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Seawater-transferred rainbow trout (Oncorhynchus mykiss) has become an attractive aquaculture product in recent years. Industrial interest is mainly due to its resistance to infectious pancreatic necrosis and its adaptability to brackish water, which allows farming in otherwise unexploited locations. However, most practices for the aquaculture of this species have been imported from Atlantic salmon (Salmo salar) without evidence data supporting their suitability for the species. These include protocols to induce their preadaptation to seawater (smoltification). As a result, following seawater transfer, large numbers of fish die (around 10%) or become growth-stunted (GS; 10-60%). Therefore, species-specific rearing protocols and seawater-readiness biomarkers are needed. In the present PhD thesis, the effects of different photoperiod and temperature protocols for rearing in freshwater were assessed on the development of smoltification traits and subsequent seawater performance. This was achieved by using an array of molecular tools to measure osmoregulation-, growth- and haematopoiesis-related genes, proteins and hormones. Moreover, the discovery of potential seawater-readiness biomarkers and the study of smoltification, seawater adaptation and GS fish development were performed using several mass spectrometry proteomic and lipidomic approaches. Results suggest that winter light signals are inadvisable for the species, while all tested summer signals produced similar good results. Moreover, increased temperature protocols not only failed to improve smoltification and growth but potentially compromised the immune system of the fish. Overall, continuous light seems an advisable light regime, irrespective of temperature. Moreover, new promising potential biomarkers for seawater-readiness were identified using proteomics, while also suggesting a previously unknown role of these proteins in smoltification and seawater adaptation. Finally, GS development was shown to be related to low insulin-like growth factor 1 levels following seawater transfer. Moreover, other related factors to the phenotype were higher stress levels, possibly caused by bullying by bigger fish, and hepatic anomalies related to oxidative stress.

Marked shifts in the life history traits of fish have been reported in many exploited fish stocks, with a particular trend towards decreasing size and age at maturity. Though other environmental and behavioural factors have been implicated, the key driver of these changes links to fishing pressure, through both the direct selective effects of fishing itself, and indirectly through the manipulation of important biological and environmental factors. Although reproduction itself has been well described in teleosts, the mechanisms of environmental and endogenous entrainment of maturation remain unclear and it was the principal aim of this thesis to improve current understanding of these systems in gadoids. Photoperiod has been identified as the strongest environmental cue for entraining seasonal behaviours, including seasonal reproduction. Over the last decade, several of the key drivers involved in the photoneuroendocrine cascade have been elucidated in mammals and birds, with the Eya3 pathway merging as an important mechanism for entraining maturation. However, little is yet known of their influence on maturation in fish. In the first study, the photoperiodic regulation of the Eya3-Tshβ-Dio2 cascade was analysed in Atlantic cod exposed to either continuous light (reproductive inhibition) or simulated natural photoperiod (reproductive stimulation) from July to December. Monthly expression was measured through QPCR, demonstrating a strong activation of pituitary Eya3 under declining photoperiod. As this coincided with the onset of secondary gametogenesis, these results suggest that Eya3 may play a stimulatory role in the photoneuroendocrine cascade of Atlantic cod. Although photoperiod represents the most reliable and noise free proximate signal to entrain the reproductive process, it is clear that a minimum growth and energetic state must be reached for maturation to progress. This directed the second line of study – a series of diet restriction trials on haddock and cod designed to investigate the influence of naturally occurring lipid levels in the diet on growth and reproduction, define the “critical window” in which fish assess their energetic state and how this is analysed before commencing secondary gametogenesis, and to assess whether Eya3 is regulated by the growth axis in cod. The results of these experiments indicate that overall size around the autumn equinox is the most accurate indicator of maturation commitment in cod. Additionally, Eya3 expression was elevated in maturing fish indicating a role for this marker in linking the energetic signal with entrainment of the reproductive axis. Finally, an analysis of the physiological and genetic stock structure of cod from the North Sea IVa stock region and westward into VIa was performed. The results of this analysis support previous genetic studies, indicating further fine-scale structuring of these stocks, reflective of the structure indicate by the differences in maturation strategies of the component populations. The results identified both environmental and harvest related pressures which may be driving the current stock structure. The results of these studies greatly improves our understanding of the key drivers and mechanisms regulating maturation in cod, highlighting new avenues for future research.

