The experiments of this thesis were concerned with the involvement of abscisic acid (ABA) in the control of stomatal closure following water stress. The hypothesis that this control is exerted in a truly hormonal manner was introduced by considering the evidence implicating wilt-induced ABA in certain post-stress stomatal responses in relation to present concepts of phytohormone action. The results are presented in seven sections, each dealing with a specific aspect of the investigation. Section 1 was concerned with the development and characterisation of experimental systems whereby the effects of ABA on stomata of isolated leaf epidermis segments of Commelina communis could be studied. In Section 2, 14C-labelled ABA was presented to epidermis tissue floating on buffer solution. The subsequent uptake of radioactivity was assayed quantitatively and qualitatively using the techniques of scintillation spectrometry, TLC, and soluble-compound microautoradiography. Section 3 involved the use of 86Rb+ as a tracer for K+, the major cation involved in stomatal movements. 86Rb+ was taken up by tissue into the guard cell region, and both the pattern of this uptake and the rate of subsequent efflux was found to be affected by ABA treatment. The fourth section described the development of a whole-leaf incubation system which was then used to examine the effects and movement of 14C-ABA supplied via the transpiration stream. Autoradiographic evidence indicating guard-cell uptake of the honnone corroborated the conclusions of Section 2. In addition, it was possible to obtain 'upper limit estimates' of the sensitivity of Commelina stomata to ABA utilising the ability of this plant to yield epidermal peels. Thus, it was found that significant stomatal closure could be effected by as little as amol ABA per stomatal complex. Complete closure (within 40 min) apparently required over 230 amol per complex. Section 5 provided a measure of the ability of Commelina leaf tissue to synthesise ABA during a short-term (0-5 h) water stress. Levels of the hormone estimated by GLC-ECD rose from 31 to 178 ng per gramme fresh weight after 3 h incubation of tissue which had lost 10% of its fresh weight. Investigation of optimal ABA purification procedures was also carried out. An attempt was made in Section 6 to examine the ultrastructure of Commelina epidermis. Plasmodesmatal junctions between cells were observed in all cell walls except those between guard cells and subsidiary cells. The relevance of this finding in relation to intercellular transport routes and mechanisms was discussed. The seventh and final section described the transport of radioactivity from ABA in the phloem sap of Ricinus communis; Double-label techniques were used to study co-transport of ABA and sucrose. The hormone appeared to be readily translocated. GLG-MS was used to identify free ABA in Ricinus phloem sap. In the General Discussion the results were considered in relation to the concept of the hormonal nature of wilt-induced ABA action, and areas of research deserving of further attention were identified.

Johnston, who discovered the mosquito auditory organ at the base of the antennae 150 years ago, speculated that audition was involved in mating behaviour. Indeed, the Johnston's organ (JO) is now known to detect the whine of flying mosquitoes. Analysis of sound recordings of flight tones from tethered, flying, mosquitoes revealed that opposite-sex pairs, when within their acoustic near-fields, attempt to frequency-match the harmonic components of their flighttones. Same-sex pairs actively avoid frequency-matching. Mosquitoes of the species Toxorhynchites brevipalpis, where the flight-tone frequencies of males and females are similar, attempt to match the fundamental frequency of their flight-tones. Haemophilic, vector-carrying mosquitoes Culex quinquefasciatus and Anopheles gambiae ss, where the fundamental frequency of the male flight tone is about 1.5 times that of the female, frequency-match harmonic components of their flight tones. Usually the male's 1st harmonic with the 2nd harmonic of the female flight-tone. In Burkina Faso, where two morphologically similar molecular forms aggregate in the same swarms but rarely hybridise, frequency-matching of flight-tones may perform a pre-mating barrier and a form of subspecies recognition. We discovered that frequency-matching occurred significantly more frequently between same-form male-female pairs of flying, tethered mosquitoes, than when each member of the pair was of a different molecular form. The bandwidth and tuning of sound-evoked flagellum vibrations and the JO's electrical responses to this mechanical input were measured using laser interferometry and extracellular electrodes, respectively. For the first time we showed that distortion products, recorded from the flagellum and JO, could provide the neural basis for frequency-matching at frequencies beyond the range of the JO's electrical responses. We also discovered that spontaneous oscillations of the antennae are produced by physiologically-sensitive mosquitoes. Through temperature-control and injection of pharmacological agents into the JO, evidence is presented advocating dynein as the molecular motor responsible for powering these oscillations.

