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The water-to-air respiratory transition of amphibiotic dragonflies Lee, Daniel Jingyu 2019

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   THE WATER-TO-AIR RESPIRATORY TRANSITION OF AMPHIBIOTIC DRAGONFLIES   by  Daniel Jingyu Lee  B.Sc. (Hons.), The University of British Columbia, 2016    A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE   in   The Faculty of Graduate and Postdoctoral Studies   (Zoology)     THE UNIVERSITY OF BRITISH COLUMBIA  (Vancouver)    April 2019   © Daniel Jingyu Lee, 2019  ii  The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, a thesis/dissertation entitled:  The water-to-air respiratory transition of amphibiotic dragonflies  submitted by Daniel Jingyu Lee  in partial fulfillment of the requirements for the degree of Master of Science in Zoology  Examining Committee: Philip G.D. Matthews, Zoology Supervisor  William K. Milsom, Zoology Supervisory Committee Member   Supervisory Committee Member Robert Shadwick, Zoology Additional Examiner     Additional Supervisory Committee Members: Colin J. Brauner, Zoology Supervisory Committee Member  Supervisory Committee Member      iii  Abstract  The transition from water-breathing to air-breathing is perhaps one of the greatest achievements in animal evolution, as it allowed them to colonize land and occupy new environmental niches. Numerous studies have investigated the respiratory adaptations that must have accompanied this respiratory transition in vertebrates and crustaceans, and have reached the conclusion that water-breathing animals have adapted to low levels of blood CO2 partial pressure (PCO2) and HCO3- while air-breathers have adapted to high levels of PCO2 and HCO3-, and that all animals making this transition follow this trend. However, the insects originated on land as air-breathers, and certain lineages subsequently evolved water-breathing capacities to become aquatic. As a result, the insects must have faced and overcome different challenges during their invasion of water compared to vertebrates and crustaceans that were ancestrally water-breathing and secondarily became air-breathers. However almost nothing is known regarding the respiratory transition of insects, and it remains to be seen whether the conclusions based on vertebrates and crustaceans are applicable to insects. This thesis is the first to explicitly investigate the respiratory physiology of insects during the transition from water to air, in order to examine how similar or different it is to that of vertebrates and crustaceans making the same transition. By measuring the total CO2 (TCO2) content of dragonfly nymphs and adults, it was revealed that the magnitude of TCO2 increase from water-breathing to air-breathing is very minor in these insects compared to that experienced by vertebrates and crustaceans. In addition, quantifying the acid-base status of dragonfly hemolymph showed that the change from water-breathing to air-breathing elicits modifications of the hemolymph chemistry that are not seen in vertebrates and crustaceans. The data presented in this thesis provide strong evidence that the respiratory transition of dragonflies from water to air is different from that observed in vertebrates and crustaceans, and questions the current consensus that all animals experience the same shift in blood PCO2 and HCO3- during the transition from water to air.       iv  Lay summary  Despite a wealth of information regarding how the blood chemistry of vertebrates and crustaceans changes as they transition from breathing water to breathing air, very little is known in this regard for insects. Insects are particularly interesting because unlike the vertebrates and crustaceans, they were initially air-breathers and secondarily became water-breathers, and as a result might have experienced different challenges and changes to their blood chemistry. This thesis aimed to study how the blood chemistry of insects changes as they transition from water-breathing to air-breathing, and the results show that insects experience noticeably different changes to their blood chemistry compared to vertebrates and crustaceans.                        v  Preface A version of Chapter 2 has been published [Lee, D.J., Gutbrod, M., Ferreras, F.M., and Matthews, P.G.D. Changes in hemolymph total CO2 content during the water-to-air respiratory transition of dragonflies. J Exp. Biol. doi: 10.1242/jeb.181438, 2018]. I was the primary author and was responsible for the conception of experiments, collection and analysis of all data, and writing of the manuscript. Gutbrod, M. and Ferreras, F.M provided experimental equipment, and provided editorial feedback. Matthews, P.G.D. was the supervisory author, and was involved in the conception of experiments and revision of the manuscript.   I wrote all chapters of this thesis, with editorial feedback from my supervisory committee, Drs. Philip G.D. Matthews, Colin J. Brauner, and William K. Milsom. In Chapter 3 I was responsible for the conception of experiments, and the collection and analysis of all data. Dr. Matthews was the supervisor, and was involved in the conception of experiments.                   vi  Table of Contents  Abstract ......................................................................................................................................... iii Lay Summary ............................................................................................................................... iv Preface .............................................................................................................................................v Table of Contents ......................................................................................................................... vi List of Tables ................................................................................................................................ ix List of Figures .................................................................................................................................x List of Symbols and Abbreviations ............................................................................................ xi Acknowledgements ..................................................................................................................... xii 1 Introduction .................................................................................................................................1 1.1 The insect evolution ...............................................................................................................1 1.2 Water and air as respiratory media .........................................................................................2 1.3 The respiratory transition of vertebrates and crustaceans ......................................................2 1.4 The respiratory transition of insects .......................................................................................4 1.5 Research questions .................................................................................................................5 2 Changes in hemolymph total CO2 content during the respiratory transition of insects ......6 2.1 Introduction ............................................................................................................................6 2.2 Materials and Methods ...........................................................................................................7 2.2.1 Animals............................................................................................................................7  2.2.1.1 Dragonfly nymphs ...................................................................................................7  2.2.1.2 Dragonfly adults.......................................................................................................8  2.2.1.3 Marbled crayfish ......................................................................................................8  2.2.1.4 Body mass ................................................................................................................9 2.2.2 Classification of dragonfly nymph developmental stage ................................................9 2.2.3 Measuring hemolymph TCO2..........................................................................................9 vii   2.2.3.1 Verification of experimental approach ..................................................................11 2.2.4 Measuring hemolymph PCO2 ........................................................................................11  2.2.4.1 Verification of experimental approach ..................................................................13 2.2.5 Statistical analyses .........................................................................................................13 2.3 Results ..................................................................................................................................14 2.3.1 Total hemolymph CO2 ...................................................................................................14 2.3.2 Effect of emersion on TCO2 ..........................................................................................15 2.3.3 Hemolymph PCO2 .........................................................................................................15 2.4 Discussion ............................................................................................................................15 2.4.1 Dragonfly TCO2 in water-breathing nymphs and air-breathing adults .........................15 2.4.2 Dragonflies compared to other animals .........................................................................17 2.4.3 Verification of experimental protocol ...........................................................................18 3 Quantifying the hemolymph acid-base status of water-breathing and air-breathing insects ............................................................................................................................................28 3.1 Introduction ..........................................................................................................................28 3.2 Materials and Methods .........................................................................................................29 3.2.1 Animals..........................................................................................................................29 3.2.2 Preparation of hemolymph samples ..............................................................................29  3.2.2.1 Dragonfly nymphs .................................................................................................29  3.2.2.2 Dragonfly adults.....................................................................................................29 3.2.3 Hemolymph CO2 solubility (α) .....................................................................................29 3.2.4 Carbonic acid apparent dissociation constant (pKapp) ...................................................31  3.2.4.1 Dragonfly nymphs .................................................................................................31  3.2.4.2 Dragonfly adults.....................................................................................................32 3.2.5 Relationship between pKapp and pH ..............................................................................32 viii  3.2.6 pH-HCO3- diagram ........................................................................................................32  3.2.6.1 Non-HCO3- blood buffer line .................................................................................32  3.2.6.2 PCO2 isopleth .........................................................................................................33 3.2.7 Statistical analyses .........................................................................................................33 3.3 Results ..................................................................................................................................33 3.3.1 Hemolymph CO2 solubility ...........................................................................................33 3.3.2 Carbonic acid dissociation constant (pKapp) ..................................................................34 3.3.3 Acid-base status .............................................................................................................34 3.4 Discussion ............................................................................................................................35 3.4.1 Hemolymph CO2 solubility and pKapp ...........................................................................35 3.4.2 Hemolymph acid-base status .........................................................................................36 3.4.3 Dragonflies compared to other animals .........................................................................37 3.4.4 Verification of