Recent years have seen considerable advances in the study of biological rhythms and the underlying molecular mechanisms that drive the daily and seasonal physiology of vertebrates. Amongst teleosts the majority of work in this field has focused on the model species the zebrafish to characterise clock genes and the molecular feedback loop that underpins circadian rhythms and physiology. Daily profiles of clock gene expression in a wide variety of tissues and cell types are now relatively well described. However the zebrafish is a tropical species that does not display distinct seasonality and therefore may not be the species of choice to investigate the entrainment of circannual physiology. In contrast, Atlantic salmon is a highly seasonal teleost that displays considerable temporal organisation of most physiological processes. In salmonids photoperiod is widely known to synchronise physiology to the environmental conditions and as such photoperiod manipulation is routinely used by the salmon industry throughout the production cycle to control and manipulate spawning, smoltification and puberty. Previous studies in salmonid species have already identified a set of clock genes that are linked to these seasonal physiological processes. However, to date, the molecular mechanisms regulating daily and seasonal physiology are largely unknown despite the strong commercial relevance in the Atlantic salmon. In the Atlantic salmon, Davie et al (2009) was the first to report the photoperiod dependent circadian expression of clock genes (Clock, Bmal and Per2 and Cry2) in the brain of the Atlantic salmon. In the same investigation the expression of clock genes was reported in a wide variety of peripheral tissues, however 24h profiles of expression in peripheral tissues were not characterised. In order to examine further the role of seasonal photoperiod on the circadian expression of clock genes, the present work first aimed to characterise diel profiles of Clock, Per1 and Per 2 expression in the brain together with plasma melatonin levels in II Atlantic salmon acclimated to either long day (LD), short day (SD), 12L:12D (referred to as experiment 1 throughout) and SNP (referred to as experiment 2 throughout). Photoperiod dependent clocks were also investigated in peripheral tissues, namely in the fin and liver. Results showed circadian profiles of melatonin under all photoperiods. In experiment 1 both Clock and Per2 displayed significant circadian expression in fish exposed to LD. This is in contrast to previous results where rhythmic clock gene expression was observed under SD. In addition, clock gene expression differed in response to experimental photoperiod in the liver, and diel rhythm differed to that of the brain. No rhythmic expression was observed in the fin. Levels of plasma melatonin exhibited a circadian rhythm peaking during the nocturnal phase as expected. However the amplitude of nocturnal melatonin was significantly elevated under LD (experiment 1) and the SNP long day photoperiod and 2010 autumnal equinox samples (experiment 2). Overall results from these experiments suggested that the control of clock gene expression would be photoperiod dependent in the brain and the liver however photoperiod history is also likely to influence clock gene expression. Interestingly, the gradual seasonal changes in photoperiod under SNP did not elicit similar profiles of clock gene expression as compared to experimental seasonal photoperiods and clock gene expression differed between experimental photoperiod and SNP treatments. In experiment 2 significant seasonal differences were also observed in the amplitude of individual clock gene expression. The mechanisms underlying this and potential impact on seasonal physiology are unknown. Developmental changes such as the smoltification process or abiotic factors such as temperature or salinity should be further investigated. In mammals previous work has focused on the molecular switch for photoperiod response and regulation of thyroid hormone bioactivity via deiodinase mediated conversion of T4 to the biologically active form T3. In mammals and birds expression of key seasonal molecular markers i.e. Tsh, Eya3 and Dio2, are up-regulated hours after exposure to the first LD and III persist under chronic LD conditions. In order to confirm the involvement of these genes in the seasonal photoperiodic response in salmon, a microarray study was first carried out. Results displayed transcriptome level differences in the seasonal expression of a wide variety of target genes including Eya3 and Dio1-3 in relation to LD and SD photoperiod suggesting that these genes may have a conserved role in salmon. qPCR validations of selected genes of interest were then performed (Dio1, Dio2 and Dio3, Eya3 and Tshover diel cycles in fish exposed to LD and SD photoperiod (autumn acclimated fish). In addition an unrelated qPCR study was undertaken in salmon parr acclimated to LD, 12L12D and SD photoperiod (spring acclimated fish)(Dio2, Eya3 and Tsh. Consistent with findings obtained in other vertebrate species, circadian expression of Dio2 was observed under LD. However expression of Eya3 and Tsh appeared to be dependent on photoperiod history prior to acclimation to the experimental photoperiods as already suggested for clock gene expression in this thesis. This is potentially a consequence of direct regulation by clock genes. To our knowledge, this is the first report on the expression of key molecular components that drive vertebrate seasonal rhythms in a salmonid species. The thesis then focused on another key component of the photoneuroendocrine axis in fish, the pineal organ. In the Atlantic salmon, as in other teleosts the photoreceptive pineal organ is considered by many to be essential to the generation, synchronisation and maintenance of circadian and seasonal rhythms. This would be primarily achieved via the action of melatonin although direct evidence is still lacking in fish. In salmonids the production of pineal melatonin is regulated directly by light and levels are continually elevated under constant darkness. In non salmonid teleosts the rhythmic high at night/ low during day melatonin levels persists endogenously under constant conditions and is hypothesised to be governed by light and intra- pineal clocks. The aims of the present in vitro and in vivo trials were to determine if circadian clocks and Aanat2 expression, the rate limiting enzyme for melatonin IV production, are present in salmon, test the ability of the pineal to independently re-entrain itself to a different photoperiod and establish whether the candidate clock genes and Aanat2 expression can be sustained under un-entrained conditions. Expression of clock genes was first studied in vitro with pineal organs exposed to either 12L:12D photoperiod, reversed 12D:12L photoperiod and 24D. Clock gene expression was also determined in vivo, in fish exposed to 12L:12D. Results were then contrasted with an in vitro (12L:12D) investigation in the European seabass, a species displaying endogenous melatonin synthesis. Results revealed no rhythmic clock gene (Clock, per1 and per2) expression in isolated salmon pineals in culture under any of the culture conditions. In the seabass, Clock and Per1 did not also display circadian expression in vitro. However rhythmic expression of Cry2 and Per1 was observed in vivo in the salmon pineal. This suggested some degree of extra-pineal regulation of clocks in the Atlantic salmon. In terms of Aanat2 no rhythmic expression was observed in the Atlantic salmon under any experimental conditions while rhythmic expression of Aanat2 mRNA was observed in seabass pineals. This is consistent with the hypothesis that in salmonids AANAT2 is regulated directly at the protein level by light while in other teleosts, such as seabass, AANAT2 is also regulated by clocks at a transcriptional level.

Photoperiod manipulation is routinely used in the aquaculture industry with the aim to enhance growth by manipulating the timing of reproduction in several commercially important temperate fish species. However, there are clear gaps in our understanding of how photoperiod is perceived by the circadian axis and transmitted to the brain to alter reproduction. Furthermore, due to the wide range of environments inhabited by fish, it is unlikely that one single organization exists. It is therefore believed that comparative studies of temperate species “models” with tropical species such as the African catfish (Clarias gariepinus) that adapted to different environments characterized by weaker light signals can help in such an aim. A number of studies were therefore performed in this PhD project to expand our knowledge on circadian biology and environmental physiological effects in African catfish. The first aim was to characterize the circadian melatonin system in this species (chapter 3). Results clearly showed that the control of melatonin production by the pineal gland was very different in the African catfish as compared to temperate species such as salmon and trout. Indeed, melatonin production appeared to mainly depend on light stimuli perceived by the eyes as opposed to salmonids where light directly perceived by the pineal gland regulates its own melatonin production within photoreceptors. The main evidence was obtained in ophthalmectomised fish that were unable to synthesize and release melatonin into the blood circulation during the dark period. This was the first time that such a decentralized organisation, similar in a way to the mammalian system, was found in any teleost species. In vitro results also supported such findings as African catfish pineal glands in isolation were not able to normally produce melatonin at night as usually seen in all other fish species studied so far. This indirectly suggested that pineal gland photo-sensitivity might be different in this tropical species. Further studies were performed to better determine the amount of light that can be perceived by the African catfish pineal gland depending on light transmittance though the skull (where the pineal gland is located). Surprisingly, it appeared that catfish cranium act as a stronger light filter than in other species resulting in lower light irradiance of the pineal gland. This could explain, although it still needs to be further confirmed, why African catfish photic control of melatonin produced by the pineal would have evolved differently than in temperate species. The work then focused on better characterizing diel melatonin production and endogenous entrainment through exposure to continuous photic regimes (continuous light, LL or darkness, DD) (chapter 4). Daily melatonin profiles of fish exposed to 12L:12D photoperiod (routinely used in indoor systems) confirmed low melatonin production at day (<10 pg/ml) and increase at night (50 pg/ml) as reported in most vertebrate species studied to date. Interestingly, results also showed that melatonin production or suppression can anticipate the change from night to day with basal melatonin levels observed 45 mins prior to the switch on of the light. These observations clearly suggest the involvement of a clock-controlled system of melatonin secretion that is capable of anticipating the next photophase period. Furthermore, when constant light (LL) was applied, day/night melatonin rhythms were abolished as expected due to the constant photic inhibition of AANAT activity (e.g. one of the enzyme responsible for the conversion of serotonin into melatonin). However when fish were exposed to constant darkness (DD), a strong endogenous melatonin rhythm (maintained for at least 4 days and 18 days in catfish and Nile tilapia respectively) was found, demonstrating once again the presence of robust circadian oscillators in this species. The next aim of the doctoral project was then to investigate circadian behaviour of catfish through locomotor activity studies (Chapter 5). African catfish is again a very interesting “model” due to its reported nocturnal activity rhythmicity as compared to most other teleosts species. Locomotor activity is considered as a very useful tool to elucidate the mechanisms of circadian organization in both invertebrates and vertebrates circadian. Results first confirmed the nocturnal activity rhythms in the species. Furthermore, clear circadian endogenous rhythms were observed under constant light (LL) or darkness (DD) during several days before losing rhythmicity. Interestingly, the activity levels varied depending on the stocking density. Finally, the last aim of this project was to test the effects of a range of photoperiodic manipulations on growth performances, sexual development and reproductive performances in African catfish reared from eggs to puberty. Results did not show any differences at the early sages (up to 90 days post hatching) in growth performances nor mortality (high) between control 12L:12D and LL treatments. In contrast, during the juvenile-adult period (from 120 to 360 DPH), significant growth effects were observed, as previously reported in other catfish species, with fish under LL displaying lower growth rate, food consumption and feed conversion efficiency in comparison to most other treatments (12:12, LL, 6:6, 6:18, 12-LL and LL-12) especially 12l:12D. However, no major effects of the photoperiodic treatments were observed with all fish recruited into puberty and developing gonads although differences in the timing of gametogenesis could be observed, especially a delay (circa 2 months) in females exposed to short daylength (6L:18D and 6L:6D). As for egg quality, egg diameter was the only parameter to differ between treatments (slightly larger in egg batch from LL treated females). Overall, none of the photoperiodic regime suppressed maturation in African catfish as opposed to some temperate species. The work carried out during this PhD project clearly advanced our understanding of circadian rhythmicity, light perception and effects of photoperiod on physiology in a tropical species. Future studies are now required to further characterise the circadian system and link it to evolutionary trends within vertebrates.

