Detection of selection 22 Amino acid substitutions 23 Previous research on adaptations to nectarivory 23 Outline of the thesis 24 Chapter II. Genomic basis of metabolic adaptations in hummingbirds 26 Section I: Screen for hummingbird-specific gene losses 26 Overview 26 Results 26 Assembly of the long-tailed hermit genome 26 Identification of hummingbird-specific gene losses 27 Hummingbird muscle expresses no FBPases 29 Discussion 30 Section II: Experimental exploration of the metabolic role of the FBP2 loss 31 Overview 31 Results 31 Expression of FBPase encoding genes in cell lines 31 Inhibition of FBPases in QM7 32 Testing the FBPase inhibitor with glycogen assays 33 Testing the FBPase inhibitor with Seahorse Glycolytic Rate Assay 37 Generation of FBP2 knockout in QM7 38 Infection - transfection strategy 38 Electroporation strategy 40 Detection of FBP2-encoded protein with immunostaining 42 Knockout of FBP2 upregulates glycolysis in avian myoblast cells 44 FBP2 downregulation upregulates OXPHOS 44 FBP2 knockout increases the number of mitochondria 44 Genes important for mitochondrial biosynthesis and function are upregulated in hummingbirds 46 FBP2 knockout is associated with increased lipid deposition 49 Discussion 49 Section III: Hummingbird tissues analysis 50 Overview 50 Results 51 Glycogen content in hummingbirds tissues 51 Histological comparison 51 Biochemical quantification of total glycogen 52 Lipid content in hummingbird tissues 53 Discussion 54 Section IV: Evolution of glucose metabolism genes in hummingbirds 55 Overview 55 Results 55 Positive selection in glycolytic genes in hummingbirds 55 Copy number of glucose transporters in hummingbirds 57 Expression of glucose transporters in hummingbirds 58 Discussion 59 Chapter III. Convergence in the evolution of nectarivory 60 Overview 60 Results 61 Sequencing and assembly of new genomes of nectarivorous birds 61 Annotation of protein-coding genes 62 Gene families expansion-contraction 64 Signatures of positive selection related to nectarivory 65 The rate-controlling glycolytic enzyme evolved faster in honeyeaters 70 Discussion 72 Chapter IV. General discussion and outlook 72 Future work 75 Chapter V. Methods 77 Methods I: Screen for hummingbird-specific gene losses 77 Genome assembly 77 Modeling and masking repeats 77 Generating pairwise genome alignments 77 Detecting gene losses 77 Gene loss dating 77 Methods II: Experimental exploration of the metabolic role of the FBP2 loss 78 Cell culture 78 Guide RNA design 78 Infection and transfection of QM7 myoblasts 78 Electroporation of QM7 myoblasts 79 Genotyping of QM7 cell pools 79 RNA isolation 80 Real-time qPCR 80 Seahorse glycolytic rate assay 80 Cell number quantification 80 Mitochondrial number estimation 80 Biochemical glycogen qualification in cells 81 Fluorescent glycogen qualification in cells 81 Lipid quantification in cells 81 Western blot 82 Homology modeling 82 RNA sequencing 82 Analysis of transcriptomic data 83 Methods III: Hummingbird tissues analysis 83 Tissue preparation 83 Paraffin embedding and sectioning 83 Glycogen staining tissues 84 Freezing tissues and cryosectioning 84 Lipid staining tissues 84 Imaging histological sections 84 Biochemical glycogen qualification in tissues 84 Methods IV: Changes in glucose metabolism genes in hummingbirds 85 Positive selection analysis 85 Methods: Convergence in the evolution of nectarivory 85 Protein-coding gene annotation 85 Positive selection analysis 86 Estimating time-calibrated phylogeny 87 Gene families expansion-contraction analysis 87 Homology modeling 87 Appendix 8

Aerospace medicine and biology: A continuing bibliography with indexes, supplement 128, May 1974