experimental protocol ...........................................................................38 4 General discussion and conclusions ........................................................................................44 4.1 The respiratory transition of dragonflies ..............................................................................44 4.2 Significance and implications ..............................................................................................45 4.3 Limitations and future directions .........................................................................................46 References .....................................................................................................................................48           ix  List of Tables  Table 2.1. Average body mass data for individuals used in each experiment   .............................19  Table 2.2. Hemolymph TCO2 of pre-final Aeshnid nymphs exposed to two different emersion durations  ........................................................................................................................................20  Table 3.1. Individual and mean CO2 solubility of dragonfly nymphs, adults, and 0.1 mol l-1 HCl ........................................................................................................................................................39  Table 3.2. Hemolymph pKapp of early-final, late-final, and adult Anax junius .............................40                      x  List of Figures  Fig. 2.1. Photographs of pre-final (left), early-final (center), and late-final (right) Aeshnidae dragonfly nymphs ..........................................................................................................................21  Fig. 2.2. Illustration of the setup used to measure in vivo hemolymph PCO2 ...............................22  Fig. 2.3. Individual and mean hemolymph total CO2 of marbled crayfish Procambarus fallax f. virginalis (n = 6), pre-final (n = 7), early-final (n = 11), late-final (n = 8) Aeshnid nymphs, adult Anax junius (n = 8) and Aeshna multicolor (n = 8), early-final Libellula quadrimaculata nymphs (n = 8), and adult Libellula quadrimaculata (n = 7) and Libellula forensis (n = 7) ......................23  Fig. 2.4. Comparison of hemolymph PCO2 in marbled crayfish Procambarus fallax f. virginalis (n = 5) and early-final Aeshnid nymphs (n = 6) ............................................................................24  Fig. 2.5. Representative graph showing hemolymph PCO2 (kPa) against time (h) for early-final Aeshnid nymphs (blue line) and marbled crayfish Procambarus fallax f. virginalis (black line) 25  Fig. 2.6. Hemolymph TCO2 in the nymphs and adults of Aeshnidae and Libellulidae dragonfly families (current study) ..................................................................................................................26  Fig. 2.7. Hemolymph PCO2 (kPa) in early-final Aeshnidae nymphs, and in the marbled crayfish Procambarus fallax f. virginalis (current study) ...........................................................................27  Fig. 3.1. Illustration of the 3D-printed microtonometer  ...............................................................41  Fig. 3.2. pH-HCO3- diagrams for early-final (a; n = 5) and late-final Anax junius nymphs (b; n = 4), Anax junius adults (c; n = 1), and Aeshna multicolor adults (d; n = 3) ....................................42  Fig. 3.3. Comparison of the non-HCO3- blood buffer lines in early-final (n = 5) and late-final Anax junius nymphs (n = 4), Anax junius adults (n = 1), and Aeshna multicolor adults (n = 3) ...43       xi  List of Symbols and Abbreviations  α CO2 solubility ANCOVA Analysis of covariance ANOVA Analysis of variance AUC Area-under-the-curve ºC degrees Celsius CO2 Carbon dioxide f fraction of... H+ Hydrogen ion HCO3- Bicarbonate ion N2 Nitrogen O2 Oxygen PCO2 Partial pressure of carbon dioxide pKapp Carbonic acid dissociation constant PO2 Partial pressure of oxygen PTFE Polytetrafluoroethylene STPD Standard temperature and pressure TCO2 Total carbon dioxide V̇CO2 CO2 flow rate VCO2 CO2 volume V̇in Incurrent flow rate          xii  Acknowledgements  First and foremost, I would like to sincerely thank my supervisor Dr. Philip Matthews for all his support during the three years of my Masters degree. He provided everything I needed to complete my thesis project, and imparted much of his knowledge onto myself. Without all his advice and consultation, this thesis would not be possible. I would also like to thank my committee members, Drs. Colin Brauner and William Milsom, for always being willing to help address any concerns and provide valuable feedback during my project.  The UBC Botanical garden, UBC Museum of Anthropology, as well as Dr. Dolph Schluter allowed me to collect insects on their property, and for that I am extremely grateful.  During my stay in the Matthews lab there were many people who became part of the lab, some that are still here and others that moved on. Thank you to everyone who was a part of my journey. I am also very grateful to the Comparative physiology group for helping me along the journey, and allowing me to be a part of such a phenomenal group of scholars.   Last but not least, I was only able to get this far due to the immense amount of support from my family. Words cannot describe how much they have done for me, and I can only hope that I can return even a fraction of what I have received from them. Without them, I would not be who I am today.             1  1 Introduction  1.1 The evolution of insects  The insects are an ancient group of animals that arose approximately 479 million years ago, having shared their most recent common ancestor with the Remipedia (Misof et al., 2014). This places the insects among some of the earliest terrestrial animals, and their appearance on land rivals that of the first terrestrial plants (Misof et al., 2014). Insects were the first to evolve powered flight (Engel and Grimaldi, 2004; Misof et al., 2014) which allowed them to disperse and colonize a wide range of ecological niches, and they also evolved a developmental metamorphosis which led to the separation and specialization of larval and adult stages (Truman and Riddiford, 1999). These two features were critical milestones during insect evolution, and today, insects have become the single most speciose group of organisms, comprising over 50% of all described species (Grimaldi and Engel, 2005).  The extraordinary diversity within the Insecta has cemented its position as the dominant animal group in terrestrial environments (Grimaldi and Engel, 2005). However, the insects' evolutionary success is not confined to land, as multiple lineages have independently invaded both freshwater and (some) seawater environments. It is currently thought that between 100,000 and 1 million described species have adopted aquatic lifestyles (Lancaster and Downes, 2013), and of them, the Odonata, Ephemeroptera, Plecoptera, Megaloptera, and Trichoptera represent five insect orders that are predominantly amphibiotic: breathing water as nymphs or larvae before metamorphosing into air-breathing adults (Pritchard et al., 1993). These water-breathing life stages are of particular interest, since the ancestral insect almost certainly had the ability to exchange respiratory gases directly with the atmosphere using an air-filled tracheal system derived from invaginations of the cuticle; in other words, the ancestral insect was an air-breather (Pritchard et al., 1993). Thus, insects have secondarily evolved water-breathing capabilities from an air-breathing ancestry, rather than vice versa as is the case in the vast majority of animals. The respiratory consequences associated with transitioning from water to air have been thoroughly investigated in vertebrates and crustaceans (both lineages made ancestral water-to-air respiratory transitions) (e.g. Dejours, 1989). However, information is severely lacking from insects that have made the opposite evolutionary transition: from air back to water.  2  1.2 Water and air as respiratory media  Perhaps the two most important characteristics of water and air that affect how animals exchange gases with these respiratory media are the differences in their oxygen (O2) and carbon dioxide (CO2) solubilities. While the volume of O2 in air is directly proportional to its fractional composition of the total atmosphere, the volume of O2 dissolved in water is dependent on its solubility and partial pressure (Rahn, 1966). The much lower solubility of O2 in water compared to air (Truchot, 1990) means that water typically contains at most 5% of the O2 found in an equivalent volume of air (Ultsch, 1996). As a consequence, aquatic animals are forced to ventilate large volumes of water in order to extract sufficient amounts of O2 to support their aerobic metabolism. However, the solubility of CO2 in water is approximately 28 times higher than that of O2 (Dejours, 1989). This means water has a very high capacitance for CO2, and facilitates its excretion in water-breathing animals by acting as a CO2 sink (Ultsch, 1996).  The disparity between O2 and CO2 solubility in water have profound consequences on the blood CO2 content of water-breathers. Water-breathing animals have greatly enhanced overall CO2 excretion across their gills due to their high ventilation, such that the blood CO2 partial pressure (PCO2) necessary to drive CO2 out of these animals is very low, and theoretically cannot (and does not appear to) exceed 0.8 kPa (6 torr) in air-saturated water (Truchot, 1987; Ultsch, 1996). In contrast, the higher O2 solubility in air reduces the ventilation demand in air-breathers. The combination of low ventilation in air and equal CO2 solubility in air and water therefore means that the blood PCO2 in these animals is substantially higher than in water-breathing animals in order to have the same excretion rates (Truchot, 1987). The high blood PCO2 of air-breathing animals causes greater hydration of gaseous CO2 into HCO3- and H+, leading to increased blood PCO2 and HCO3- content (Ultsch, 1996) and blood acidosis (Truchot, 1990), and all air-breathing animals must therefore cope with this dramatic change in acid-base status.  1.3 The respiratory transition of vertebrates and crustaceans  The ability to breathe air has evolved multiple times within the ancestrally water-breathing vertebrates (Randall et al., 1981), and without exception each lineage to make this transition has faced and overcome a rise in blood PCO2 and HCO3-. For example, obligate water-breathing fish typically have an arterial blood PCO2 that is less than 0.8 kPa (6 torr) (Dejours, 3  1989; Truchot, 1987), whereas obligate air-breathing fish have a much higher arterial PCO2. For example, the air-breathing fish Electrophorus electricus and Synbranchus marmoratus, have a blood PCO2 of 3.9 and 3.5 kPa (29 and 26 torr) respectively (Garey and Rahn, 1970; Heisler, 1982), while a similar dramatic rise in blood PCO2 can be seen in Lepidosiren paradoxa when it switches from aquatic to aerial ventilation (Johansen and Lenfant, 1967). As these fish depend heavily on air-breathing to obtain enough O2 (Garey and Rahn, 1970; Johansen, 1966; Johansen and Lenfant, 1967), they demonstrate an air-breathing induced rise in blood PCO2 levels similar to those seen in terrestrial air-breathing animals. Blood HCO3- content necessarily follows the same trend as blood PCO2, with water-breathers having substantially lower concentrations (Truchot, 1987) than air-breathers (Garey and Rahn, 1970; Heisler, 1982). A classic example of the rise in blood PCO2 and HCO3- associated with a water-to-air transition can be seen in the amphibians which recapitulate the vertebrate's evolutionary journey from water onto land, but do so during their development. The development from water-breathing tadpole to air-breathing adult frog is accompanied by a four-fold increase in PCO2 and a five-fold increase in HCO3- (Erasmus et al., 1970), illustrating the typical increase in blood PCO2 and HCO3- as a result of breathing air. However, this substantial increase in blood PCO2 does not appear to affect blood pH, as measurements have shown that the in vivo blood pHs in the tadpole and adult frog are very similar (Erasmus et al., 1970), indicating that regardless of water-breathing or air-breathing, blood pH is regulated within a specific range.   