Despite teleosts being the largest and most diverse group of vertebrates, fish models currently used to study photoperiodic effects on fish physiology have been limited to a few species, most of which are temperate seasonal breeders. The overall aim of this work was to expand our knowledge on circadian biology and environmental physiological effects in Nile tilapia (Oreochromis niloticus niloticus), a continuous breeding species of tropical-subtropical origin. The circadian light axis of Nile tilapia is described with regards to melatonin production. Circadian melatonin profiles of fish under 12L:12D photoperiods were observed to be low at day and high at night, suggesting melatonin to be an entraining signal as observed in all other vertebrates. When constant light (LL) was used, such day and night fluctuations where abolished. However when fish where exposed to constant darkness (DD) a strong robust endogenous melatonin rhythm was found, suggesting the presence of circadian oscillators in this species. Importantly, this endogenous rhythm was observed to be maintained for at least three weeks under darkness and proved to be circadian in nature. Moreover, although the melatonin system was able to produce day and night melatonin rhythms when exposed to a different (6L:6D) photocycle, the oscillator appeared to not be entrainable to such a short photo cycle when exposed to DD, as melatonin levels remained high. When comparing the circadian organization of different teleost species including Nile tilapia, preliminary studies showed at least three divergent circadian light organizations in teleosts. Nile tilapia was characterised by a pineal gland far less sensitive than in other fish species as demonstrated through in vitro studies. Furthermore, pineal melatonin production was clearly dependent on the light perceived by the eyes as ophthalmectomy resulted in basal plasma melatonin levels during the dark period. These findings are the first to be reported in a teleost and could be comparable to the circadian light organization of higher vertebrates such as mammals. The onset of puberty of Nile tilapia was studied with regards to the newly discovered Kiss1/GPR54 system. Such a system has recently been discovered in mammals and found to be the primary switch of the brain-pituitary-gonadal (BPG) axis. The results of this study not only suggest a link between the Kiss1/GPR54 system and the onset of III puberty in this tropical batch spawning teleost, that would be a highly conserved feature across vertebrates, but also that the transcriptional mechanisms regulating GPR54 expression could be directly or indirectly influenced by light. Finally, a study was conducted on the effects of different intensities of continuous light (LL) on the growth and sexual development of Nile tilapia up to first maturation. The results showed a significant growth response of fish in all LL treatments compared to control fish. Importantly, this confirmed that LL enhances growth in this species and suggests that it is the light regime more than the intensity which is having an effect. This work thus provides important basic knowledge of the light entrainment pathway and circadian melatonin rhythms in Nile tilapia. Of special importance is the discovery of a strong endogenous melatonin oscillator and a novel circadian organization in fish which would seem to be homologous to that observed in higher vertebrates. Moreover, this work provides evidence that the newly discovered Kiss1/GPR54 system has a similar role in fish as has been found in mammals and that such a system could be directly or indirectly regulated by light. If so, Nile tilapia and other fish species could become important models in the chronobiology and reproduction fields. Finally, this work not only increases our basic and applied knowledge of this species, but also broadens our understanding of the circadian light axis in teleosts and its mediatory effects on reproduction.

Photoperiod manipulations are widely used throughout the Atlantic salmon (Salmo salar) farming industry as a means of producing a product of uniform quality all-year round. However, farmers still remain sceptical over their effectiveness to regulate growth and maturation during the on-growing stage. Furthermore, reports of a characteristic growth-dip following light exposure suggest that light may negatively affect the physiological performance of fish in the short-term. Thus, this thesis investigates the effects of light characteristics (spectral quality, intensity and photoperiod) on growth and maturation of salmonid fish and addresses some of the uncertainties surrounding photoperiod use currently reported within the industry. Rainbow trout (Oncorhynchus mykiss) are seemingly an ideal model species for examining photoperiod effects on growth. Consequently, the application of constant light exposure (LL) at two different intensities (28W and 16W) during two different thermal conditions (summer and winter) was examined on individually tagged fish. Feed intake and growth appeared to be related to the ambient water temperature and did not appear to be affected by intensity or photoperiod, although the onset of constant light did appear to initially affect growth rate. This may indicate that LL has a limiting effect on the growth of trout or that the prevailing water temperature at which light is applied may override the photoperiodic effect. Furthermore, the lack of enhanced growth in trout exposed to LL, unlike that demonstrated for other salmonids, suggest that there may be a species-specific response to environmental variables. Thus, questions regarding photoperiod effects should be limited to the species in question. The main source of variation in results observed under photoperiod manipulations stems from the salmon industry. Atlantic salmon post-smolts were reared in seawater tanks and either maintained under a natural photoperiod (NP) or exposed to a simulated natural photoperiod (SNP), constant light superimposed on the natural light (NPLL) or constant light only (LL). Artificial light onset, irrespective of photoperiod, resulted in an apparent trend for a reduced appetite lasting up to 60 days. Furthermore, the onset of constant light resulted in a significant chronic elevation of plasma cortisol levels and changes to growth and thyroid hormone levels, providing direct evidence that constant light exposure induces stress. In addition, fish exposed to SNP failed to exhibit a stress response despite a low feed intake. However, differences in the plasma melatonin levels during twilight times, as compared to NP, suggest that gradual changes in the natural light intensity throughout the day, particularly around dawn and dusk, may be important for synchronizing daily events. No differences in growth were observed between the NP and NPLL regimes, although fish reared in an enclosed regime (SNP and LL) exhibited a significantly lower weight gain than fish in an open environment (NP and NPLL). This further highlights the impact that the rearing environment has on the growth performances of fish and the need for commercially run trials. Advances in lighting technologies and a greater understanding of how light is transformed through the water column have focussed research on the spectral sensitivity of fish. Therefore the lighting efficiency of novel blue narrow bandwidth LED lighting units through the water column and their effects on growth and maturation performances of salmon reared in commercial production cages were compared against the standard metal halide units currently utilized throughout the industry. LL application, irrespective of intensity or spectrum, reduced the numbers of fish maturing as compared to fish reared under a natural photoperiod. However, this was greatest under the standard metal halide units reflecting a greater light penetration and perception as determined by plasma melatonin levels. The metal halide groups exhibited the greatest relative weight gain over the trial period as compared to control fish. No evidence was observed for a growth-dip under metal halide light, although blue lit treatments exhibited an initial significant reduction in food consumption, suggesting a possible welfare issue. Nevertheless, the prototype blue LED units showed possible potential for commercial application by penetrating the water depth at half the distance of the metal halide units for only one eighth the power and one fifth the brightness. However, further tests of these prototype spectral units are required to examine the potential welfare and physiological growth and reproductive effects. These studies have shown that the efficacy of artificial light regimes is largely dependent upon the effectiveness of the light source through the underwater environment and its perception by fish, providing a sufficient intensity is emitted exceeding the physiological threshold level for the species cultured. Moreover, whilst the onset of artificial light may elicit a stress response and demonstrate a trend for a suppression of appetite for salmon reared in experimental tanks, no compelling evidence for a suppression of appetite or growth was found under normal commercial cage conditions. This suggests that the growth-dip observed within the industry may in part be a combination of a physiological response to the onset of light further exaggerated by the farmer’s perception and altered judgement in feeding. In addition, the results obtained from this study have helped to standardize the use of light regimes within the industry. Nevertheless, further studies are necessary to fully elucidate the underlying mechanisms which may govern growth and maturation in fish following the onset of light exposure.

