Philip Matthews

Many evolutionarily distant species of insect display an episodic pattern of breathing termed a discontinuous gas exchange cycle (DGC), defined by bursts of ventilation interspersed with long apnoeic periods. Internal O₂ and CO₂ are not tightly regulated during DGCs as PO₂ and PCO₂ fluctuate significantly. It is as of yet unknown what mechanisms drive the emergence of DGCs, although one hypothesis states that DGCs arise from an unstable ventilatory control system that is unable to quickly respond to fluctuations in internal PO₂ and PCO₂ resulting in alternating cycles of ventilation and apnoea. Essentially, this hypothesis suggests that a temporal lag present between chemoreception of CO₂ and a ventilatory response results in CO₂ levels oscillating around a ventilatory CO₂ threshold as ventilation is turned on and off. This hypothesis is tested in this thesis by implanting PO₂ and PCO₂ optodes into the hemocoel of Madagascar hissing cockroaches, to measure hemolymph PO₂ and PCO₂ fluctuations in vivo during periods of continuous and discontinuous ventilation. Additionally, rates of CO₂ release were measured using a flow-through respirometry setup, and ventilatory frequency was measured using an infrared phototransistor. The stable hemolymph PCO₂’s measured during continuous ventilation were assumed to represent the CO₂ threshold stimulating gas exchange, and these levels were compared with the CO₂ fluctuations during DGCs elicited in decapitated cockroaches. Cockroaches were also exposed to hypoxia (low O₂) and hypercapnia (high CO₂) in order to artificially manipulate hemolymph PO₂ and PCO₂. Decapitated Madagascar hissing cockroaches were observed maintaining DGCs with internal O₂ and CO₂ levels outside of the assumed threshold values. Results suggest that the DGCs displayed by Madagascar hissing cockroaches are not the result of PO₂ and PCO₂ oscillating around fixed ventilatory thresholds. However, it was observed that patterns of DGCs, such as ventilatory burst duration, interburst duration, and ventilation pattern were altered by exposure to hypoxia and hypercapnia.

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The watery sap within the xylem vessels of vascular plants is thought to exist under high tensions (i.e., negative pressures) that routinely exceed -1 MPa, as well as being very nutrient poor. While this should make xylem sap an energetically unfavourable source of nutrition, some Hemipteran insects within the suborder Auchenorrhyncha feed on it exclusively, extracting copious quantities of this liquid using a muscular cibarial pump. However, neither the strength of the insect’s suction, and thus the maximum xylem tensions that the insect can feed at, nor the energetic cost of xylem feeding, have been determined. Here I used adult meadow spittlebugs (Philaenus spumarius) to address this gap in knowledge. First, the maximum suction they could generate was calculated from biomechanical principles using morphological data obtained from micro-CT scans of their cibarial pump. Second, the metabolic rates (MR) of adult P. spumarius were measured while feeding on hydroponically-grown plants (Vicia faba, Pisum sativum, and Medicago sativa) with known xylem tensions, while their rate of xylem sap extraction and cibarial pumping frequency (fpump) were obtained from simultaneously recorded video footage. Furthermore, during these feeding experiments, the plants were exposed to the osmolyte polyethylene glycol (PEG), revealing how the insects changed their feeding behaviour in response to increasing xylem tensions. These findings indicate that the cibarial pump is capable of generating an average maximum tension of -1.3 MPa. This is higher than any xylem tension recorded from their food plants using the Scholander-Hammel pressure bomb method. In addition, while it was calculated that the xylem sap likely contains sufficient sugars to sustain the energetic requirements of pumping, the total MR of the feeding insect could be satisfied only by assuming contributions from amino acids.

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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.

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Implantable fibre-optic probes are commonly used to measure the oxygen partial pressure (PO₂) within the haemolymph and tissues of insects, but they are highly invasive and traumatic. Furthermore, they can only measure the PO₂ of one spot of the insect’s body at a time. The objective of this thesis was to develop Fluorescent Implantable Elastomer Tags (FIETs) as an alternative to fibre-optic probes. These FIETs were characterized in terms of their uniformity in size, response to PO₂ and photodegradation. I assessed their viability for in vivo measurements by testing them in an autofluorescent system in situ. I constructed a microfluidic chip to produce the FIETs, and characterized their uniformity. To establish the FIETs response to PO2, they were exposed to oxygen (O₂) gas in nitrogen, ranging from 0 to 0.2 atm O₂. Holding the FIETs within steady-state environments of 0, 0.1 and 0.2 atm O2 and constantly illuminating them for 60 seconds with the excitation light source determined the degree of photodegradation. The FIETs were tested within an autofluorescent system by creating an O₂ gradient within a block of 0.5% (w/v) agar. My results indicate that 72% of the emulsions produced by the microfluidic chip are highly uniform when 1% sodium dodecyl sulfate (SDS) in water is used as the continuous phase. In comparison, only 55% of emulsions are highly uniform when 5% Kolliphor in water is used. FIET diameters ranged from 110 – 401 μm for 1% SDS and 67-120 μm for 5% Kolliphor. The FIETs exhibit a linear response to PO₂ (R²=0.963), which is improved when fluorescence is normalized to fluorescence in anoxia (R²=0.983). Photodegradation occurred over 60 seconds, causing a 31.6%, 6.1% and 359.7% drift in measured PO₂ within 0.2, 0.1 and 0.02 atm O₂ respectively. The FIETs were able to detect an O₂ gradient within 0.5% agar. These results suggest that the FIETs are a viable option for measuring O₂ in insects in vivo, although improvements can be made to the uniformity and photostability of the FIETS. Future work should focus on the FIETs response to confounding factors such as temperature.

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