Crustaceans also appear to show the same trend of increasing blood PCO2 when they transition from water-breathing to air-breathing. The blood PCO2 of water-breathing species is indeed lower than 0.8 kPa (6 torr), and that of their air-breathing counterparts are well above this level (Howell et al., 1973). The HCO3- data is less conclusive as it shows no consistent change between water-breathing and air-breathing. However, this likely reflects the varied regulation mechanisms and life history traits of the animals studied (Howell et al., 1973), and the data still demonstrate the rise in blood PCO2 during aerial gas exchange. Species that have adopted a terrestrial life style tend to show higher blood PCO2 and HCO3- content, with the coconut crab Birgus latro possessing levels of 0.8 kPa (6.2 torr) and 14.1 mmol l-1, respectively (Cameron and Mecklenburg, 1973). These animals cannot survive in water and so this data is representative of the most terrestrial crustacean (Cameron and Mecklenburg, 1973). Similarly, bimodally breathing species show pronounced differences in blood PCO2 and total CO2 (the sum of all 4  forms of CO2; TCO2) between water-breathing and air-breathing. The intertidal crab Carcinus maenas shows a four-fold increase in PCO2 and TCO2 content during air-breathing compared to water-breathing (Truchot, 1975), and a similar change in both parameters can be seen to occur in other crab species during air-breathing, including Hemigrapsus nudus (Burnett and McMahon, 1987; Morris et al., 1996), Cardisoma carnifex (Cameron, 1981), Pachygrapsus crassipes, and Eurytium albidigitum (Burnett and McMahon, 1987).  Comparing the respiratory CO2 change during the transition from water to air across phylogeny shows that water-breathing animals in general have lower blood PCO2 and HCO3- content compared to the air-breathers (Dejours, 1989), and collectively, decades of research has led to the consensus that water-breathing animals have adapted to a low blood PCO2 and HCO3- content, while air-breathers have adapted to higher levels of PCO2 and HCO3- due to the different gas exchange properties of water and air (Rahn, 1966; Truchot, 1987; Ultsch, 1996).   1.4 The respiratory transition of insects  Despite a good understanding of the changes in blood PCO2 and HCO3- that occur as a consequence of the transition from water to air in vertebrates and crustaceans, almost nothing is known about how amphibiotic insects transition between water-breathing and air-breathing. Furthermore, very little is known regarding the typical range of blood PCO2 and HCO3- levels even in most air-breathing insects (with the exception of grasshoppers/locusts (e.g. Harrison et al., 1990; Harrison, 1988) and moth pupae (e.g. Buck and Keister, 1958; Levy and Schneiderman, 1966)) let alone regarding water-breathing insects (Cooper, 1994; Sutcliffe, 1962). Unlike the vertebrate and crustacean species studied to date, the insects made an ancestral transition from air to water rather than vice versa (Pritchard et al., 1993), meaning that water-breathing juvenile life stages must have secondarily evolved from an air-breathing ancestor. This ancestral insect likely breathed air using spiracles, which were subsequently lost and replaced by tracheal gills as water-breathing insects evolved (Tillyard, 1915). The gills in these water-breathing insects are formed from the epidermis, and as a result have a thin layer of cuticle over the epithelium (Tillyard, 1915). This cuticle layer may present an additional diffusion barrier to gas exchange in these animals as opposed to the epithelial gills of ancestrally water-breathing vertebrates and crustaceans. Therefore, the ancestral evolution of water-breathing insects suggests that these animals might have had to adapt to lower blood PCO2 levels over evolutionary time, contrary to 5  the challenge faced by ancestrally water-breathing animals, and have evolved gas exchange organs that may present challenges that water-breathing vertebrates and crustaceans do not face. The insects provide a rare opportunity to assess how the shift in blood PCO2 and HCO3- between water-breathing and air-breathing may be confounded by evolutionary constraints, and to investigate whether there is only a single pattern in the water-to-air respiration transition in animals.  1.5 Research questions  The overall goal of this thesis is to quantify the shifts in hemolymph CO2 and acid-base status that occur during the respiratory transition in amphibiotic insects, in order to test whether they display the same pattern as has been observed in vertebrates and crustaceans, despite their differences in evolutionary history. To accomplish this task, the thesis has been separated into two parts: Chapter 2: Quantifying the change in hemolymph CO2 content between water-breathing and air-breathing insects.  This chapter will focus on measuring and comparing the hemolymph TCO2 content of insects to observe the effect of water-breathing versus air-breathing. I hypothesize that the physicochemical properties of the respiratory medium will determine the hemolymph TCO2 in insects. Based on this hypothesis, I predict that water-breathing insects will have a significantly lower TCO2 content compared to air-breathing insects. Chapter 3: Comparing the hemolymph acid-base status of water-breathing and air-breathing insects.  This chapter will focus on describing the various aspects of acid-base status in insect hemolymph to observe if and how they change between water-breathing and air-breathing. As this chapter is exploratory in nature, there are no associated experimental hypotheses. However, the research goal is to measure acid-base parameters in insects and compare them to data from vertebrates and crustaceans.     6  2 Changes in hemolymph total CO2 content during the respiratory transition of insects  2.1 Introduction   Insects were among the first terrestrial animals to appear some 479 million years ago (Misof et al., 2014). Their emergence likely coincided with their evolving an air-filled tracheal system that allowed them to breathe air directly (Pritchard et al., 1993). However, while insects have come to inhabit all terrestrial environments, some lineages have also secondarily reinvaded the aquatic environment, evolving the ability to exchange respiratory gases directly across their cuticle with the surrounding water. In particular, the Ephemeroptera, Megaloptera, Odonata, Plecoptera, and Trichoptera are all predominantly amphibiotic (Pritchard et al., 1993). Thus, insects are one of the few examples of an ancestrally air-breathing animal lineage that has repeatedly evolved different abilities to breathe water across multiple orders. But how the respiratory physiology of these amphibiotic insects change as they transition from water to air is almost completely unknown.  Studies investigating the water-to-air respiratory transition have traditionally examined vertebrates and crustaceans, lineages that are both ancestrally water-breathing. This research has revealed that, as a result of O2's low solubility in water, water-breathers have a high convective requirement, and this, in combination with water's approximately 28 times greater solubility for CO2 than for O2 (Rahn, 1966), enhances their CO2 excretion and results in a low blood PCO2 (Ultsch, 1996). From this, it may be concluded that water-breathing animals must have lower blood PCO2 and HCO3- than their air-breathing counterparts (Dejours, 1989; Erasmus et al., 1970; Howell et al., 1973). A rise in blood CO2 content associated with the respiratory transition from water to air can be seen across evolutionary time, i.e., from water breathing to obligate air-breathing vertebrates (Howell, 1970) and crustaceans (Howell et al., 1973), as well as across ontogeny in the case of vertebrate amphibians (Erasmus et al., 1970).   However, the universality of these findings should not necessarily be assumed to hold true for the insects which derived their water-breathing life stage from an air-breathing ancestor. The potential confounding effect of evolutionary history on the transition between water and air has previously been neglected and deserves investigation in order to test whether the effect of respiratory medium on blood CO2 levels is truly independent of life history, phylogeny, and ventilation mechanism (Truchot, 1987). 7   Members of the suborder Anisoptera (order Odonata), particularly species of the large-bodied Aeshnidae, are an ideal group in which to investigate how transitioning from water to air affects the hemolymph CO2 levels of an insect. Fossil evidence suggests that members of the Odonata evolved their amphibiotic lifecycle some time prior to the divergence of the Zygoptera (damselflies) and Protoanisoptera (dragonflies), which had occurred by the lower Permian (Wootton, 1988). Thus, the nymph life stages of this group have been living as water-breathers for at least 300 million years. The gas exchange structures of the Anisoptera are also well studied, with the nymphs breathing water using a tidally-ventilated rectal gill which they then discard when they undergo metamorphosis, while the air-breathing adult dragonflies that emerge from the exuviae of the final instar nymphs share the same open tracheal system as all other terrestrial insects (Tillyard, 1915).  To determine whether amphibiotic insects undergo the same dramatic increase in hemolymph PCO2 and HCO3- content as observed in other animal lineages that transition from water to air (Dejours, 1989; Truchot, 1987), hemolymph samples from the aquatic and terrestrial life stages of Aeshnid and Libellulid dragonflies were analyzed for TCO2 content. TCO2 was chosen as the parameter of measurement, since it is defined as the sum of all forms of CO2 (which are primarily gaseous CO2 and HCO3-) and can easily be measured from small volume solutions. An increase in TCO2 must be correlated with an increase in PCO2 and HCO3-, and provides a reliable means of assessing overall blood CO2 content. In addition, a novel optical PCO2 microsensor was used to measure in vivo PCO2 directly in the dragonflies' hemocoel. As a procedural control, the same techniques were used to obtain measurements of TCO2 and in vivo PCO2 from freshwater marbled crayfish in order to provide results that could be compared with previously published values from other crustaceans in the literature.  2.2 Materials and Methods 2.2.1 Animals  Aeshnid and Libelullid dragonfly nymphs were captured from ponds at the University of British Columbia, Point Grey campus using aquatic sweep nets. Anax junius and Aeshna multicolor dragonflies were the most common adult Aeshnid species flying around these ponds, and subsequent identification using photographs of the captured nymphs revealed that they were 2.2.1.1 Dragonfly nymphs 8  indeed a mix of Anax and Aeshna species (Cannings and Stuart, 1977). However not all sampled nymphs were photographed, and as it was not possible to further identify them to the species level, these nymphs were treated as a single Aeshnid group. The Libellulid dragonfly nymphs were all identified as being Libellula quadrimaculata (Cannings and Stuart, 1977).     Captured nymphs were brought back to the lab and housed individually in 11×11×17 cm square glass aquaria filled with dechlorinated Vancouver tap water. Each aquarium contained a pea gravel substrate. These aquaria were connected to a re-circulating system which consisted of a pump inside a 76 l sump that supplied a gentle flow of water in through the top of each aquarium. A drain port on one side of each aquarium directed the overflow water through a filter before discharging it back into the sump. The water in the sump was completely replaced with fresh dechlorinated Vancouver tap water as required. Nymphs that were small enough to enter the drain ports were housed in 600 ml polypropylene containers with a pea gravel substrate. The water in these containers was completely replaced with fresh dechlorinated Vancouver tap water once a week. The nymphs were maintained at lab temperature (20 – 23°C), 12:12 daylight cycle, fed on a variety of aquatic and terrestrial invertebrates, and allowed to acclimate for at least two weeks prior to measurement. All nymphs were starved for 24 h prior to measurement. The same dechlorinated Vancouver tap water was used in all subsequent experiments.  2.2.1.2 Dragonfly adults Four species of dragonflies from two families were caught using aerial nets between June and August 2016 - 2017 from around the same ponds where the nymphs were collected. They were Anax junius, Aeshna multicolor (Aeshnidae), Libellula quadrimaculata, and L. forensis (Libellulidae). Captured adults were immediately placed in blacked-out glass jars that were covered with aluminum foil and lined with fine plastic mesh on the inside. The adults remained at rest in the jars for at least 2 h prior to measurement. Following the experiments, they were released back into the wild.      Parthenogenic marbled crayfish (Procambarus fallax f. virginalis) were purchased from a commercial supplier (JLAquatics, Vancouver, British Columbia, Canada) and acclimated to lab conditions (20 – 23°C, 12:12 daylight cycle) in a 20 l volume aquarium filled with dechlorinated 2.2.1.3 Marbled crayfish 9  Vancouver tap water and fed ad libitum on frozen bloodworms and spirulina tablets for one week prior to measurement. All individuals were starved for 24 h prior to measurement.   All animals were weighed to 0.01 mg on an electronic balance (XPE205DR, Mettler-Toledo Inc., Missisauga, ON, Canada) directly before use in experiments (Table 2.1).  