Sexual maturation during commercial culture of Atlantic cod (Gadus morhua L.) represents a significant production bottleneck restricting the profitability of the industry. Such problems in other species have traditionally been addressed by artificial manipulation of photoperiod cycles, however little research exists in this field in cod. This thesis therefore investigates the interactions between artificial photoperiod manipulation, sexual maturation and somatic growth in this species. In the first experiment, populations of Atlantic cod (hatched, spring 1999) were maintained on either a simulated natural photoperiod (SNP) or continuous illumination (LL) from approximately 15 months post hatch (MPH) (July 2000) in an enclosed tank system. Growth performance was recorded monthly along with observations of reproductive activity over the subsequent 2 years (up to July 2002). At both 2 and 3 years of age the entire population raised under SNP matured and spawned, during which time mean weight reduced by 13% and 24% respectively. No spawning individuals were recorded at 2 years of age in the LL population and only 18% were observed to spawn at 3. However, observations of both changes in gonadal morphology (observed via ultrasound scanning) and a suppression in growth rate at 2 years of age in the LL population alluded to a maturation “dummy run” regulated by an endogenous clock. Despite this phenomenon, the LL treatment realised a 39% and 43% improvement in wet weight following 1 and 2 years of exposure to LL respectively. When the diel cycle of plasma melatonin was compared between the treatments in February 2001 (23MPH) the SNP population displayed an A-profile diel rhythm ranging between 20 and 50 pg/ml while the LL treatment did not display any rhythm. In the second experiment of this work, two populations of cod (hatched, spring 2001) were reared in commercial open cage systems, one of which experienced continuous additional artificial illumination between July 2002 (15MPH) and October 2003 (30MPH) provided by four, 400W submerged lighting units. Growth and maturation were assessed in both populations throughout. In March 2003 (24MPH) it was apparent that spawning individuals were present in both the SNP and LL populations though a significantly lower number of spawning individuals in the LL treatment suggested that the peak in spawning activity was delayed by about 1 to 2 months. With both populations apparently maturing at 2 years of age, there was no significant difference in weight between the populations at the end of the trial. In comparison to experiments I and IV of this work, these results would suggest that in comparison to salmonids for example, Atlantic cod appear to have a heightened sensitivity to light allowing individuals to differentiate the ambient photoperiod signal from the application of continuous artificial light. In the third experiment, 6 populations of approximately 20 tagged individuals (hatched spring 1999) were maintained, from December 2000 to July 2002, under either SNP, LL or one of four, out of season “square wave” photoperiod regimes (repeating cycles with a 12 month period, consisting of a 6 month window of LL followed by six months of short day lengths [SD, 7L:17D] which had been staggered to start over a six month period). Each individual was monitored monthly for maturation status. Out of season “square wave” photoperiods were demonstrated to successfully entrain maturation and hence significantly alter the spawning profiles in these populations. Application of LL from December 2000 failed to inhibit maturation in the spring of 2001 and, in fact, advanced the spawning season by 1 month while those that experienced SD from the same date showed significant extension of the subsequent spawning season. Interestingly, the males maintained on LL throughout the experiment matured both in the spring of 2001 and one year later in the spring of 2002 while females under the same treatment only matured and spawned in 2001. In the fourth experiment, a total of 830 tagged individuals were raised either under SNP or one of 7 photoperiod treatments, consisting of 5 groups transferred from SNP to LL at 3 monthly intervals between 6 and 18 MPH where they remained and a further two groups maintained on LL from 6 to 15MPH and 6 to 21 MPH respectively before being returned to SNP. Both the gonadic and somatic axes were monitored at the physiological and endocrinological level at three monthly intervals from 6 to 27 MPH. The results demonstrated that it is the falling autumnal photoperiod signal after the summer solstice, more specifically after October, that is responsible for recruiting individuals to enter the sexual maturation cycle. Furthermore, in all treatments where this signal was masked i.e. those which experienced LL starting at or prior to 15MPH, except for some restricted spermatogenic activity in the males testis observed at 27MPH, there was no significant reproductive activity and growth was improved by up to 60% at 27 MPH. While providing evidence for direct photic stimulation of somatic growth, the growth results were also correlated with the measurement of plasma IGF-I and demonstrated its potential as a tool to assess growth rates in the species. Plasma melatonin measured at 15MPH, as in experiment I, was suppressed in all populations which were under LL photoperiods. By identifying the photoperiod “window of opportunity” which recruits individuals into the sexual maturation cycle, this work was able to conclude that the application of LL from the summer solstice prior to maturation is the most efficient photoperiod strategy to be adopted by the aquaculture industry to realise maximum growth potential from their cultured stocks.