Insects are ectotherms and have limited ability to regulate body temperature above or below ambient and are consequently greatly affected by temperature. The aphid \(Myzus\) \(persicae\) has an extensive distribution throughout Europe from Scandinavia to Southern Spain, representing three distinct climatic regions: sub-Arctic, temperate and Mediterranean. The aphid also has genetically distinct clones within its holocyclic (sexual) and anholocyclic (asexual) life cycles. This raises the possibility that aphids are regionally-adapted to distinct climatic zones along the latitudinal cline of its European distribution. Genetically distinct clones of \(M. persicae\) were collected from Sweden, UK and Spain. Indices of temperature tolerance (upper and lower lethal temperature50, coma temperatures and mobility thresholds) were determined for each aphid clone at different rearing temperatures. Acclimation at 10°C for one generation increased cold tolerance by depressing lower lethal, chill movement and chill coma temperatures when compared to 20°C and 25°C and further enabled mobility to be maintained to lower temperatures. Acclimation at 25°C for one generation increased heat tolerance by raising upper lethal, heat movement and heat coma temperatures when compared to 10°C and 20°C. Acclimation at 10°C also acted to raise upper lethal temperatures, indicating that the physiological processes conferring heat tolerance are induced at both high and low temperatures. Data did not support intergenerational acclimation to higher or lower temperatures. Lower thermal limits were more plastic than upper limits, enabling tolerance ranges to be increased following acclimation at 10°C, but reduced on acclimation at 25°C. Rates of change varied between clones, suggesting that certain clones could be more affected by climate change. A relationship between thermal tolerance range and latitude was not supported by data on thermal traits investigated with the exception of heat coma temperature. This suggests that clonal mixing across Europe is extensive and prevents local adaptation, although long term populations could persist in the Mediterranean allowing increased heat tolerance. Clonal type, as identified by microsatellite analysis, did show a relationship with thermal tolerance, suggesting that clonal types could respond independently to climate change, affecting relative proportions of clones within populations.

Over the decades, the question why did neural organisation emerge in the way that it did? has proved to be massively elusive. Whilst much of the literature paints a picture of common ancestry the idea that a species at the root of the tree of nervous system evolution spawned numerous descendants the actual evolutionary forces responsible for such changes, major transitions or otherwise, have been less clear. The view presented in this thesis is that via interactions with the environment, neural organisation has emerged in concert with the constraints enforced by body plan morphology and a need to process information eciently and robustly. Whilst these factors are two smaller parts of a much greater whole, their impact during the evolutionary process cannot be ignored, for they are fundamentally signicant. Thus computer simulations have been developed to provide insight into how neural organisation of an articial agent should emerge given the constraints of its body morphology, its symmetry, feedback from the environment, and a loss of energy. The first major finding is that much of the computational process of the nervous system can be ooaded to the body morphology, which has a commensurate bearing on neural architecture, neural dynamics and motor symmetry. The second major finding is that sensory feedback strengthens the dynamic coupling between the neural system and the body plan morphology, resulting in minimal neural circuitry yet more ecient agent behaviour. The third major finding is that under the constraint of energy loss, neural circuitry again emerges to be minimalistic. Throughout, an emphasis is placed on the coupling between the nervous system and body plan morphology which are known in the literature to be tightly integrated; accordingly, both are considered on equal footings.