2.2.1.4 Body Mass  2.2.2 Classification of dragonfly nymph developmental stage  In this study, the dragonfly nymphs were divided into three stages: pre-, early-, or late-final instar. Nymphs with small, incompletely developed wing-buds were collectively termed pre-final instars (Fig. 2.1). Early-final instar nymphs were characterized by large wing-buds with both pairs of wings lying flat on the dorsal surface. In addition, the hind wing-buds were collapsed on top of the fore wing-buds such that the fore wing-buds were not visible. Late-final instars were identified by wing-buds that spread apart such that both the fore and hind wing-buds were clearly visible, in addition to possessing more pronounced venation. These 'late-final' changes occurred in the absence of a molt and were accompanied by cessation of eating, indicating that the nymphs were beginning to prepare for metamorphosis (Corbet, 1962).   2.2.3 Measuring hemolymph TCO2  Hemolymph TCO2 content was measured using a custom-built gas sparging system coupled with a two-channel LI-7000 infra-red CO2 gas analyzer (LI-COR, Lincoln, Nebraska, USA). Compressed N2 from a gas cylinder (99.998% pure, Praxair, Mississauga, Ontario, Canada) was connected to a 0 - 100 ml min-1 mass flow controller (GFC17, AALBORG, Orangeburg, New York, USA) set to a flow rate of 20 or 100 ml min-1 STPD. This N2 stream then passed through cell A of the two-channel LI-7000 to provide a constant zero CO2 reference. The cell A outlet was then connected to the bottom of a custom-made gas sparging column that consisted of a cylindrical glass chamber (4.7 ml internal volume). Gas entering from the bottom of the column passed up through a sintered glass disc, then bubbled through an acid solution: 1 ml of 0.01 N HCl mixed with 1 μl Antifoam 204 (Sigma-Aldrich, St. Louis, Missouri, USA) to prevent excessive frothing during sample injections. After passing through the acid solution, the N2 gas and any liberated gaseous CO2 was passed through cell B of the infra-red CO2 gas 10  analyzer (IRGA) for analysis. The CO2 concentration (μmol CO2 mol-1 N2) in the N2 stream was logged at 5 Hz using a desktop PC running LI-7000 software (V. 2.0.3, LI-COR, Lincoln, Nebraska, USA). A gas-tight injection port with a PTFE-lined septum mounted on the side of the sparging column allowed samples to be injected directly into the acid solution for analysis.  To generate a TCO2 calibration curve, a series of sodium bicarbonate standard solutions were prepared by dissolving 1.26 g of NaHCO3 in 500 ml of distilled water to create a 30 mmol l-1 stock solution. This stock solution was then diluted to produce additional standard solutions of 10, 15, 20, and 25 mmol l-1. Then a 26s-gauge 10 μl Hamilton syringe was primed with CO2-free distilled water (that had been purged with 99.998% pure N2) by repeatedly pumping the plunger while the needle tip was kept submerged in the water, thereby expelling any air from the needle lumen (hereafter referred to as 'primed'). This primed syringe was then used to withdraw and inject a 5 μl sample of each standard solution into the gas sparging column through the PTFE-lined septum. The resulting reaction between the bicarbonate and HCl led to the liberation of gaseous CO2, which was recorded by the IRGA as a CO2 pulse in μmol mol-1. The CO2 pulse was first converted to instantaneous V̇CO2 (ml min-1) using the formula:   V̇CO2 = CO2/1000000 × V̇in, (1) where CO2 is the CO2 pulse trace (μmol mol-1), and V̇in is the incoming flow rate (ml min-1, STPD).  The area under the recorded CO2 pulse was then integrated against time using the area approximation formula:    VCO2 = [(Y2 + Y1)/2] × (X2 − X1), (2) where VCO2 is the volume of CO2 liberated (ml), Y2 and Y1 are instantaneous V̇CO2 (ml min-1) and X2 and X1 are time (min). The CO2 volume (ml) was first converted to litres, then divided by the molar volume constant 22.414 l mol-1. The resulting moles of CO2 was converted to millimole then divided by the volume of sample injected to produce the final TCO2 mmol l-1 value. This series of standard solution injections was repeated 2 to 3 times, and the true CO2 value was fitted against the average measured value in order to create a calibration curve.   Hemolymph TCO2 was measured using samples extracted directly from the insect's hemocoel. Nymphs were removed from their aquaria and quickly patted dry with paper towels, after which they were gently restrained in a groove cut into an expanded polystyrene block. A 26s-gauge 10 μl Hamilton syringe was again primed, and the needle tip was inserted into the nymphs between the dorsal abdominal tergites of segments 7 and 8 to either the left or right side 11  of the midline depending on the position of the nymphs. 5 μl of hemolymph was withdrawn from the animal, and the extracted sample was injected immediately into the gas sparging column. Sodium bicarbonate standard solutions were run before and after each series of hemolymph sample injections to provide direct references to known values, and CO2-free distilled water blanks were run between each injection to ensure no residual CO2 remained in the syringe. The average time that the nymphs spent out of water was 1 min.  The hemolymph TCO2 of adult dragonflies was measured using the same protocol as above, except that the adults were restrained ventral-side down on an expanded polystyrene board using insect pins, and a primed syringe was used to pierce the mid-dorsal surface of the mesothorax to extract a 5 μl hemolymph sample directly from the aortic diverticula (Jensen, 1976).   To determine whether the removal of nymphs from water during the extraction procedure had any effect on hemolymph TCO2, measurements were taken from two additional groups of nymphs. One group had the hemolymph extracted as quickly as possible, while the other group had the hemolymph extracted after being left in air for 2 min. 2.2.3.1 Verification of experimental approach  As a procedural control for the TCO2 protocol, hemolymph TCO2 was measured from marbled crayfish using the same technique, thereby providing data that could be compared to published TCO2 values for other crustaceans. The crayfish were removed from the aquarium, quickly patted dry using paper towels, and held in place with one hand while a primed 26s-gauge 10 μl Hamilton syringe was inserted into the hemocoel surrounding the heart. 5 μl of hemolymph was obtained and analyzed for TCO2 as described in Chapter 2 Section 2.2.3. The average time that the marbled crayfish spent out of water for each extraction was 0.5 min.   2.2.4 Measuring hemolymph PCO2   The syringe-mounted PCO2 microsensors (NTH-CDM1, PreSens, Regensburg, Bavaria, Germany) were calibrated in a 0.154 M NaCl solution equilibrated with various known PCO2s. A 100 ml glass bottle was filled with the saline solution and an aquarium air stone connected to a gas mixing system was threaded through one of two holes in the bottle's lid. The bottle was then placed in a temperature-controlled water bath (F33-ME, Julabo, Seelbach, Baden-Württemberg, 12  Germany) set to 21°C. The PCO2 microsensor was inserted into the calibration bottle through the second hole in the lid, and its sensor tip was extended out from the hypodermic needle into the solution. A series of PCO2s were generated using two mass flow controllers (MC-500SCCM-D/5M, Alicat Scientific, Tucson, Arizona, USA) to combine a 99.998% N2 compressed gas with a 5% CO2 bal. N2 compressed gas (Praxair, Mississauga, Ontario, Canada). Flow rates were verified using a Bios DryCal Definer 220-L primary flow meter that had been calibrated using NIST standards (Mesa Laboratories, Inc., Lakewood, Colorado, USA). Gas mixing software (Flow Vision V. 1.3.13.0, Alicat Scientific, Tucson, Arizona, USA) running on a PC controlled the mass flow controllers to generate 0, 0.5, 1, 2, and 3 kPa PCO2 gas mixtures at a total combined flow rate of 500 ml min-1 STPD. The PCO2 sensor was allowed to equilibrate for 1 h at 0 kPa PCO2 and for 30 min at all other PCO2s. PCO2 readings were recorded once every 5 min using a pCO2 micro CO2 meter (PreSens, Regensburg, Bavaria, Germany) and pCO2 Micro View (V. 1.0.0, PreSens, Regensburg, Bavaria, Germany). Following calibration, the sensors were back-checked at 0.5 and 1 kPa PCO2 before being placed in a saline solution for storage until use. All sensors were used within one hr following calibration.  Calibrated PCO2 probes were implanted into early-final Aeshnid nymphs that had been starved for at least 24 h (Fig. 2.2). Before implantation, the nymph was first cold-stunned in ice water for 4 min. Following removal from the ice water, its dorsal surface was quickly patted dry with paper towels. The nymph was then attached to a plastic harness using a low viscosity polyvinylsiloxane dental impression material (President light body, Coltene Whaledent, Cuyahoga Falls, Ohio, USA). The concave bottom surface of the harness was coated with the impression material which was then pressed down over the wing-buds of the nymph and allowed to cure. During the curing period, the nymph's abdomen was kept submerged in dechlorinated Vancouver tap water to allow them to breathe. The harness was then attached to a micromanipulator (M3301, World Precision Instruments, Sarasota, Florida, USA) and positioned on top of a Plexiglas platform inside a 25×16.7×8 cm polypropylene container such that the ventral abdominal surface of the nymph was approximately 3 mm above the platform. The polypropylene container was then filled with water until the tip of the nymph's abdomen was submerged. A 1 ml hypodermic syringe with a 20-gauge needle was used to make a small hole in the middle of the cuticle between the first and second thoracic tergites. This was done by using a syringe mounted on a second micromanipulator. The syringe then was removed and replaced 13  with a needle-mounted PCO2 microsensor. This allowed the sensor to be inserted through the hole, approximately 2 mm into the nymph's hemocoel, while monitoring the PCO2 reading to ensure a successful implantation. The hole and sensor were then sealed in place using the dental impression material. The polypropylene container was then filled to the brim with dechlorinated Vancouver tap water, continuously bubbled with room air, and covered with an opaque mesh to reduce the visual agitation of the nymph. The container was maintained at 21°C by being placed within a larger acrylic water bath, which was kept thermally stable using a temperature-controlled water bath (F33-ME, Julabo, Seelbach, Baden-Württemberg, Germany). The hemolymph PCO2 was recorded every 5 min. The experiment lasted for 24 h, after which the sensor was retrieved and checked in CO2-equilibrated saline for signal drift. It was not possible to measure in vivo PCO2 in the Libellulid nymphs due to their small size which did not allow enough room for both the harness and the microsensor.  The PCO2 readings recorded using the pCO2 Micro View software were exported into Microsoft Excel for analysis. The raw % CO2 traces were first converted to PCO2 by converting each value to a fraction, then multiplying each value by 101.3 kPa. The PCO2 values were then plotted against time and the values from 12 to 24 h were averaged to calculate mean PCO2 for each individual. These individual means were then averaged together to calculate the grand mean PCO2 for the nymphs.     Given that in vivo PCO2 was measured using an experimental PCO2 sensor in this study, it was considered prudent to also measure hemolymph PCO2 in an animal group that has been measured previously to provide a comparison between our measured values and those in the literature. Marbled crayfish were selected for this purpose. The protocol for measuring PCO2 in these crustaceans was largely the same as the one used for dragonfly nymphs except that following the 24 h starvation period, the individuals were cold-stunned in ice water for 20 min, and after removal from the ice water a 20-gauge needle was used to make a small hole in the carapace approximately 3 mm to the left of the mid-dorsal line. The crayfish were then attached to the plastic harness while ensuring the hole was not occluded by the impression material, and after transfer to the measurement setup, the PCO2 sensor tip was inserted into the pre-existing hole and sealed in place, again using dental impression material. 2.2.4.1 Verification of experimental approach 14  2.2.5 Statistical analyses  Data was analyzed in R ver. 3.4.1 (R Core Team, 2017). A one-way ANOVA was performed to test for any statistical differences between the mean TCO2 of dragonfly nymphs, adults, and marbled crayfish, and two sample t-tests were performed to test for any changes in the mean TCO2 of the two treatment groups exposed to either short or long emersion, as well as for any changes in the mean PCO2 of dragonfly nymphs and marbled crayfish. Data are shown as mean ± standard error (s.e.m) unless otherwise stated.    