Photoperiod is an important signal involved in the timing and co-ordination of many processes such as growth and reproduction in salmonids. Both growth and reproduction appear to be controlled by endogenous rhythms, which under natural conditions, is entrained by the seasonal changes in daylength, that is accurately reflected by the diel pattern of melatonin. This thesis investigates the influence of photoperiod on growth and reproduction in rainbow trout (Oncorhynchus mykiss), and examines the effect on plasma insulin-like growth factor-I (IGF-I). These studies aim to further our scientific understanding of the endocrine mechanisms controlling growth and reproduction and transfer this knowledge to commercial trout farming practices. Studies at fry (<5g) and fingerling (>25g) stages investigated the effect of exposure to constant long-days (LD18:6) and constant light (LL) on growth performance. In all experiments, there was a significant growth enhancing effect following exposure to extended light regimes relative to those under ambient or constant short-day photoperiods. Growth enhancement appeared to be caused through direct photo-stimulation or a phaseshift in an underlying endogenous rhythm dependent on the timing of photoperiod application. Measurement of plasma IGF-I accurately reflected growth rate during juvenile development. Furthermore, a clear autumnal increase in IGF-I was observed that was apparently up-regulated by long-day photoperiods. Additionally, it was shown that melatonin implants significantly reduced growth rate below that of unimplanted controls irrespective of photoperiod, but did not reduce circulating IGF-I levels suggesting that elevated melatonin levels masked the perception of daylength but did not act directly on the somatotropic axis to control growth. As size or one of its correlates (growth rate, energy balance or nutritional status) is regarded as an important determinant of the ability to undergo puberty, the interaction of natural (SNP) and advancing (ADV: long-short day) photoperiods with growth, IGF-I and reproduction was investigated in virgin female broodstock. Under SNP 63% of the population attained maturity while only 29% spawned in the ADV regime. Under SNP both size and growth rate in late spring/early summer appeared to determine whether an individual may initiate maturation. Conversely, under ADV, condition factor appeared to be a better predictor. A complete seasonal relationship between plasma IGF-I, daylength and temperature was demonstrated for the first time in rainbow trout under natural conditions, and provides direct evidence for the relationship between maturation and IGF-I. IGF-I levels showed a negative correlation with testosterone as fish initiated maturation. Furthermore, IGF-I levels accurately reflected growth rate prior to the initiation of vitellogenesis, suggesting that IGF-I may provide an endocrine signal between the somatotropic and reproductive axes that nutritional status, growth rate and/or size is sufficient to initiate maturation. In addition, maturing individuals under both photoperiods typically expressed higher circulating IGF-I levels than those that remained immature and may reflect a greater opportunity for IGF-I to act on the pituitary to stimulate gonadotropin production. In this sense, the observation of elevated levels for 3 months under SNP compared to only 1 month under ADV may reflect a reduction in the window of opportunity to initiate maturation under advancing photoperiods and hence explain the reduction in fish spawning. Finally, using a commercially available ELISA kit provided evidence of a possible detection method for measurement of a leptin-like peptide as absolute levels differed significantly between mature and immature fish. This provides preliminary evidence for a possible involvement of a leptin-like peptide in rainbow trout reproduction and energy homeostasis. Given the above evidence of a growth enhancing effect of photoperiod in covered systems, the transfer of constant light (LL) regimes to uncovered commercial trout farming practices was also examined. The application of LL regimes during either fry grow-out in tanks using floating lights or ongrowing to harvest size during winter in cages using submersible lights was investigated. In all trials conducted, the exposure to LL in autumn significantly enhanced winter growth rate and feeding efficiency by up to 30% and 25% respectively relative to those maintained under ambient light. Furthermore, the effect of light intensity was clearly shown to be an important requirement in successfully applying photoperiod regimes onto the ambient photoperiod in order to enhance commercial production in "uncovered systems". These studies clearly indicate the importance of photoperiod in influencing growth and reproduction in rainbow trout. Furthermore, the use of plasma IGF-I as an indicator of growth provides a practical tool for studying growth-photoperiod-reproduction interactions in this species. However, further studies are necessary to further our understanding of the endocrine pathways governing physiological mechanisms, especially growth and reproduction. Overall, this work has provided important information to improve both scientific understanding and commercial development although it is clear that substantial research is still required.

Colostrum quality is critical to calf health as colostrum provides immunoglobulins (Ig) that are critical for a calf's immune system. Despite close management of factors known to affect colostrum production, 23% of dairy cows are still producing inadequate volume or quality of colostrum, which causes calf death and poor performance. Therefore, the objectives of this dissertation are to investigate factors that affect colostrum production and methods to improve colostrum yield and quality. Based on previous literature showing that photoperiod impacts milk yield post-calving in dry cows and that light intensity and temperature-humidity index (THI) impact colostral Ig content, I hypothesized that photoperiod and THI during the dry period impact colostrum yield and quantity in Holstein and Jersey cows. The first study evaluated the isolated effect of photoperiod on colostrum production. Dry cows were housed in a temperature-controlled barn and exposed to either short-day photoperiod (SDPP) of 8 h of light per day or long-day photoperiod (LDPP) of 16 h of light per day for the entire dry period until calving. Altered photoperiod had no effect on colostrum yield, Ig content or other components of colostrum. However, Jersey cows had a higher Brix score, fat, protein, IgA and IgM. After calving, milk production was not affected by photoperiod treatment, likely due to cows being exposed to an irregular lighting scheme. The second study evaluated the combined effects of photoperiod and THI during the dry period on colostrum production and broke cows into a bottom (1), middle (2) and top (3) third based on their photoperiod exposure. Holstein cows produced more colostrum than Jersey cows in each photoperiod category. For both breeds, photoperiod category 1 cows produced less colostrum than cows in photoperiod category 2 and 3. Brix score did not differ by breed but differed by farm and photoperiod category with farm 1, photoperiod category 3 cows having increased Brix score compared to farm 2, photoperiod category 1 and 2 cows. Colostrum components for Jersey cows did not differ by photoperiod category. However, colostrum volume, Brix score, protein and SNF were all impacted by THI and (or) photoperiod variables in predictive modeling. This indicates that colostrum yield and quality in Holstein and Jersey cows are similarly impacted by both photoperiod exposure and THI exposure during the last two months of pregnancy. Therefore, farmers can utilize short-day photoperiod during the dry period during times of moderate THI to improve milk production post-calving without negatively impacting colostrum production. However, future studies are needed to tease out THI and photoperiod impact on colostrum on a large scale in order to improve dry cow management and colostrum production.

Hemp (Cannabis sativa

Humulus lupulus is an economically important horticultural crop grown globally in narrow bands of latitude and climate. The H. lupulus female inflorescence are used in brewing to add flavours, aromas and bitterness to beer. Within the New Zealand context, the majority of H. lupulus are grown in the Nelson and Tasman regions, however there is increasing interest to expand the growing region further south. New Zealand Humulus breeders also face unique challenges, with the inability to easily breed from the majority of current elite cultivars, limited genetic diversity available in the country and limited information available of the drivers of most aspects of cone quality and agronomic traits. A rapid cycling breeding program has the potential to aid some of these unique challenges and accelerate the potential rate of genetic gain for the New Zealand Humulus industry. A rapid cycling breeding program aims to maximise the potential rate of gain within a given timeframe by reducing the length of time between generations, increasing the number of generations within that given time. This can be achieved by manipulating plant growth and development by artificially replicating natural seasonal cues that plants utilise to trigger key development growth phases, for example, photoperiod and temperature. While some research has been completed on the flowering controls and triggers in H. lupulus there are still a number of unknowns that need to be addressed in order to establish a rapid cycling breeding program, especially within the context of the New Zealand industry and cultivars. The initial purpose of this research program was to explore the possibilities of growing H. lupulus, indoors in a highly controlled environment, and describe what a potential rapid cycling breeding program could look like. While this research indicated that it would be possible to grow H. lupulus from root straps indoors, out of season with a mean reproductive cycle of 95 days, limited potential genetic gain would have been possible through this form of propagation. Theoretically, the largest potential rate of change in genetic gain can be made through seed to seed (seed germination to seed harvest) propagation. However, plants grown from seedlings in the highly controlled environment did not flower, which was believed to be related to the low level of available photosynthetic active radiation in the growth room. Subsequently, the potential to manipulate plant growth and development with plant growth regulators in order to accelerate generational cycling was investigated. The use of plant growth regulators had no effect on plant growth and development and were deemed not feasible for use to accelerate breeding cycles. Throughout this research it became clear that even if a potential rapid cycling breeding program was possible through seed to seed propagation the ability to complete two cycles within a calendar year would be reliant on the ability to germinate the seed in a timely manner. A seed germination methodology was developed, which maximised the germination percentage of the seed, in the shortest period of time. This methodology involved the use of 70 percent sulphuric acid to scarify, and remove the outer layer of lupulin from the seed, a 15 day chilling period at four degree Celsius and the addition of one millimolar of gibberellic acid into the imbibition solution. Further work was initiated to explore the potential to further reduce the chilling time required to germinate a significant amount of the seed, using the phytoene desaturase inhibitor fluridone, which showed promise to increase the germination percentage of H. lupulus seed with less chilling. However, this work was discontinued due to unavoidable and irremediable fungal issues. In conclusion, Humulus lupulus is an important horticultural crop with expanding growing regions. New Zealand H. lupulus breeders face unique challenges such as limited genetic diversity and limited information on agronomic and cone quality traits. This research program has shown that while it is possible to grow H. lupulus indoors as apart of a rapid cycling breeding program, only a limited increase in potential genetic gain would be possible. The use of plant growth regulators was also found to be not feasible for accelerating breeding cycles. Germinating seed in a timely manner is crucial for a potential rapid cycling breeding program, and a seed germination methodology was developed to maximize germination percentage in the shortest period of time. However, further research on the use of phytoene desaturase inhibitors to reduce the chilling time required for germination was discontinued due to fungal issues.