Angiogenesis- growth of capillaries from a pre-existing network- can be induced in cold-acclimated fishes, where torpor onset and increased oxygen availability, suggests that the primary stimulus for angiogenesis is not metabolic. It was hypothesised that cold-induced angiogenesis was due to increased blood viscosity, therefore endothelial mechanotransduction of high shear stress, and that warm-induced capillary rarefaction was due to reduced shear stress. The reversal of elevated shear stress by vasoconstriction using the nitric oxide synthase inhibitor, L-NNA, and cyclooxygenase inhibitor, indomethacin had different effects. L-NNA administration hinted towards capillary regression at low temperatures but there was a trend towards increased capillarity at intermediate and high temperatures, whereas indomethacin had no effect. Neither warm acclimation nor vasodilatation using the α-adrenoceptor antagonist, prazosin, had an effect on capillarity. Investigation of the effects of NO on heart rate at high temperature showed NO may reduce heart rate at high temperature. However, this does not explain the trend towards an increase in capillarity with L-NNA at high temperature. Evidence is presented for the absence of eNOS in fishes suggesting either nNOS-derived NO or prostanoids are responsible for vascular tone. Microarray analyses were used to identify signalling pathways that would explain the discrepancies, but proved inadequate to reveal significant endothelial responses to cold acclimation.

Immune responses in insects include components and mechanisms that are highly evolutionarily conserved. In addition to providing insight into the insects themselves, knowledge of the conserved mechanisms involved in insect immunity can offer valuable insight that is broadly relevant to a wide variety of other species. Three aspects of insect immune cell signalling have been studied here. Cell signalling responses have been investigated in two insect cell lines following treatment with double stranded (ds) RNA, a common intermediate of viral replication. It has been established that both cell lines investigated, from the fruitfly Drosophila melanogaster and vector mosquito Aedes albopictus, do not exhibit activation of mitogen activated protein kinases (MAPKs) or NF-KB proteins as a direct response to dsRNA. Secondly, a detailed analysis of the mechanisms of transcriptional regulation has been carried out on the Drosophila drosomycin gene, a key factor both in terms of its function during the immune response and in terms of its role during previous characterisation of Drosophila immune signalling. The drosomycin promoter was found to be regulated independently by the Toll and IMD signalling pathways via distinct sequence elements. Finally, investigation of the responses of an A. albopictus cell line to treatment with bacterial cell wall components has revealed key differences in the mechanisms involved in immune-induced regulation of transcription compared with the model established in Drosophila. A role for p38 MAPK has been identified in the negative regulation of transcription of A. albopictus cecropin Ai, an inducible antimicrobial peptide gene.