2.3 Results 2.3.1 Total hemolymph CO2   TCO2 measurements were made on pre-final, early-final, late-final Aeshnid nymphs, early-final L. quadrimaculata nymphs, and adults of Anax junius, Aeshna multicolor, L. quadrimaculata, and L. forensis, as well as on marbled crayfish (Fig. 2.3). The pre-final and early-final Aeshnid nymphs had statistically the same TCO2 of 15.6 ± 1.2 and 16.0 ± 0.7 mmol l-1, respectively (One-way ANOVA, F = 36, df = 8, p < 0.001). However, there was a significant increase in TCO2 to 21.3 ± 1.3 mmol l-1 in the late-final Aeshnid nymphs. This high TCO2 was not statistically different from the values seen in either species of adult Aeshnid. The Anax junius and Aeshna multicolor adults had TCO2 levels that were not significantly different (23.2 ± 0.3 and 22.1 ± 1.3 mmol l-1, respectively), but Anax junius individuals had noticeably less variation in their measured TCO2 compared to all other groups. The early-final L. quadrimaculata nymphs had a mean TCO2 of 19.6 ± 1.0 mmol l-1 which was not significantly different from the values in all the water-breathing and air-breathing Aeshnid groups. However, the early-final L. quadrimaculata TCO2 was significantly lower than the mean TCO2s of both adult Libellulid species. Adults of L. quadrimaculata and L. forensis had TCO2s that did not differ significantly (29.2 ± 1.3 and 30.6 ± 1.4 mmol l-1 respectively). However, both were significantly higher than the values from Anax junius, Aeshna multicolor, and the late-final Aeshnid nymphs. Marbled crayfish had a mean TCO2 of 8.4 ± 1.0 mmol l-1 which was lower than all dragonflies, aquatic or terrestrial.    15  2.3.2 Effect of emersion on TCO2   TCO2 measurements were made on two groups of pre-final Aeshnid nymphs (Table 2.2). The average emersion duration for group 1 was 0.5 min, while the average duration for group 2 was 3.2 min. The lowest and highest values in the 0.5 min group were 17.7 and 28.9 mmol l-1 respectively, while the corresponding values in the 3.2 min group were 14.0 and 24.7 mmol l-1. Comparing the mean TCO2 values showed that there was no significant difference between the two groups (Two sample Student's T test, df = 4, p = 0.9).   2.3.3 Hemolymph PCO2   PCO2 measurements were made on early-final Aeshnid nymphs and marbled crayfish (Fig. 2.4). The mean PCO2 of early-final nymphs was 0.9 ± 0.1 kPa, which was significantly higher than the 0.38 ± 0.05 kPa mean PCO2 of marbled crayfish (Two sample Student's T test, df = 9, p = 0.002). The PCO2 always started high for both the dragonfly nymphs and marbled crayfish (mean 2.1 ± 0.4 and 1.0 ± 0.1 kPa respectively) (Fig. 2.5). As the experiment progressed, the PCO2 began to decrease such that all animals had reached a stable plateau by 12 h into the experiment. Therefore, the PCO2 readings from 12 h to the end of the experiment were considered to be physiologically relevant and were used to calculate mean hemolymph PCO2 for each animal.   2.4 Discussion 2.4.1 Dragonfly TCO2 in water-breathing nymphs and air-breathing adults  Findings from the current study indicate that hemolymph TCO2 increases when dragonflies undergo the transition from breathing water as nymphs to breathing air as adults. This is not entirely unexpected, and suggests that the nymphs are pressed by their need to extract sufficient amounts of O2 from water, which results in high ventilation rates and greater CO2 excretion in water relative to air. However, the observed increase in TCO2 does not coincide with the nymph leaving the water, but precedes metamorphosis. As such, the aquatic late-final Aeshnid nymphs have a hemolymph TCO2 that is significantly higher than both the pre- and early-final instars, but not significantly different from the air-breathing adult (Fig. 2.3). One possible explanation for this unexpected result is that as late-final instar nymphs begin to modify their physiology in anticipation of metamorphosis, the conductance of their rectal gill declines 16  and/or they become less reliant on their gills for gas exchange. This is plausible, as the mesothoracic spiracles of final instar nymphs become functional prior to metamorphosis, providing them with the ability to breath air (Corbet, 1962; Gaino et al., 2007) during periods of emergence from water (Corbet, 1962) The nymphs of the primitive Petaluridae family are known to possess functional thoracic spiracles during all their later instars (Green, 1977; Wolfe, 1953). This trait, combined with amphibious habits, allows them to hunt on land in a manner that is reminiscent of the ancestral Odonata (Corbet et al., 1960). Interestingly, the air-breathing final instar Petalurid nymphs of Uropetala carovei possess hemolymph TCO2 values of 28.0 ± 6.0 mmol l-1 (Bedford and Leader, 1975), a value that is far higher than the values measured from all Aeshnid nymphs and adults, but similar to the values recorded from the adult Libellulidae (29.2 to 30.6 mmol l-1; Fig. 2.6).  Comparing Anax junius and Aeshna multicolor to L. quadrimaculata and L. forensis shows that the adult Libellulidae have significantly higher hemolymph TCO2s than the adult Aeshnidae. Differences between these two dragonfly families can also be seen from the water-breathing nymph stages, where, although not statistically significant, the TCO2 of the early-final Aeshnid nymphs is lower than the value from the early-final L. quadrimaculata. A previously reported value for hemolymph HCO3- concentrations in a mixed-age sample of Aeshna grandis (Aeshnidae) nymphs (15.0 ± 2.5 mmol l-1) (Sutcliffe, 1962) is also in good agreement with the Aeshnid nymph TCO2 values presented here (Fig. 2.6), suggesting family-specific differences in internal CO2 levels. Understanding the phylogeny, ecology, and behaviour of these two common dragonfly families can be helpful in interpreting the above trend. The Aeshnidae and Libellulidae families are evolutionarily distant (Misof et al., 2001; Saux et al., 2003), and have diverged substantially in their morphology and behaviour. Nymphs belonging to the Aeshnidae are typically active hunters that crawl through aquatic vegetation, while Libellulid nymphs are sluggish and live on the substrate, often in warmer, eutrophic waters (Cannings and Stuart, 1977). Thus, the Libellulid nymphs are more likely to be exposed to hypoxia and hypercapnia than the Aeshnids. The adults of these two families, too, show markedly different flight behaviours, with the Aeshnidae spending most of their time on the wing, while the Libellulidae fly intermittently from a selected perch, chasing after prey and conspecifics (Corbet, 1962). These different flight strategies are reflected in the dragonflies' circulatory systems, where Anax junius and Aeshna multicolor Aeshnids have much larger hearts and hemolymph volumes than the similarly-sized 17  Libellulid Libellula saturata (Heinrich and Casey, 1978). Thus, the comparatively poor hemolymph circulation in the Libellulidae may well impede CO2 excretion compared to the Aeshnidae, resulting in elevated TCO2 levels. A phylogenetic approach examining the relationship between hemolymph TCO2 and adult flight strategies across a wide range of dragonfly families would be a powerful way to test the hypothesis that internal TCO2 is mechanistically linked to flight behaviour within the dragonflies.  2.4.2 Dragonflies compared to other animals  The dragonfly nymph values presented here are comparable to those few published hemolymph HCO3- values from the aquatic larvae of other developmentally amphibiotic insect lineages. In particular, dragonfly nymph TCO2 is similar to the HCO3- concentration seen in larvae of the alderfly Sialis lutaria (Shaw, 1955), but it is substantially higher than that of the larval caddisfly Limnephilus stigma (Sutcliffe, 1962) (Fig. 2.6). Both caddisfly and alderfly larvae breathe water using external tracheal gills, but while caddisfly larvae are generally apneustic, alderfly and other Megalopteran larvae possess functional spiracles along the length of their body, allowing them to breathe air (Barclay et al., 2005). When compared to air-breathing insects, the CO2 content in the dragonfly nymphs is similar to the aquatic (but air-breathing) water boatman Cenocorixa blaisdelli (Cooper et al., 1987), but is noticeably higher than the values measured from terrestrial grasshopper and locust species such as Melanoplus, Romalea, and Schistocerca (Gulinson and Harrison, 1996; Harrison et al., 1990; Harrison, 1988; Harrison, 1989; Krolikowski and Harrison, 1996).  While hemolymph TCO2 is a useful indicator of blood-gas composition that includes both HCO3- and dissolved gaseous CO2 contributions, the direct measurement of in vivo hemolymph PCO2 in dragonfly nymphs further suggests that these insects do not have the same low PCO2 as seen in most other water-breathers. For example, while lower than the PCO2 of various air-breathing Orthopterans (Gulinson and Harrison, 1996; Harrison et al., 1990; Krolikowski and Harrison, 1996), the early-final Aeshnid nymphs have a significantly higher hemolymph PCO2 compared to the water-breathing marbled crayfish (Fig. 2.4), and is also much higher than the CO2 tension in rainbow trout Oncorhynchus mykiss (Eddy et al., 1977) (Fig. 2.7) and other 'typical' fish (Ultsch, 1996).  18   In addition to an overall higher internal TCO2 and PCO2 in the water-breathing dragonfly nymphs relative to other water-breathing animals, the increase in TCO2 that occurs during their transition from water- to air-breathing is fairly modest: an increase of only 45% and 48% from early-final instar to adult in the Aeshnidae and Libellulidae, respectively. Ancestrally aquatic animals making this same transition show a far higher increase. For example, the tadpole to bullfrog transition of Rana catesbiana results in a 445% increase in blood HCO3- (Erasmus et al., 1970), while the crab Carcinus maenas experiences a rise of 258% when moving from water to air (Truchot, 1975). These trends suggest that, as a consequence of adapting their ancestrally air-breathing respiratory system to function in water, dragonfly nymphs have evolved gas exchange strategies that limit CO2 excretion and result in greater internal CO2 accumulation. One possible explanation could lie in the efficiency of the rectal gill as a gas exchange organ. However, it has also been recognized that insects lack carbonic anhydrase in their hemolymph and gills (Harrison, 2001; Kohnert et al., 2004). As a result, a disequilibrium between CO2 and HCO3- could arise in the hemolymph (Cooper, 1994) which may impede CO2 excretion across the gills.  2.4.3 Verification of experimental protocol  One criticism of the protocol used in the current study is the removal of dragonfly nymphs from water during hemolymph extraction, during which time they are unable to excrete CO2. This may lead to an artificially elevated TCO2 which would confound the measurements in this study. However, this is unlikely to be the case, as the nymphs that had been out in air for 0.5 min did not have significantly different TCO2 compared to those out in air for 3.5 min (Table 2.2). Given that the actual extraction only lasted 1 min, the measured TCO2 is unlikely to be artificially elevated. The marbled crayfish TCO2 also suggests the protocol did not confound the measurements, as they were treated and sampled in the same way as the nymphs, and their TCO2 is both significantly lower than the dragonfly nymphs and is also comparable to values from other freshwater crayfish such as the European crayfish Astacus astacus (Jensen, 1990).       19  Table 2.1. Average body mass data for individuals used in each experiment  Experiment Species/group n Mass (g ± s.e.m) Hemolymph TCO2 Procambarus fallax f. virginalis 6 1.6 ± 0.2 Pre-final Aeshnid 7 0.47 ± 0.03 Early-final Aeshnid 11 0.98 ± 0.05 Late-final Aeshnid 8 1.42 ± 0.02 Early-final Libellula quadrimaculata 8 0.37 ± 0.02 Anax junius† 8 0.95 ± 0.03 Aeshna multicolor† 8 0.57 ± 0.03 Libellula quadrimaculata† 7 0.37 ± 0.02 Libellula forensis† 7 0.52 ± 0.03 Time-wise TCO2 0.5 min air exposure 3 0.35 ± 0.04 3.2 min air exposure 3 0.39 ± 0.02 Hemolymph PCO2 Procambarus fallax f. virginalis 5 1.4 ± 0.2 Early-final Aeshnid 5* 0.60 ± 0.01 *Mass available for 5 out of 6 individuals, † indicates adult dragonflies   20  Table 2.2. Hemolymph TCO2 of pre-final Aeshnid nymphs exposed to two different emersion durations Emersion duration (min) Individual TCO2 (mmol l-1) 0.5 1 2 3 Average 17.7 18.0 28.9 21.5 ± 3.7 3.2 4 5 6 Average 23.5 24.7 14.0 20.7 ± 3.4 Where applicable, values are shown as means ± s.e.m (n = 3). The average TCO2 in the two emersion groups were not significantly different from each other (Student's T test, p = 0.9).  21    Fig. 2.1. Photographs of pre-final (left), early-final (center), and late-final (right) Aeshnidae dragonfly nymphs. These three instar stages were differentiated based on their wing-bud morphology, with pre-final instars having short underdeveloped wing-buds, early-final instars having long and collapsed wind-buds, and late-final instars having long and spread apart wing-buds. The grid is 5 × 5 mm.        22   Fig. 2.2. Illustration of the setup used to measure in vivo hemolymph PCO2. A micromanipulator (1) held a calibrated PCO2 microsensor (2) that was implanted into a dragonfly nymph (3). The nymph was held in place above an acrylic platform (4) by a plastic harness (5). Measurements from the PCO2 microsensor were recorded by a pCO2 micro CO2 meter (6).             