This research aimed to understand the growth and development of hemp (Cannabis sativa L.) crops and determine their impact on hemp fibre quality. An experiment was sown using four cultivars, ‘Ferimon’, ‘Kompolti’, ‘Futura 75’ and ‘Carmagnola’, on three dates (22/10/2020 (SD1), 11/11/2020 (SD2) and 09/12/2020 (SD3)) in Canterbury, New Zealand. The duration of important phenophases was quantified using temperature and photoperiod. Components of dry matter (DM) growth patterns were calculated and explained by differences in light interception, canopy architecture and radiation use efficiency (RUE). Secondary fibre (F2) production was related to key physiological drivers to determine optimum harvest stage. The facultative photoperiod response meant the duration of the vegetative phase, from emergence to flowering, was described in thermal time (Tt), and was progressively longer (P

The key to the survival of every organism on the planet is the optimal timing of essential life cycle events. This allows them to first avoid unfavourable periods, which would be harmful to the metabolic functioning or directly threatening the survival of a species (e.g. drought or freezing events). Second, it allows them to synchronize sexual reproduction among individuals of the same species to ensure gene flow in a population. In the case of plants, prominent examples of such ‘phenological events’ include the emergence of leaves in spring as well as flowering, fruit ripening and the abscission of leaves in autumn (senescence). Sensing the optimal time for vegetation onset and completing the phenological cycle requires regular feedback with the environment to adjust investments in vegetative growth, reproduction or preparation of the dormancy – the state in which trees can persist cold periods during winter in temperate and boreal latitudes. The mechanisms for predicting the onset of spring include daylength (photoperiod; PP) and temperatures as the two driving factors. The accumulation of low temperatures ('chilling') followed by an accumulation of a certain amount of thermal forcing in combination with critically PP, at least in some species, are such 'predictive mechanisms' or at least models, that proved to be well usable. The optimal timing of the growing season onset (e.g. leaf-out) marks a trade-off between maximizing the growing season length including competition for resources (e.g. light, nutrients, water) and reducing the risk of damaging late spring frost events. Global warming has changed this trade-off established over a long evolutionary history, increasing the risk of damaging late frosts in many regions of the world, due to a more rapid advancement of leaf emergence compared to the advancement of last spring frost events. This has recently led to large-scale defoliation of the forest canopy in many regions worldwide, which in turn has massive impacts on water, nutrient, and carbon cycling as well as on the overall food web of the forest ecosystem. This PhD aims to address the following three objectives, each presented in one experimental study corresponding to one chapter: i) (Re-)assess the effective chilling temperature range and their efficiency to release temperate deciduous trees from winter dormancy. ii) Disentangle the effects of PP and temperature during the growing season on the timing of senescence and assess the leaf emergence under limiting chilling conditions for provenances from the southern and northern distribution ranges. iii) Quantify the classic trade-off involved in leaf-out timing, i.e. estimating the cost of freezing damages versus the potential gains associated with advanced leaf emergence. An additional chapter is dedicated to a project which aims to foster the exchange of phenological knowledge between researchers, Swiss rangers and the public to promote environmental awareness. In the first study of my thesis (chapter 2), I placed 1,020 twig cuttings of six common European tree species in climatic chambers and exposed them to various chilling temperatures and durations. The experiment revealed a much wider chilling range than was previously assumed, spanning temperatures from minimum -2 up to 10 °C with freezing temperatures revealing equal or highest efficiencies in releasing the dormancy of deciduous trees. Moreover, I showed that species strongly differ in their chilling requirement and that the chilling mediated dormancy progression and release is characterized by three indicators, namely the maximum dormancy depth (a measure for the inability of a species to flush without prior chilling), the temperature-dependent rate of dormancy release (shape of the forcing-chilling-curve) and the minimum forcing requirement after saturating chilling (corresponding to the asymptote of the forcing-chilling-curve). IV In the second study (chapter 3), I exposed 324 saplings of three deciduous tree species originating from the northern and southern distribution ranges to a combination of 3 PP x 2 temperature treatments between the spring and fall equinoxes. Additionally, I extended the warming treatments to exclude critical portions of the chilling range during the subsequent dormancy period. This experiment revealed that a pure PP effect (not altering the amount of light energy for photosynthesis) was evident, generally advancing the timing of senescence in birch and beech (but not in oak) under shorter PPs. The exclusion of temperatures below 5 or 10 °C throughout winter dormancy resulted in consistent advancement of the budburst timing in oak, but weakened or prevented further advances in birch and beech with the latter showing first signs of incomplete leaf development under the most limiting chilling conditions. Provenances from the northern distribution range revealed both earlier senescence as well as budburst dates compared to the southern provenance in birch and beech, reflecting higher temperature sensitivities at higher latitudes. Oak, in contrast, showed the reverse trend with the southern provenance flushing earlier compared to the northern one. In the third study (chapter 4), I used 960 saplings of four deciduous tree species to artificially induce a wide range of realistic leaf-out dates and to subsequently apply a damaging frost event. Except for hornbeam, mortality after the induced damaging frost was negligible for all species. The timing of leaf-out significantly affected the regreening and growth of frozen and non-frozen saplings, with the lowest performance found at the most delayed leaf-out date. I demonstrate that species have different ways to withstand and recover from frost damage, reflecting their risk-taking strategy. Strategies to recover included a clear prioritization of the recovery of sugar reserves over growth (all species), the deployment of dormant reserve buds (oak), regrowth from the stem basis (cherry) and delayed leaf senescence (beech). The ability to recover from frost damage thus becomes an increasingly important functional trait for species persistence in the coming decades, as the frequency of extreme climatic events such as late spring frosts or droughts is expected to increase for wide areas across Europe. In addition to these research goals, I have initiated an Agora Project (still ongoing) entitled ‘PhenoRangers’ (chapter 6). Within this framework, a network has been established to collect high quality data for research to assess how the seasonal activity of different taxa and trophic levels will change in the future (scientific objective). At the same time, this network serves as a basis for communication between researchers, Swiss rangers, and the public to increase awareness of climate change and promote positive attitudes about how to address environmental problems at an individual level (communication objective).