The fact that fish possess a sense of smell has been known for some time. Fabricus (1780) described how lampreys and sharks would follow rotting meat and Mono, (1744) demonstrated that fish would react to a worm in the water and show adaptation to its odour. Bateson (1890) showed feeding responses in both elasmobranchs and teleosts to hidden food and juices squeezed through cloth. These reactions are present in both seeing and blind fish. Copeland (1912)and Parker (1914) shoved that dogfish were able to localize hidden food by olfaction, thus proving that it could be a directional sense. Initially the study of chemosense had been performed by observing the occurrence of a definite feeding reaction either as a result of detecting food odour or to extend the type of stimulus studied when the reaction had been conditioned to another stimulus (Göz, 1941; Teichmann, 1959). Conditioning is a long process and requires a suitably hardy species. In many cases where a stimulus not connected with feeding is used to condition a feeding reaction we may learn of sensory acuity in absolute terms but nothing of the natural use of the reaction. To examine more subtle reactions to stimuli. especially those not related to feeding, specialized apparatus had to be developed. This allowed workers to look at how a response would be used by the fish in nature. As well as showing reactions to food and prey organisms fish have been shown to use olfaction in social behaviour both with other species and conspecifics of both sexes. Göz (1941) managed to condition a single, blinded, minnow Phoxinus phoxinus to show a feeding reaction to the odour of another fish species Ictalurus nebulosus not the prey of the minnow. This took many weeks using even the easily-trained minnow. Because of the difficulties and limitations many workers have adopted more direct approaches, Wrede (1932) found blinded minnows (a shoaling species) preferred to visit a compartment in an aquarium where the odour of a conspecific lingered rather than a control compartment. Hemmings (1966a) used a more complex preference trough derived from that of Shelford and Allee (1914) and found that the shoaling, freshwater roach (Rutilus rutilus) showed a preference for the end which had the odour of other roach. By analysis of movement in the trough he showed that this preference was due to increased turning rate. Investigating these pheromones in char, D6ving, Nordeng and Oakley (1974) used electrophysiological methods, recording electrical impulses from the olfactory tract, and found the char was able to identify racial differences in this social identifier. Doving,Enger and Nordeng (1973) proposed a component of mucus to be the pheromone. Interest in amino acids as a possible stimulus to feeding in marine organisms began when Steven (1959) found that glutamic acid produced a feeding reaction in two species of tropical marine fish. Case and (iwilliam (1961) found that a range of amino acids would stimulate a blinded crab to feed when applied to the cheiae This reaction was confirmed electrophysiologically on isolated dactyl preparations, responses being obtained from the dactyl receptors to a range of amino acids. Many workers including Hara et al (1973), Hashimoto et al (1968), Haynes et al (1967) and Suzuki and Tucker (1971) have found similar reactions from the olfactory nerves of teleosts. The long, easily accessible olfactory tract in some teleost species makes them ideal subjects for electrophysiological investigation. The technique of monitoring nerve impulses enables a large number of amino acids to be rapidly tested; the thresholds obtained however may not be those which will stimulate a feeding reaction and hardy species are needed. Most work on chemosense in teleosts has been performed on adult and juvenile fish; this is not surprising since rearing beyond the non-feeding yolk sac stage of many important marine species has only been successful in the last decade. The histology and morphology of the development of the olfactory system has also received little study since Holm (1894) described this process in Salmo salar. He showed that in Salmo salar, which took 90 days from fertilization to hatch, there was no nervous connection between epithelium and brain at 60 days post fertilization but one was found at 83 days post fertilization. The olfactory nerve appeared as the groove closed. Attention has been drawn to this lack of knowledge by Hasler (1957), Johnson and Brown (1962) and Branson (1963). In many of the teleosts so far studied the olfactory system is undergoing development not only for the whole of larval life but beyond into juvenile development. Larval development is a valuable time to study this, and indeed any organ system, since it is changing in physical structure, increasing in complexity and possibly changing in acuity and function. The role of a larva can be in many cases to give the early stages of an organism a different ecological niche from the adult, preventing intraspecific predation and competition for food. Therefore its senses may be used for different purposes to the adult, (for example the use of chemosense and touch in the settlement of Balanus nauplii (Crisp, 1974) a system obviously of no use to the adult). In the case of a nektonic shoaling fish such as the herring with a planktonic non-shoaling larva, there would be a possibility of larvae dispersing over a large area prior to onset of shoaling behaviour. There would obviously be some value in keeping larvae together in loose groups and it is likely chemosense may play some part in this. Aggregations of planktonic marine organisms are a well documented phenomena (Barnes and Marshall, 1951; Weibe & Holland, 1968) from the longevity of these aggregations it seems unlikely that this is caused by physical boundaries of water parcels. It seems possible that chemosense acts to keep aggregations intact and in some organisms is retained in adult shoaling life, perhaps to maintain aggregations when the shoals disperse at night (Harden-Jones, 1962). Feeding patterns in larvae and adults can also differ; for example herring and plaice larvae will snap at food organisms in the water column, taking selected individual prey; in the case of herring almost stalking. As adults, herring mainly feed by filtering, although evidence from other filter feeding fish suggest a proportion of particulate feeding will also occur depending on prey size and density (O'Connell, 1972), and plaice move along the sea bed eating epibenthic organisms, mollusc siphons and sedentary worms, Both modes of feeding require good vision. Newly hatched herring (Blaxter and Jones, 1967) and plaice (Blaxter, 1968a) have very different eyes from the adult, the eye developing throughout larval life. In Sardinops caerulea, Schwassmann (1965), the eye is a very rudimentary structure when feeding begins. Although the feeding act is visual, the volume which can be searched using sight alone is small (in herring 0.3- 2.0 litres per hour and in plaice 0.1-1.8 litres per hour (Blaxter and Staines, 1971). It seems possible that olfaction could assist in the search for food either by directionally guiding the fish larva to a concentration of food organisms or restricting energy-requiring searching behaviour to periods when food organisms can be detected by presence of their odour. With these possibilities in mind the outline of study below was adopted.