This special bibliography lists 282 reports, articles, and other documents introduced into the NASA scientific and technical information system in April 1974

Environmental Physiology of Flight in Migratory Birds

Migratory birds complete amazing journeys between their breeding and wintering grounds. Each journey comprises a series of flights that last hours to days, followed by stopovers where fuel stores are replenished. Despite the long flights undertaken by migratory birds, where respiratory water losses are high for extended periods of time, birds are not dehydrated after flight. My studies demonstrate that birds maintain hydration by modulating rates of endogenous water production in response to rates of water loss. In resting, water restricted house sparrows (Passer domesticus) I used quantitative magnetic resonance body composition analysis (QMR) and hygrometry to demonstrate that stressed resting birds increase the rate of lean mass (protein) catabolism to liberate water and maintain osmotic homeostasis. I then flew Swainson’s thrushes (Catharus ustulatus) in a climatic wind tunnel under high- and low-humidity conditions for up to 5 hours. Flight under dry conditions increased the rate of lean mass loss, endogenous water production and plasma uric acid concentrations. This demonstrated that atmospheric humidity influences fuel composition in flight and suggest that protein deposition and catabolism during migration are a metabolic strategy to maintain osmotic homeostasis during flight. Next, I investigated the metabolic response to flight in the American robin (Turdus migratorius). These birds have high rates of endogenous water production early in flight due to a high contribution of carbohydrate and protein to energy during the transition to fat oxidation, and do not require additional protein catabolism to maintain water balance. Migratory birds may reduce excretory water losses to avoid dehydration in flight. I investigated kidney function in fed, rested and flown Swainson’s thrushes and found no decrease in glomerular filtration rate during flight, however they rely on increased water reabsorption to reduce excretory water losses in flight and at rest. Finally, the effect of diet on mitochondrial metabolism was investigated. I demonstrated that the performance increases often attributed to high dietary polyunsaturated fatty acids are likely due to reduced rates of production of reactive oxygen species by mitochondria. Together, these studies advance our knowledge of the metabolic response to the environment in the context of bird migration

Lindude põgenemiskäitumine erineva kisklusriskiga olukordades: terviklikum käsitlus