Following emergence, perennial ryegrass is a vegetative juvenile and goes through primary (PI) and secondary induction (SI) phases before becoming reproductive and exhibiting floral initiation (FI). Experimental and anecdotal evidence shows the duration of primary induction is influenced by temperature while photoperiod (Pp) may influence the duration of both primary and secondary induction. The range of flowering responses shown by perennial ryegrass genotypes may be characterized by different responses to Pp and temperature. The aim of this work is to understand and quantify the range of observed responses. The influence of temperature and Pp on the development of perennial ryegrass was investigated using genotypes from different centres of origin grown under controlled environment and field conditions. Initial screening showed many accessions that originate from ~33 to 46° latitude became reproductive in constant 18°C, 20 h Pp, while few flowered in constant 18°C, 14 h Pp. These results demonstrate that flowering occurred in response to a long Pp, without prior PI. Progress through PI was assessed at 4, 8, 12 and 18°C, in both 8 and 17 h Pp. Plants were subsequently transferred to non-primary inducing conditions, 18°C, 17 h Pp, that would fulfil the SI requirements, at two weekly intervals for up to 12 weeks. Flowering was determined as when 50% of plants produced seed heads following SI. FI was determined by tracking accumulated organ number (primordium plus leaves) relative to Haun Stage (HS). The expected pattern was represented by three straight lines. The first period represents vegetative growth with a rate of ~2 primordium/HS. The second stage began at FI when the rate of primordium production increased. The final period was when primordium production ceased, which was represented by no further increase in the number of primordium/HS. FI and terminal spikelet (TS) were calculated as the inflection points for this relationship. Maximum temperatures for completing PI in an 8 h Pp and 17 h Pp (respectively) were 18°C and 18°C for ‘Medea’, 12°C and 4°C for ‘Kleppe’, 12°C and 8°C for ‘Grasslands Nui’ and 12°C and 12°C for ‘Grasslands Impact’. Thus, a function was required to reduce the upper limit of effective temperatures as Pp increased. No treatment achieved FI prior to exposure to long photoperiods. Thus, optimum temperatures for progression through PI (Vsat) were defined from treatments exposed to short days (8h). Vsat was defined as when the number of leaves produced post transfer reduced to 4HS from the shortest treatment duration. For ‘Medea’, this was 18°C and Vsat occurred after 28 days exposure, ‘Grasslands Impact’ required 56 days at 12°C, while ‘Grasslands Nui’ and ‘Kleppe’ required 70 days between 4 and 12°C. Vbase, the minimum exposure required at the optimum temperature for 50% of plants to flower, was 0 days for Medea, 23 days for ‘Grasslands Impact’ and 46 days for ‘Kleppe’ and ‘Grasslands Nui’. After Vsat plants continued to produce primordium at the vegetative rate until they were transferred to 17 h Pp, when FI occurred. This confirmed exposure to a long Pp was an obligate requirement for flowering. Additionally, no plant achieved FI prior to obtaining HS4-5, which sets a base HS for photoperiod perception while the minimum number of main stem leaves was nine. In the field, all four genotypes achieved primary induction from all autumn sowing dates prior to the shortest day. Subsequently FI was triggered by lengthening Pp at a genotype specific base Pp (Ppbase) of ~10.5 h for ‘Medea’, Grasslands Nui’ and ‘Grasslands Impact’ and 12 h for ‘Kleppe’. When FI occurred at Ppbase, Grasslands Nui’, ‘Grasslands Impact’ and ‘Kleppe’, produced ~6.5 leaves compared with ‘Medea’ that produced 5.5 while all genotypes reduced towards four leaves when FI occurred at the saturating Pp (Ppsat). Ppsat were 14 h for ‘Medea’, 15.7 h for ‘Kleppe’, 15.6 h for ‘Grasslands Nui’ and 17 h for ‘Grasslands Impact’. Therefore, combinations of Ppbase and the slope of Pp response separated the genotypes and described the time from FI to final leaf emergence. Concurrently the relationship between the number of leaves to emerge post FI multiplied by the phyllochron determined the date of final leaf emergence. Since ~2HS remained to emerge at TS for all sowing dates, the mechanism reducing the duration from FI to final leaf emergence is a reduction in the HS duration from FI-TS as Pp increases. Two modelling techniques were calibrated to incorporate vernalisation and Pp responses. These predicted competence to flower and final main stem leaf emergence to within 6 days for four genotypes sown on five dates between early autumn and late spring.

Flowering plants produce two different kinds of flowers: chasmogamous, open flowers that promote cross-pollination, and cleistogamous, small mechanically sealed flowers that force self-pollination. Typically, plants produce either chasmogamous or cleistogamous flowers. However, some species contain a chasmogamous/cleistogamous mixed breeding system and develop both flowers at different locations along the plant or at different times of the season. The temporal separation of chasmogamous and cleistogamous flowers is hypothesized to be influenced by the environment. However, the specific environmental cues and genetic mechanisms that signal for the induction and differentiation of each flower type remain largely unknown. To analyze the environmental variables driving separate, seasonal development of chasmogamous and cleistogamous flowers in Viola pubescens, bud counts and measurements of light quantity, canopy cover, photoperiod, temperature, soil moisture, and soil pH were collected over a native population in 2016 and 2017. Statistical analyses confirm that the two flowers develop in response to differing environmental factors and distinguish which factors are significantly associated with chasmogamous versus cleistogamous bud presence, the number of buds produced, and specific threshold values at which each bud type is most likely to develop. To enable genetic investigations, the V. pubescens draft genome was assembled using DNA and RNA sequencing data from eight diverse tissues, which together generated 38,081 gene models including 81 novel cyclotide genes. Gene expression was visualized for each tissue and highlighted transcriptional networks specific to chasmogamous and cleistogamous flowers. Functional annotation of the flower-specific genes indicate that chasmogamous flowers are enriched with genes involved in environmental sensing and repressing precocious development, while cleistogamous flowers contain up-regulated genes involved in DNA topology and microRNA mediated gene silencing. To further explore the role of microRNAs in chasmogamous versus cleistogamous development, a paired miRNA/mRNA-Seq experiment was conducted on shoot apical meristem tissue harvested just prior to chasmogamous (pre-CH) and cleistogamous (pre-CH) budding. From the sequencing data, 2,007 miRNAs were identified and putatively annotated based on homology to known miRNAs. miRNA expression analyses revealed 22 significantly differentially expressed miRNAs between pre-CH and pre-CL tissues. Using a modified Ssearch algorithm, 2,584 gene targets were predicted for the 22 miRNAs, and 400 of the gene targets were significantly differentially expressed between pre-CH and pre-CL tissues. Gene Ontology enrichment and homolog analyses of the differentially expressed gene targets support that chasmogamous flowering involves a tightly regulated network of environmental sensing and gene signaling. This network may act to both repress precocious chasmogamous flowering and promote the transition to cleistogamy through miRNA-mediated silencing of genetic machinery that is no longer supported by current environmental conditions and/or needed for cleistogamous development. The cumulative results of this work will guide future analyses that may resolve the entire regulatory networks involved in chasmogamous and cleistogamous flowering and lead to the development of technologies that enable the preferential induction of each flower.