The hypothesis that midge sex pheromones could be chemically more diverse in structure than previously thought was tested in the context of four midge species of importance to UK horticulture: pear leaf midge, Dasineura pyri; pear midge, Contarinia pyrivora; blackcurrant midge, D. tetensi; and blackberry midge, D. plicatrix. The major component of the pheromone of D. pyri was identified as (2R, 13R, 8Z)-2, 13-diacetoxy-8-heptadecene. Four isomers were separated by HPLC and in field tests the first eluting isomer only was attractive to male midges. Analysis of volatile collections from female C. pyrivora showed two consistent responses from male midges and they were identified as 2,7-diacetoxyundecane and 7-acetoxyundecane-2-one. The field testing with isomers of 2,7-diacetoxyundecane separated by HPLC revealed that the first and the third eluting isomers were attractive. The racemic 7-acetoxyundecane-2-one was active as well as the first eluting isomer from HPLC. Two EAG active components were detected in D. tetensi female volatile collections. The major component was identified as (Z)-2,12-diacetoxy-8-heptadecene and after separation of stereoisomers by HPLC the third eluting isomer has shown to be attractive to male D. tetensi in the field. The structure for the minor component was proposed as a keto-acetate homologue of the corresponding major component. Preliminary work carried out on identification of the female sex pheromone of D. plicatrix indicated two responses from conspecific males. These were shown to be 15-carbon acetates with the acetate function at C-2, probably with two and one double bonds respectively.

No description available.

In mammals endogenous, self sustained oscillators, known as circadian clocks, have evolved as a result of day night cycles, with a period close to 24 hours, and are involved in many physiological processes; such as sleep wake cycles, metabolic and hormonal activity. The suprachiasmatic nucleus (SCN), is the central oscillator, and is synchronised to the external environment by light, via the eye. It has been demonstrated that peripheral clocks, too, contain the circadian oscillator, with tissues such as the lung, liver, heart and kidney as well as many isolated cell types remaining rhythmic, in culture, for many days. However, these peripheral oscillators require a signal from the central oscillator in order to co-ordinate a synchronised time. Leading candidates in the relay of this information are the circulating glucocorticoid hormones corticosterone (rodents) or cortisol (man), which are known to have potent effects on the peripheral clock, both in-vivo and in-vitro. Further to this, glucocorticoids have been used for many decades to suppress the symptoms of inflammation, a by product of many human diseases.This thesis aims to address the temporal regulation of the peripheral clock by the endogenous glucocorticoid, corticosterone, using a transgenic mouse harbouring a luciferase conjugated clock reporter, and circadian reporter cell lines. It also aims to address the relative contribution of the two closely related nuclear hormone receptors, the glucocorticoid and mineralocorticoid receptors. A further aim of the work with glucocorticoid signalling was to design a flow-though culture system, in order to address the effects of the endogenous pulsatile release of glucocorticoids on the peripheral oscillator. This thesis also aims to characterise the inflammatory response in relation to its circadian characteristics; its relationship with corticosterone and the effect of inflammation on the central clock components. Finally, this thesis aims to investigate a potential input/output of the clock, a member of the family of C/EBP transcription factors, C/EBP alpha, and whether it is under endogenous circadian control and regulated by glucocorticoids.Work in this thesis has shown that glucocorticoids dynamically regulate the peripheral clock at all phases of the circadian cycle and that this regulation occurs mainly through the glucocorticoid receptor; yet the mineralocorticoid receptor does have a function in the immediate response to glucocorticoid administration. Furthermore, as a result of the initial temporal profile after corticosterone addition, on the clock protein PERIOD2, I have shown transient regulation of the clock through Caveolin-1 based signalling. There is also a significant circadian component to the inflammatory response, which appears, at least in part, to be REV-ERB alpha mediated, and the inflammatory response also has profound effects on circadian gene expression in the periphery. A functional flow-through system was designed and a working model produced, albeit with technical difficulties, to address glucocorticoid pulsing and circadian timing but much more work is needed for effects to be fully understood. C/EBP alpha appears not to be under circadian regulation nor under direct glucocorticoid regulation, at least in peripheral models used here.