Väitekirja elektrooniline versioon ei sisalda publikatsiooneSuurema hirmutunde korral panustavad loomad enam aega valvsusele ja ohtu silmates põgenevad varem. Inimene võib oma tegevustega tahtmatult loomade hirmutunnet suurendada, põhjustades halbu käitumisotsuseid ja populatsiooni arvukuse langust. Loomade hirmutunde hindamiseks mõõdetakse enamasti kas nende valvsust või põgenemiskaugust. Doktoritöös uuriti, kas üheainsa käitumispõhise indikaatori mõõtmisest siiski piisab, et lindude hirmutunnet ja põgenemisega seotud kulutusi veenvalt hinnata. Lisaks selgitati, kas linnud jätkavad kiskja jälgimist veel ka põgenemise ajal ja milliseid lisategureid tuleks põgenemiskäitumise uurimisel arvesse võtta. Doktoritöö raames uuriti standardiseeritud viisil kümnete linnuliikide põgenemiskäitumist mitmel pool üle Euroopa, et tuvastada üldisi trende lindude käitumismustrites. Põgenemiskäitumise poolest olid linnalinnud mitmes mõttes julgemad kui maalinnud ning laiuskraadi kasvades muutusid linnud vähem ettevaatlikuks. Üllatuslikult olid linnalinnud valvsamad kui maalinnud ning vastupidiselt varem eeldatule viivitasid valvsamad linnud mõlemas elupaigas põgenemisega kauem. Need tulemused seavad valvsuse tavapärase kasutatavuse lindude hirmutunde kirjeldamiseks kahtluse alla. Tööst ilmnes veel, et lisaks enamasti uuritavale põgenemiskaugusele tuleks mõõta ka põgenemishetkele järgnevaid käitumismustreid. Need täiendavad mõõtmised annavad terviklikuma ülevaate põgenemisega seotud ajalistest ja energeetilistest kulutustest, mis omakorda aitab kaasa põgenemisotsuste mõistmisele. Näiteks leiti, et põgenemisele kulutatud aeg sõltub hirmutunde dünaamikast põgenemise ajal. See on esimene eksperimentaalne tõend, et linnud jätkavad kiskja jälgimist ka pärast põgenemise alustamist. Doktoritöö tulemused toetasid ka varasemaid uuringuid, mis on leidnud, et põgenemisotsuseid mõjutavad ka näiteks liigiomane kehamass, peidupaiga lähedus ja seltsingu suurus. Doktoritööst saadud teadmised aitavad paremini ennustada, kuidas lindude hirmutunne võib inimtekkeliste häiringutega seoses muutuda, mis omakorda aitab kaasa looduskaitseliste meetmete planeerimisele.Animals with higher levels of fearfulness will spend more time being vigilant and will escape earlier after having spotted a potential threat. Human-caused disturbances can involuntarily increase fearfulness in animals, which can result in inaccurate behavioural decisions that can lead to population declines. The most common measures of fearfulness in animals are vigilance and flight initiation distance. Present thesis examined whether the use of a single behavioural indicator is enough to assess fearfulness and costs related to escape. The thesis also investigated whether birds continue to monitor predators during escape, and which other factors should be considered when studying escape behaviour. Tens of bird species were studied across Europe in a standardized way to find general patterns in the behaviour of birds. Urban birds were characterized by a more relaxed escape behaviour than rural birds. In addition, birds took longer to become alert to threats as latitude increased. Surprisingly, urban birds were more vigilant than rural birds, and, contrary to the prevalent theory, vigilant birds delayed escape more. These results raise doubt whether vigilance should be used as an indicator of fearfulness in birds. The thesis also highlighted that in addition to measuring flight initiation distance, it is important to measure subsequent behavioural decisions. Doing so provides a more complete view of the energetic and opportunity costs of escape, which helps to understand escape-related decisions. For example, it was found that escape duration depends on how the perceived risk of predation changes while fleeing. That is the first experimental evidence that birds continue to monitor predators after initiating escape. Last, the evidence from the thesis complements previous research that, for example, has found body mass, distance to refuge, and group size to influence escape decisions in animals. The knowledge gained from the thesis improves predictions about the impact of human-caused disturbances on fearfulness in birds, which in turn benefits decision making in wildlife management.https://www.ester.ee/record=b529839

THE PHENOTYPIC FLEXIBILITY OF THERMOGENIC CAPACITY: FROM PHYSIOLOGICAL MECHANISM TO EVOLUTIONARY IMPLICATIONS

Individuals face many selection pressures that change throughout their lives. Phenotypic flexibility, the ability to flexibly and reversibly modify a trait value, is one way an individual can optimally match its phenotype to the prevailing environmental conditions. In this dissertation, I used juncos as a lens to understand the causes of variation in flexibility within physiological systems and among individuals. In my first chapter, I investigated how Dark-eyed Juncos (Junco hyemalis) alter mechanisms of heat production and heat conservation to cope with variation in ambient conditions. My results demonstrate the ability of birds to adjust thermoregulatory strategies in response to thermal cues and reveal that birds may combine multiple responses to meet the specific demands of their environment. To further explore the thermoregulatory strategies available to juncos, in my second chapter, I assess their potential use of non-shivering thermogenesis. My results indicate that muscular non-shivering thermogenesis is not an important mechanism of avian thermoregulation, potentially as a consequence of a tradeoff between the many demands placed on avian muscles. In my third chapter, I measured 20 additional physiological traits to explore the mechanistic basis of flexibility in complex phenotypes. I show that the relationships among traits contributing to whole-organism performance varied with the environmental context. Moreover, whole-organism flexibility in thermogenic performance was correlated with only a handful of subordinate phenotypes. In my fourth chapter, I identified drivers of variation in flexibility among juncos. To do this, I integrated measures of population genetic variation with assays of thermogenic performance and indices of environmental heterogeneity for individuals across the genus Junco. I find that native temperature heterogeneity correlates both with population genetic variation and the degree of thermogenic flexibility exhibited by an individual. In my fifth chapter, I present a review that considers the evolutionary implications of phenotypic flexibility and contrast those with developmental plasticity. I hypothesize that because these two processes experience selection distinctly, confer stability to populations differentially, and will likely evolve at different rates. Collectively, this work helps us understand the role of flexibility in adaptation and species’ resilience to environmental change