23   Fig. 2.3. Individual and mean hemolymph total CO2 of the study species. From left to right: marbled crayfish Procambarus fallax f. virginalis (n = 6), pre-final (n = 7), early-final (n = 11), late-final (n = 8) Aeshnid nymphs, adult Anax junius (n = 8) and Aeshna multicolor (n = 8), early-final Libellula quadrimaculata nymphs (n = 8), and adult Libellula quadrimaculata (n = 7) and Libellula forensis (n = 7). Points with different letters are significantly different (One-way ANOVA, p < 0.001, Tukey's HSD). Data are presented as mean ± s.e.m.           24   Fig. 2.4. Comparison of hemolymph PCO2 in marbled crayfish Procambarus fallax f. virginalis (n = 5) and early-final Aeshnid nymphs (n = 6). * represents significant difference (Student's T test, P = 0.002). Data are presented as mean ± s.e.m.               25   Fig. 2.5. Representative graph showing hemolymph PCO2 (kPa) against time (h) for early-final Aeshnid nymphs (blue line) and marbled crayfish Procambarus fallax f. virginalis (black line). Time 0 was the start of the experiment. In all individuals, the PCO2 started high, then slowly stabilized to a lower level by 12 h, and the steady state level was maintained for the rest of the 24 h experiment period.              26   Fig. 2.6. Hemolymph TCO2 in the nymphs and adults of Aeshnidae and Libellulidae dragonfly families (current study). This data is compared with the hemolymph HCO3- content in nymphs of additional dragonfly species (1Sutcliffe (1962), 2Bedford and Leader (1975)), other water-breathing and air-breathing insects (3Sutcliffe (1962), 4Shaw (1955), 5Harrison et al. (1990), 6Gulinson and Harrison (1996), 7Harrison (1988), 8Krolikowski and Harrison (1996), 9Harrison (1989), 10Cooper et al. (1987)), decapod crustaceans (11Truchot (1975)), and amphibians (12Erasmus et al. (1970)). Blank bars represent air-breathing animals/life stages, while diagonal dashes represent water-breathing or potentially bimodally-breathing animals/life stages. The various dragonfly nymph developmental stages are represented by shades of gray. Values are represented as means.       27   Fig. 2.7. Hemolymph PCO2 (kPa) in early-final Aeshnidae nymphs, and in the marbled crayfish Procambarus fallax f. virginalis (current study). This data is compared with the blood PCO2 (kPa) of a water-breathing fish (1Eddy et al. (1977)), and hemolymph PCO2 of air-breathing Orthopterans (2Harrison et al. (1990), 3Krolikowski and Harrison (1996), 4Gulinson and Harrison (1996)). Values are represented as means.          28  3 Quantifying the hemolymph acid-base status of water-breathing and air-breathing insects  3.1 Introduction  The respiratory consequences of an animal switching from water-breathing to air-breathing have primarily been studied on vertebrates (e.g. Erasmus et al., 1970; Garey and Rahn, 1970; Just et al., 1973; Lenfant and Johansen, 1968), and within the vast invertebrate lineage, only crustaceans have received notable attention (e.g. Howell et al., 1973; Truchot, 1975). However, numerous insect groups have secondarily evolved amphibiotic lifestyles, thus providing a rare opportunity to see how evolutionary history affects how an organism modifies its respiratory system to function as it moves from water to air. Chapter 2 examined hemolymph TCO2 content changes during development in the amphibiotic Anisoptera, revealing that the water-to-air respiratory transition of Aeshnid and Libellulid dragonflies is accompanied by a comparatively minor increase in hemolymph TCO2, unlike those experienced by vertebrates and crustaceans. The finding that water-breathing dragonfly nymphs have an unusually high hemolymph TCO2 content was unexpected, and is possibly due to their secondarily derived water-breathing capabilities.  The observation that TCO2 changes significantly across dragonfly development begs the question: what is happening to the acid-base status of the hemolymph? The hemolymph acid-base status is of particular interest, as it describes the equilibrium between PCO2, HCO3-, and pH, and can show if and how these parameters change across the water-to-air transition. However, little information is available regarding the acid-base status of the insects, and the few measurements available in the literature have generally been restricted to a few select species (Matthews, 2017). Therefore, studying the hemolymph acid-base status of dragonflies not only provides further insight into their respiratory transition, but also begins to expand our knowledge of previously unstudied insects.   To quantify the hemolymph acid-base status of dragonflies across development, the hemolymph CO2 solubility and apparent dissociation constant of carbonic acid (pKapp) were measured in Aeshnid dragonflies. In addition, the non-bicarbonate blood buffer lines and PCO2 isopleths were calculated to describe and compare the hemolymph acid-base status across development. 29  3.2 Materials and Methods 3.2.1 Animals  Dragonfly nymphs and adults were captured and housed as described previously (Chapter2: Section 2.2.1). The nymphs were classified as early- and late-final Anax junius while the adults were Anax junius and Aeshna multicolor.  3.2.2 Preparation of hemolymph samples  Hemolymph was obtained from dragonfly nymphs using the same protocol as described in Chapter2 (Section 2.2.3). However, for this study a 22s-gauge 25 μl Hamilton syringe with its needle lumen primed with distilled water was used to extract the hemolymph. Priming was necessary in order to remove the air from the needle lumen as well as to prevent clogging of the syringe. Hemolymph was collected into an ice-cooled 1.5 ml Eppendorf tube to minimize clotting, and was centrifuged at 1520 g for 10 min to separate any cellular and tissue debris from the supernatant. 3.2.2.1 Dragonfly nymphs   Dragonfly adults were restrained as described in Chapter 2 (Section 2.2.3), then the 8th abdominal tergite was removed using a razor blade and scissors to expose the underlying hemocoel (Heinrich and Casey, 1978). The gut and heart were pushed to the side using an insect pin, and the hemolymph from the wound was then slowly aspirated into a length of 30-gauge PTFE tubing connected to a 26s-gauge 10 μl Hamilton syringe. The hemolymph was then collected into an Eppendorf tube and processed as described above. 3.2.2.2 Dragonfly adults  3.2.3 Hemolymph CO2 solubility (α)  To calculate hemolymph CO2 solubility (α), 30 μl of hemolymph was collected from an individual insect. This was then centrifuged as described above, before 20 μl of the supernatant was removed and placed in a 6 mm × 50 mm test tube together with 20 μl of 0.1 mol l-1 HCl. The hemolymph-HCl solution always had a pH below 3, indicating that all HCO3- had been converted to CO2 (Harrison, 1988). A micro-combination pH electrode mounted in a 16 ga hypodermic needle (MI-414B, Microelectrodes Inc., Bedford, New Hampshire, USA) was used 30  for all pH measurements. It was connected to a pH amplifier (FE165, ADInstruments, Colorado Springs, Colorado, USA) and a Powerlab analogue-to-digital converter (PL3504, ADInstruments, Colorado Springs, Colorado, USA). The pH electrode was calibrated using three pH standards (pH 4, 7, 10), and pH measurements were recorded using Labchart (V.8.1.10400.0, ADInstruments, Colorado Springs, Colorado,USA)  A custom-made microtonometer was used to equilibrate the hemolymph-HCl sample with gas mixtures of known PCO2 (Fig. 3.1). This 3D-printed microtonometer was capable of rotating two test tubes simultaneously, and consisted of a 1000:1 geared-down motor (Micrometal gearmotor # 1596, Pololu, Las Vegas, Nevada, USA) that turned a 24 mm diameter wheel with two grooves around its perimeter. An elastic band in each groove looped around the middle of a test tube which was loosely clamped in a frame beneath the motor assembly. The rotation of the motor caused the test tube to rotate in place at 42 rpm, allowing the hemolymph-HCl mixture to smear across the inner wall. Mixing of the sample was further enhanced by a 7 × 2 mm PTFE-coated stir bar placed into each test tube. During equilibration the microtonometer was held at an angle 20º from horizontal with the test tubes partly submerged in a water bath that was temperature-controlled to 20ºC (F33-ME, Julabo, Seelbach, Baden-Württemberg, Germany). Two 0 - 500 ml min-1 mass flow controllers (MC500SCCM-D/5M, Alicat Scientific, Tucson, Arizona, USA) were controlled by a gas mixing software (Flow Vision V. 1.3.13.0, Alicat Scientific, Tucson, Arizona, USA) to combine 99.998% N2 and 100% CO2 compressed gases (Praxair, Mississauga, Ontario, Canada) into a 20 kPa PCO2 gas mixture flowing at a combined total flow rate of 400 ml min-1 STPD. Flow rates were verified using a Bios DryCal Definer 220-L primary flow meter that had been calibrated using NIST standards (Mesa Laboratories, Inc., Lakewood, Colorado, USA). The 20 kPa gas mixture was split into four streams using an air-line aquarium manifold (Accuair 4-way aquarium gang valve, J.W. Pet Company Inc.), and each stream was humidified by bubbling it through a 2 ml volume of 20ºC water before being delivered to the surface of a hemolymph-HCl mixture by a length of 21 ga Tygon tube. The microtonometer motor was connected to a 6 V power supply, and the hemolymph-HCl mixture was equilibrated with the 20 kPa PCO2 gas mixture for 30 min. After the equilibration, the test tube was removed from the microtonometer and slotted into a holding rack inside the water bath. The gas line was placed back into the tube to continue flushing the headspace with the same 20 kPa PCO2 gas mixture, while the top of the tube was loosely plugged with a cotton ball to hold 31  the tube in position and prevent outside air from reaching the hemolymph sample. A 5 μl sample of the CO2-equilibrated  hemolymph-HCl mixture was extracted and analyzed for TCO2 using the protocol outlined in Chapter 2.2.3. However the only difference was that rather than the previous area approximation formula, the area-under-the-curve (AUC) function in the statistical software R ver. 3.5.0 (R Core team, 2018) was used to calculate the volume of liberated CO2. The TCO2 (mmol l-1) and PCO2 (kPa) were used to calculate CO2 solubility using Henry's law: αmix = [TCO2]/PCO2, where αmix is the CO2 solubility of the hemolymph-HCl mixture.   The above process was repeated 3 times using 40 μl samples of the 0.1 mol l-1 HCl used to acidify the hemolymph samples, as both a technical replication and to calculate the CO2 solubility of this acid solution. Finally, the following equation was used to calculate the true hemolymph CO2 solubility: αhemo = fhemo−1(αmix − fHClαHCl), where fhemo and fHCl are the proportions of hemolymph and HCl in the mixture respectively (Harrison, 1988).   The same protocol was used for both dragonfly nymphs and adults, however for Aeshna multicolor, 30 μl of hemolymph was pooled from two individuals as it was not possible to obtain the required amount from a single adult.  3.2.4 Carbonic acid apparent dissociation constant (pKapp)  The protocol for calculating the pKapp of hemolymph was identical to that used in the above experiment, except for the following differences: 80 μl of hemolymph was collected from an individual, and 68 μl of the centrifuged supernatant was placed in a test tube. 2 μl of 0.7 mol l-1 NaF was added to the hemolymph to inhibit any enzymes or reactions that could spontaneously alter pH (Harrison, 1988), then the test tube was placed into the microtonometer setup. The two mass flow controllers were used to combine 99.998% N2 and 5% CO2 bal. N2 compressed gases (Praxair, Mississauga, Ontario, Canada) to create 0.5, 1, 2, 3, 4, and 5 kPa PCO2 gas mixtures. The hemolymph sample was equilibrated to each PCO2 for 30 min, and following the equilibration period at each PCO2, both the pH and TCO2 content were measured. The pH electrode described previously was inserted directly into the test tube to measure pH. Afterwards, 3.2.4.1 Dragonfly nymphs 32  the electrode was removed and 5 μl of the hemolymph was analyzed for TCO2 as described previously. The pH, TCO2, CO2 solubility (α), and PCO2 were used to calculate pKapp by using a rearrangement of the Henderson-Hasselbalch equation: pKapp = pH − log10 [TCO2]−αPCO2αPCO2 .   The same protocol as above was used for adult dragonflies, however 60 μl of hemolymph was pooled from two Anax junius while 60 μl was pooled from three Aeshna multicolor, and 48.6 μl of the supernatant was mixed with 1.4 μl of 0.7 mol l-1 NaF. Only 0.5, 1, 3, and 5 kPa PCO2s were tested due to the limited amount of hemolymph. 3.2.4.1 Dragonfly adults  3.2.5 Relationship between pKapp and pH  It is generally accepted in the literature that blood pKapp is dependent on blood pH in vertebrates and crustaceans (Boutilier et al., 1984). As one of the objectives of this study is to quantify the pKapp of dragonfly hemolymph and compare it to those in vertebrates and crustaceans, it is therefore necessary to assess whether such a relationship exists in dragonflies. In order to test for a significant effect of pH on pKapp, pKapp was first calculated at each experimental PCO2 for all measured hemolymph samples within a dragonfly species and life stage. The calculated pKapp values for all samples were then plotted against their respective pH on a single graph, and a linear model was fitted to the data to test for a significant relationship between these two variables.   