Growth Characteristics and Lipid Metabolism of Cultured Migratory Bird Skeletal Muscle Cells

Diets rich in n-3 polyunsaturated fatty acids (PUFA) may alter the muscle metabolism of migratory birds, improving their endurance performance. I established and validated for the first time in vitro muscle models of a migratory songbird (yellow-rumped warbler, Setophaga coronata coronata) and shorebird (sanderling, Calidris alba). To evaluate the role of n-3 PUFA in improving fatty acid metabolism in migratory bird muscle, I measured metabolic outcomes following n-3 PUFA supplementation in these two avian cell types and a murine (Mus musculus, C2C12) cell line. PUFA supplementation in C2C12 cells increased metabolic transcription factor expression and increased mitochondrial respiratory chain efficiency. Migrant bird muscle cells did not display the same changes in transcriptional signaling, but sanderling cells increased basal and maximal oxygen consumption with n-3 PUFA supplementation. This research provides support for the hypothesis that n-3 PUFA increase aerobic capacity of a migrant sandpiper and efficiency in mammalian skeletal muscle

Oksüdatiivsed kaitsereaktsioonid immuunökoloogilises kontekstis: lämmastikoksiidi produktsiooni ning oksüdatiivse purske mõõtmise meetodite kohandamine ja rakendamine värvulisele

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Immuunökoloogia aluseks on põhimõte, et immunvastused on kulukad ning seetõttu omavad lõivsuhteid teiste elukäigu tunnustega. Samas pole veel selge, mis võiks olla immunvastuse kulukuse põhjuseks. Üheks võimalikuks variandiks on immuunvastuse käigus tekitatud patogeenide hävitamiseks mõeldud reaktiivsete osakeste poolt tekitatud koekahjustused. Selle teooria kontrollimisel on takistavaks saanud nii immuunökoloogias kasutusel olevate meetodite vähesus kui ka asjaolu, et immuunsüsteem on väga keeruline ning põhjalikult seotud nii endokriinse kui ka närvisüsteemiga. Antud töös on immunökoloogia sagedastele uurimisobjektidele, värvulistele lindudele, kasutamiseks kohandatud kaks meetodit, mis mõõdavad kaasasündinud immuunvastuse käigus tekitatud reaktiivseid osakesi- lämmastikoksiidi ning oksüdatiivse purske käigus tekkivaid reaktiivseid osakesi. Seejärel on neid uusi meetodeid rakendatud uurimaks immuunsüsteemi seoseid endokriinse ja närvisüsteemiga ning selleks, et testida antioksüdandi karotenoidi mõju immuunvastusele.Immunoecology is based on the principle that immune defences are costly and therefore have trade-offs with other life-history traits. At the moment, however, the reasons why immune defences are costly, are poorly understood. One of the possible versions is the collateral damage of tissues by reactive oxygen species that are produced during immune response to destroy pathogens. The controlling of this theory has turned out to be difficult for two main reasons- lack of methods used in immunoecology and the fact that immune system is very complex and has tight interconnections with nervous and endocrine systems. In the current thesis I have modified two methods for use with immunoecological model species, passerine birds. These methods measure reactive species produced during innate immune responses - nitric oxide and reactive species produced during oxidative burst. Subsequently I used these new methods to study connections between immune system and endocrine and nervous systems and to test the effects of antioxidant carotenoid to immune response