3.2.6 pH-HCO3- diagram  To generate the blood buffer line, data collected from the pKapp experiment was used. For each dragonfly species and life stage, the TCO2 measured at each experimental PCO2 was first converted to HCO3- using the following equation (Davenport, 1969): 3.2.6.1 Non-HCO3- blood buffer line [HCO3−] = [TCO2] − αPCO2. Then the calculated HCO3- and their corresponding pH values were averaged to find the mean HCO3- and pH for each experimental PCO2. Linear models were fitted to these data to produce the buffer lines, and the non-HCO3- buffer capacity was taken to be the slope of this line. 33   The PCO2 isopleths were also generated using data from the pKapp experiment, and the rearranged Henderson-Hasselbalch equation: 3.2.6.2 PCO2 isopleth [HCO3−] = 10pH−pKapp × αPCO2. For each experimental PCO2, HCO3- was calculated across a pH range of 7 to 8.3, and these sets of HCO3- against pH were plotted for each experimental PCO2 to generate the isopleths.  3.2.7 Statistical analyses  Data was analyzed in R ver 3.5.0 (R Core team, 2018). Due to the small and variable sample sizes between comparison groups, it was not possible to test for statistical differences in hemolymph CO2 solubility and pKapp across dragonfly nymphs and adults. Instead, summary statistics were used to observe the overall trends for these data. Linear models were fitted to the pKapp vs pH data to test for any statistical relationships between these variables, and were also fitted to the HCO3- vs pH data to visualize the non-HCO3- blood buffer lines and calculate the buffer capacities for the different dragonfly groups. Analysis of covariance (ANCOVA) was performed to test for any statistical differences between the non-HCO3- buffer capacities of dragonflies. Data are shown as mean ± standard error (s.e.m) unless otherwise stated.  3.3 Results 3.3.1 Hemolymph CO2 solubility  CO2 solubility was determined for samples of hemolymph from early- and late-final Anax junius nymphs, Anax junius and Aeshna multicolor adults, as well as for the solution of 0.1 mol l-1 HCl (Table 3.1). The early- and late-final Anax junius nymphs had hemolymph CO2 solubilities of 0.370 ± 0.002 and 0.358 ± 0.007 mmol l-1 kPa-1 respectively, with the individual measurements tightly clustered around their means. Anax junius adults had a mean hemolymph CO2 solubility of 0.345 ± 0.028 mmol l-1 kPa-1, while Aeshna multicolor had the lowest mean hemolymph CO2 solubility of 0.321 ± 0.035 mmol l-1 kPa-1. However, the variation between the individual measurements was also the highest in Aeshna multicolor. The CO2 solubility of the 0.1 mol l-1 HCl solution was higher than all of the hemolymph samples examined (0.390 ± 0.003 mmol l-1 kPa-1). Given the nature of the data, no attempt was made to determine the statistical relationships between these comparison groups. 34  3.3.2 Carbonic acid apparent dissociation constant (pKapp)  pKapp measurements were made on early- and late-final Anax junius nymphs, and Anax junius and Aeshna multicolor adults. Only Aeshna multicolor adults had a significant relationship between pH and pKapp. Therefore, average pKapp values were calculated for Anax junius nymphs and adults while the equation of the line was calculated for Aeshna multicolor adults.   Early- and late-final Anax junius nymphs had a mean pKapp of 6.25 ± 0.02 and 6.29 ± 0.02 respectively, with both groups having their individual data clustered evenly around the means with no evidence of outliers (Table 3.2). Unfortunately, only 1 pooled hemolymph sample was measured for Anax junius, with this sample having a pKapp of 6.23. The relationship between pH and pKapp for Aeshna multicolor was defined by the equation: pKapp = 0.075 × pH + 5.48.   3.3.3 Acid-base status  The non-HCO3- blood buffer lines and PCO2 isopleths were calculated for early- and late-final Anax junius nymphs, and Anax junius and Aeshna multicolor adults (Fig. 3.2). Assuming an in vivo hemolymph PCO2 of 1 kPa (Chapter 2.3.3), the corresponding HCO3- content from the buffer line for early-final nymphs was 12.4 ± 0.54 mmol l-1, while late-final nymphs had a higher value of 16.2 ± 0.93 mmol l-1. The hemolymph pH of early-final nymphs at 1 kPa PCO2 was also lower, with a pH value of 7.79 ± 0.02 compared to a pH of 7.95 ± 0.05 for late-final nymphs. The single pooled hemolymph sample from Anax junius adults had a hemolymph HCO3- content of 18.4 mmol l-1 and a hemolymph pH of 7.94 at 1 kPa PCO2, while Aeshna multicolor adults had a hemolymph HCO3- content of 16.1 ± 0.32 and a hemolymph pH of 7.75 ± 0.02 at 1 kPa PCO2.   Comparing the non-HCO3- buffer capacities of the dragonfly groups showed that early-final Anax junius nymphs had a value of 2.9 mmol l-1 pH-1, which was significantly lower than the buffer capacities for both late-final Anax junius nymphs (5.6 mmol l-1 pH-1; ANCOVA, F = 30.49, df = 1, p < 0.001) and Aeshna multicolor adults (5.8 mmol l-1 pH-1; ANCOVA, F = 46.81, df = 1, p < 0.001) (Fig. 3.3). Late-final Anax junius nymphs and Aeshna multicolor adults had statistically the same buffer capacities (ANCOVA, F = 0.095, df = 1, p = 0.8). The buffer capacity of Anax junius adults was 5.5 mmol l-1 pH-1, only slightly lower than the values seen in 35  Aeshna multicolor adults. However they could not be compared statistically to the other dragonflies due to a sample size of 1.  3.4 Discussion 3.4.1 Hemolymph CO2 solubility and pKapp  Findings from this study suggest that hemolymph CO2 solubility does not change as dragonflies transition from water-breathing to air-breathing. This is not surprising, since temperature was held constant during the entire experiment, and there is currently no evidence to indicate that hemolymph ionic strength or composition differs between the nymphs and adults. With the two parameters that influence CO2 solubility (Boutilier et al., 1984) being held constant, we would not expect a difference in CO2 solubility, and this is indeed shown in the present data. Therefore, the data indicate that a single mean value could be used to represent the hemolymph CO2 solubility of all dragonfly species/life stages used in this experiment. Doing so, however, requires that there are no statistically significant differences between the hemolymph CO2 solubilities of the different dragonfly species/life stages. As mentioned previously, no statistical analyses were performed on this data due to the small and variable sample sizes, and more replicates must be measured in order to properly assess the statistical relationships between these dragonfly species and life stages.  The measured solubilities of CO2 in the hemolymph of dragonfly nymphs and adults are also in good agreement with values from grasshoppers and locusts, as at 20 ºC Melanoplus bivittatus has a hemolymph CO2 solubility of 0.34 mmol l-1 kPa-1, which is, in turn, similar to values determined for vertebrate plasma (Harrison, 1988). Thus, the current data appears to support the general trend that plasma/hemolymph CO2 solubility is similar across a wide variety of animal lineages for a given temperature and ionic strength. Studies investigating whether Boutilier et al.'s (1984) generalized model for calculating CO2 solubility in vertebrates is applicable to invertebrates would provide valuable information in generalizing the acid-base status of vertebrates and invertebrates.   The pKapp of carbonic acid calculated in this study is very close to that obtained from Melanoplus bivittatus (6.24 at 20 ºC; Harrison, 1988), and also indicates that it does not change with the dragonfly's developmental stage. With temperature and ionic strength held constant, this is to be expected. However, it is surprising that pH does not appear to affect pKapp in most of the 36  dragonfly groups, since data from vertebrates indicate a nonlinear (Boutilier et al., 1984) or a negative linear relationship (Iversen et al., 2012) between pH and pKapp. None of the Anax junius groups show a pH dependence of pKapp, and while Aeshna multicolor adults show a significant effect of pH on pKapp, the statistical relationship is positively linear, unlike those previously described (Boutilier et al., 1984; Iversen et al., 2012). Studies have also shown that pH does not affect pKapp in grasshoppers and locusts (Harrison, 1988), indicating that insects do not necessarily follow the trends seen in vertebrates. However, much work is still needed to fully understand the hemolymph acid-base status of such a diverse group of animals. It is unknown why there appears to be a difference in the effect of pH on pKapp between Anax junius and Aeshna multicolor. It is possible that this result may be explained by small sample size, thus more data should be collected to properly assess any differences between dragonfly species and developmental stages.  3.4.2 Hemolymph acid-base status  Findings from Fig. 3.2 show that at a given PCO2, the hemolymph HCO3- content of late-final Anax junius nymphs is always noticeably higher than that of the early-final nymphs, and is instead very similar to those in the air-breathing adults. This is in good agreement with the hemolymph TCO2 data from Chapter 2 Section 2.3.1 which showed that the TCO2 of late-final Anax junius nymphs is statistically the same as those in the air-breathing adult Aeshnidae, and indicates that the hemolymph acid-base status of late-final nymphs is more like that of an air-breather rather than that of a water-breather. This may again be explained by the observation that late-final nymphs develop the ability to breathe air even while continuing to use their rectal gills to breathe water (Corbet, 1962; Gaino et al., 2007). The data also show that the hemolymph pH of late-final nymphs is more alkaline at a given PCO2 compared to early-final nymphs. A higher hemolymph HCO3- content and hemolymph pH at a given PCO2 is indicative of a metabolic compensation in response to a respiratory acidosis (Truchot, 1975). Considering that late-final Anax junius nymphs are capable of breathing air, and show very similar hemolymph acid-base status to the air-breathing Anax junius adults, it is likely that these nymphs are in a state of compensated respiratory acidosis, which would explain the observed HCO3- concentration and hemolymph pH. Future studies will aim to obtain in vivo measurements of PCO2 and pH across 37  dragonfly development in order to test the hypothesis that late-final nymphs are metabolically compensating for an acidosis caused by a higher hemolymph PCO2.   The non-HCO3- blood buffer capacities also show marked differences between the dragonfly developmental stages. The buffer capacity of early-final nymphs was significantly lower than that of late-final nymphs, which was, in turn, not statistically different from that of the Aeshna multicolor, and presumably Anax junius as well (Fig. 3.3). This indicates that the hemolymph of air-breathing adults, and potentially air-breathing late-final nymphs, is more resistant to respiratory acidosis than the water-breathing early-final nymphs. This agrees with the previous data showing that changes in the hemolymph acid-base status of dragonflies occur late within the final nymph stage before the onset of metamorphosis. The non-HCO3- buffer capacity is typically attributed to the presence of proteins and organic acids (Harrison, 2001; Levenbook, 1950b), but due to the lack of studies on how the composition and concentrations of various proteins and other organic molecules change during development in dragonflies, the basis for the difference in non-HCO3- buffer capacity is currently unknown.  3.4.3 Dragonflies compared to other animals  Calculating the non-HCO3- buffer capacities from amphibians and crustaceans shows that there is very little change from water-breathing to air-breathing in both lineages. In Rana catesbiana, the buffer capacities change from 9.7 mmol l-1 pH-1 during water-breathing to 8 mmol l-1 pH-1 during air-breathing (Erasmus et al., 1970), while in Carcinus maenas the change is only from 13 to 13.3 mmol l-1 pH-1 (Truchot, 1975). These relatively small changes indicate that the compensation of a respiratory acidosis during the transition from water-breathing to air-breathing is mainly achieved by increasing HCO3- in the blood (Truchot, 1975), rather than changing the non-HCO3- buffer system. In contrast, the dragonflies' buffer capacity more than doubles from water-breathing to air-breathing, suggesting that the rise in non-HCO3- buffer system may play a substantial role in defending hemolymph pH in the face of elevated hemolymph PCO2 in these animals.   When comparing dragonflies to other insects, results indicate that the non-HCO3- buffer values of dragonflies are noticeably lower than those measured from other insects. The highest buffering value of Aeshna multicolor is only a third of the average value found in grasshoppers, locusts, moth pupae, and fly larvae (Harrison, 2001). However, many of these species display 38  discontinuous ventilation (Buck and Friedman, 1958; Harrison et al., 1995) or live in hypercapnic environments (Levenbook, 1950a), both of which are expected to elevate their hemolymph PCO2. Under such circumstances, the hemolymph would require higher buffer capacity to prevent changes in pH due to the respiratory CO2, and may explain the comparatively high non-bicarbonate buffer values in these species.      3.4.4 Verification of experimental protocol  Since a custom-designed microtonometer system was used in this study, it was necessary to ensure that our technique produced values that were comparable to those found by others. While no other study has measured the hemolymph CO2 solubility of dragonflies, comparing the CO2 solubility of 0.1 mol l-1 HCl to other acid solutions showed that our values were in very good agreement with those previously reported at 20 ºC: 0.39 mmol l-1 kPa-1 in 1.0 mol l-1 HCl (Bridges and Scheid, 1982) and 0.38 mmol l-1 kPa-1 in 0.001 mol l-1 HCl (Harrison, 1988). The microtonometer system is capable of replicating literature values, and presents a practical technique that allows the assessment of acid-base status in microliter samples.                 39  Table 3.1. Individual and mean CO2 solubility of dragonfly nymphs, adults, and 0.1 mol l-1 HCl Species/developmental stage Hemolymph sample CO2 solubility (mmol l-1 kPa-1) Anax junius early-final nymph 1 2 Mean 0.368 0.372 0.370 ± 0.002 Anax junius late-final nymph 1 2 3 4 5 Mean 0.338 0.347 0.360 0.371 0.376 0.358 ± 0.007 Anax junius adult 1 2 3 4 Mean 0.262 0.365 0.372 0.381 0.345 ± 0.028 Aeshna multicolor adult 1 2 3 Mean 0.255 0.331 0.376 0.321 ± 0.035 0.1 mol l-1 HCl 1 2 3 Mean 0.385 0.392 0.394 0.390 ± 0.003 Where applicable, values are shown as means ± s.e.m       40  Table 3.2. Hemolymph pKapp of early-final, late-final, and adult Anax junius Species/developmental stage Hemolymph sample pKapp Anax junius early-final nymph 1 2 3 4 5 Mean 6.19 6.22 6.28 6.29 6.29 6.25 ± 0.02 Anax junius late-final nymph 1 2 3 4 Mean 6.26 6.27 6.29 6.35 6.29 ± 0.02 Anax junius adult 1 6.23 Where applicable, values are shown as means ± s.e.m.                   41   Fig. 3.1. Illustration of the 3D-printed microtonometer. Panels (a) and (b) show the front and rear views respectively, while panel (c) shows the three individual components of the microtonometer. Panel (d) shows the placement of the test tubes.                42   Fig. 3.2. pH-HCO3- diagrams for early-final (a; n = 5) and late-final Anax junius nymphs (b; n = 4), Anax junius adults (c; n = 1), and Aeshna multicolor adults (d; n = 3). Blue points represent the mean in vitro pH and HCO3- values at a given PCO2, while blue lines represent the corresponding non-HCO3- blood buffer lines. The buffer lines were calculated and positioned based on the series of in vitro pH and HCO3- measurements for each dragonfly comparison group. Black lines represent the PCO2 isopleths. Horizontal and vertical error bars represent standard error for HCO3- and pH, respectively.          43   Fig. 3.3. Comparison of the non-HCO3- blood buffer lines in early-final (n = 5) and late-final Anax junius nymphs (n = 4), Anax junius adults (n = 1), and Aeshna multicolor adults (n = 3). Error bars represent standard error, and buffer capacities represent the slopes of the blood buffer lines.            44  4 General discussion and conclusions  4.1 The respiratory transition of dragonflies  The Odonata arose approximately 250 million years ago (Misof et al., 2014), making them one of most basal of the winged insects (the other being Ephemeroptera; Misof et al., 2014). As a result, the current data from select Anisopteran species may be representative of the respiratory physiology of the ancestral insect. This thesis investigated the respiratory transition of the amphibiotic Aeshnidae and Libellulidae to produce much-needed data regarding how the insect's terrestrial origin may have affected their switch between water-breathing and air-breathing. My findings showed that during the transition from water-breathing nymph to air-breathing adult, there is a significant increase in the hemolymph TCO2 content (Fig. 2.2). This increase was indeed predicted, and mirrors that seen in the various vertebrates and crustaceans studied to date (e.g. Dejours, 1989; Howell et al., 1973). Interestingly, however, the actual magnitude of the TCO2 increase is minor when compared to what is experienced by other water-breathing and air-breathing animals (Fig. 2.5), and is further corroborated by the pH-HCO3- plots (Fig. 3.2). This suggests that either the nymphs possess a hemolymph with HCO3- levels that are elevated relative to other water-breathers, or the hemolymph HCO3- of adult dragonflies is reduced relative to other air-breathers. While there is currently no data available in the literature to compare with the HCO3- concentrations measured in adult dragonflies, the in vivo hemolymph PCO2 measured in this thesis (Fig. 2.3), as well as the few studies that have measured hemolymph HCO3- in dragonfly nymphs (Bedford and Leader, 1975; Sutcliffe, 1962), suggest that HCO3- is unusually elevated in the nymph, and that this may reflect the physiology of the ancestral odonate nymph.  This study is the first to measure the CO2 solubility and pKapp of carbonic acid in the hemolymph of dragonfly nymphs and adults, showing that there was no change in either parameter across development, nor was there a difference between these values and those recorded from vertebrates and crustaceans (Harrison, 1988). This indicates that the physical processes of CO2 dissolution and hydration in water is the same between the above lineages. However, the pH-HCO3- diagrams showed that as the nymphs transformed to the air-breathing adults, the rise in hemolymph HCO3- and decrease in pH due to increased PCO2 is accompanied by a significant increase in the non-HCO3- buffer capacity. This is unlike what is seen in the 45  vertebrates and crustaceans, where water-breathing and air-breathing species adapt to their respective blood HCO3-, pH, and PCO2 without altering the non-HCO3- buffer capacity (e.g. Erasmus et al., 1970; Truchot, 1975). This change in buffer capacity in the dragonflies is likely attributed to changes in the composition and concentration of proteins and organic acids in the hemolymph, which comprise the non-HCO3- buffer pool in insects (Harrison, 2001).    Overall, results from this thesis are the first to indicate that the respiratory response of the amphibiotic dragonflies during the transition from water-breathing to air-breathing follows the same general pattern as in the vertebrates and crustaceans. However, the associated specific changes in the hemolymph acid-base status are noticeably different from what has been previously observed, indicating that there are substantial differences in how ancestrally terrestrial insects have re-adapted to life in water.   4.2 Significance and implications  Despite the abundance of amphibiotic insects in freshwater ecosystems, our current understanding of the respiratory changes that occur when they transition between water and air is almost non-existent. The data presented in this thesis is the first to explicitly address this problem, and challenges the long-held assumption that all animals transitioning from breathing water to breathing air undergo the same shift in blood CO2, regardless of other factors (Truchot, 1987). Clearly, trends seen in vertebrates and crustaceans are not fully applicable to amphibiotic dragonflies, and this highlights the need for further investigation on other amphibiotic insect orders to reach a better understanding of how insects have ancestrally transitioned from air to water.  In addition, findings from this thesis indicate that previous methods of classifying dragonfly nymphs may be inappropriate. The stages of an insect's development have traditionally been based on molts or instars, and within the dragonflies, the final instar nymph represents the stage before metamorphosis. However, I found that within the final instar, there was a radical change in their morphology (Fig. 2.1), resulting in two significantly different hemolymph acid-base statuses (Fig. 2.2; Fig. 3.2; Fig. 3.3). Previous studies measuring hemolymph CO2 content in dragonfly nymphs did not recognize this (Bedford and Leader, 1975; Sutcliffe, 1962), and instead analyzed them as a single group. Their data were likely confounded by the combination 46  of two physiologically different nymph stages, and future physiological studies should distinguish between the early-final and late-final instars.   4.3 Limitations and future directions  One shortcoming of this thesis is that, although it generated novel findings, these results were based on only two different dragonfly families: Aeshnidae and Libellulidae. Given the diversity of the dragonfly lineage, it would certainly be beneficial to obtain measurements from various other families, such as the most primitive Petaluridae (Green, 1977), to determine whether the findings here are generally applicable to the respiratory transitions of all dragonflies. In addition, the various experiments described in this thesis should be repeated on representatives from the four other predominantly amphibiotic insect orders (i.e. Ephemeroptera, Plecoptera, Megaloptera, and Trichoptera). Each lineage invaded the aquatic environment independently at different periods of prehistory (Misof et al., 2014), and also evolved different types of tracheal gills and ventilation patterns. Such a comprehensive study would reveal if and how such different methods of breathing water affect an insect's respiratory physiology, and also show whether it has changed across evolutionary time.  Another limitation of this study is that all hemolymph sampling for nymphs took place outside of the water. While steps were taken to increase our confidence in the data, previous studies have reported that handling and air-exposure can lead to significant changes in blood pH in fish (Railo et al., 1985). We cannot exclude the possibility that the acid-base status of the nymph hemolymph was altered during sampling as a result of handling stress and/or being exposed to the air. To eliminate this potential source of error a cannulation technique would prove very useful in preserving the normal resting state of the insect, but unfortunately would be technically challenging to implement.  One of the biggest questions that arises from this thesis is: what is causing the observed differences in the respiratory transition between insects and other animals? Based on the current data and a survey of the literature, several hypotheses can be proposed. First and foremost, unlike vertebrates and crustaceans, insects do not appear to have carbonic anhydrase in their hemolymph (Harrison, 2001) nor their rectal gills (Kohnert et al., 2004). The absence of this enzyme in the hemolymph is likely a shared trait with the insects' closest living relative, the crustaceans. Its absence in the rectal gills may be due to an initial loss of carbonic anhydrase-47  containing gills during the origin of insects, followed by a secondary development of tracheal gills from the epidermis. Since carbonic anhydrase is critical in catalysing the conversion of CO2 to HCO3- and vice versa, its absence can create a disequilibrium between these two molecules (DeFur et al., 1980), where the uncatalyzed formation of CO2 from HCO3- will be slower than the removal of gaseous CO2 by ventilation and lead to a disequilibrium where blood HCO3- remains high even as gaseous CO2 is expelled. This can therefore lead to an accumulation of HCO3- in the blood, preventing the majority of TCO2 from being excreted. Therefore, one hypothesis is that the lack of carbonic anhydrase and the subsequent disequilibrium between CO2 and HCO3- inhibits CO2 excretion in dragonfly nymphs and elevates hemolymph TCO2 content through a retention of HCO3-. Another potential cause may lie in the nymphs' rectal gills. Although the O2 diffusing capacity of dragonfly gills has been calculated previously based on their mophology (Kohnert et al., 2004), no data exists regarding the diffusion of CO2 across this gas exchange organ. It is possible that the secondarily derived cuticular gills impair CO2 excretion, and it may be hypothesized that the ventilatory excretion of CO2 in dragonfly nymphs is diffusion limited and leads to elevated hemolymph TCO2 content. The use of internalized rectal gills in dragonfly nymphs also means that their gas exchange surfaces are tidally ventilated, whereas the gills of a typical water-breather are unidirectionally ventilated (Dejours, 1989). Tidal ventilation will inevitably lead to anatomical dead spaces which can prevent a complete renewal of the respiratory water in the branchial chamber, and lead to elevated PCO2 in the water as a consequence. 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