In vertebrates, melatonin is a hormone that is produced and secreted at night and inhibited by light. This unique “darkness-only” expression profile makes it an intellectually appealing candidate for a means of transmitting temporal information to an individual, both time of day and time of year. In passerine birds, “time of day” information is certainly transmitted via melatonin secretion. The primary producer of systemic melatonin in this family of birds is the pineal gland, and surgical removal of it causes a bird to become arrhythmic in constant conditions. I find that as pinealectomized house sparrows (Passer domesticus) become behaviorally arrhythmic in extended constant darkness the molecular clock in their peripheral tissues largely continues to cycle absent its hypothesized synchronizing cues from the pineal gland. This suggests that the peripheral tissues are either autonomous or that there is a possible secondary circadian oscillator that synchronizes these tissues, such as the avian suprachiasmatic nucleus. In passerine birds, “time of year” information is not transmitted via melatonin secretion to the primary gonads, unlike the case in seasonally breeding mammals. The duration of melatonin, longer in the winter and shorter in the spring as the photoperiod changes with the seasons, does affect secondary sexual characteristics, such as the vocal behavior of birds and the size of the associated nuclei in the brain. I find that long durations of melatonin are sufficient in preventing the photoperiodic expansion of vocal state in male house sparrows. This vocal state change in males consists of the development of a dawn and dusk chorus, as well as a switch from a vocal subtype associated with the wintertime birds flocking together to one of mate attraction and territory defense. This dynamic was independent of the size of the gonads, which were consistent those of photostimulated males. I also investigate this vocal state change in outdoor captive sparrows. This vocal state change is also present in female house sparrows, although unlike in male birds, the presence of absence of the pineal gland does not affect the timing of their vocalizations. Further, I investigate the ability for rhythmic presentations of vocalizations to influence the circadian clock, in zebra finches, Taeniopygia guttata. Aural cues have been shown to act as a weak external cue for entraining a passerine bird’s circadian clock. I presented various permutations of zebra finch vocalization: a single song played repeatedly, the same song played reversed, random tones, and live monitoring of a breeding colony entrained to a light-dark cycle. The live monitoring was the strongest aural cue, and more generally it appears that novelty and context enhance the effect of audio cues on the circadian clock. These experiments in sum suggest a role of melatonin in gating the seasonal expression of vocalization behavior in house sparrows. In male birds, a component of the seasonal dynamic is the development of a multimodal rhythm to when during the day the birds vocalize as the days lengthen consistent with spring, suggesting that the circadian clock may be involved with this diel variability. Additionally, this vocal behavior can also feedback onto the circadian clock. The pineal gland and its primary hormone melatonin function, in part, to regulate complex behavioral rhythms in passerine birds.

Using a high throughput closed respirometry method to measure oxygen consumption, I determined metabolic rates in asexual and sexual Schmidtea mediterranea and Girardia dorotocephala, as a function of temperature, taxon, stressors, reproductive mode, age, regeneration, and specific dynamic action. This study has shown that oxygen consumption can reliably be measured in planaria using optode closed respirometry, and also provided a reliable method for measuring wet mass in planaria, which has been a challenge to researchers in the past. This research revealed that oxygen consumption in S. mediterranea is 1.5-2.1X greater in the sexual strain over the asexual strain at 13-18°C. Within the sexual strain, oxygen consumption is 1.5 -2.2X greater in sexually mature adults over the sexually immature groups (hatchlings, juveniles, and regenerating sexuals). Furthermore, I was able to quantify differences in sexual morphology between these groups exhibiting significant differences in oxygen consumption. The results of this research supports a theory of higher metabolic costs with sexual maturity in S. mediterranea. Therefore, this study has established sexual and asexual S. mediterranea as simple, yet attractive models for investigating energetic costs between sexual and asexual phenotypes. This research also provided quantitative values for specific dynamic action in planaria, with a maximum increase in oxygen consumption of 160% induced by feeding, as well as metabolic relationships in planaria involving temperature, age, and regeneration. These values establish planaria as one of the simplest animal models in which common metabolic patterns, such as SDA and poikilothermic temperature sensitivity, have been demonstrated. Therefore, this research has contributed to the overall knowledge of the basic physiology in this animal, providing the framework for future metabolic studies in planaria involving environmental factors, reproduction, regeneration, development, and aging. Information from this study may supplement interpretation and understanding of modern cellular, molecular, and genomic studies in planaria.

The main aim of this research was to understand differences in the growth and development of three lucerne genotypes with different fall dormancy (FD) ratings; FD2 (dormant), FD5 (semi-dormant) and FD10 (winter-active). To do this, one field experiment was undertaken over two years; a seedling establishment phase followed by multiple regrowth cycles (October 2014 to January 2017). By the end of the seedling phase, the FD10 genotype had produced 20% more shoot and 16% more root biomass than the other two genotypes. Lucerne physiology was examined to see if the yield advantage of FD10 was maintained during subsequent regrowth cycles. After the seedling phase, a second treatment of defoliation frequency regime (DF) at 28 (DF28), 42 (DF42) and 84 (DF84) days was used to create different levels of root reserves, to examine whether treatments affected the yield and quality potential of crops. Annual shoot yields ranged from 4.4 t DM/ha in DF28 crops to 17.5 t DM/ha in DF84 crops. Most of this difference was due to changes in the rates of shoot growth in response to temperature and photoperiod (Pp). When crops were growing into an increasing Pp, growth rate was 3.5 kg DM/ha/°Cd for DF28 crops compared with 7.5 kg DM/ha/°Cd for DF42 and 8.8 kg DM/ha/°Cd for DF84 crops. The leaf stem ratio (LSR) declined by 0.82 for each one ton increase in shoot DM. The CP and ME accumulation in whole shoots or in leaf, soft stem and hard stem followed an allometric relationship. As DM increased, CP and ME increased in a similar pattern for all treatments. By third year 2016/17, crops defoliated at 42 and 84 day intervals produced 1.3 t CP/ha and 55 GJ ME/ha greater than a 28 day regrowth crop. Thus, quality was unaffected by FD ratings and explained allometrically by the leaf and stem ratio, associated with shoot DM. Physiologically, the differences in shoot yield among DF crops were explained mainly by differences in the amounts of radiation intercepted. More frequent defoliation caused lower radiation interception because short regrowth cycles reduced leaf area index (LAI). There was no difference in canopy architecture with an extinction coefficient of 0.83 for all treatments. The lower LAI in DF28 canopies was explained by a lower leaf area expansion rate (LAER). This was 50 and 62% slower during the main spring-summer growth periods than for DF42 and DF84 crops, respectively. The lower LAER in DF28 crops was caused by slower development of individual leaf area and a longer phyllochron. However, other LAI components including branching, shoot population, leaf senescence and reproductive development (flowering) were relatively consistent for all crops. The shortest defoliation interval reduced the amounts and levels of root reserves (DMroot) by 40 and 60% in relation to the DF42 and DF84 regimes. The smaller root reserves in DF28 crops were caused by a decline in the fractional DM partitioning in roots (Proot). Therefore post defoliation, a lack of root reserves reduced RUEshoot and consequently reduced biomass accumulation for DF28 crops. There was no interaction between the effects of the FD and DF treatments. Irrespective of the DF regime, the FD10 genotype produced 23% higher shoot yield in autumn. During this period, stem expansion rate of FD10 was 0.99 mm/°Cd which was faster than FD5 (0.70) and FD2 (0.53). Autumn mean leaf area was 226, 369, and 489 mm2 for FD2, FD5 and FD10, respectively. Therefore the higher autumn shoot yield of the FD10 genotype came from a faster stem elongation rate and larger leaves. However, individual leaf area was similar for all genotypes during the main spring-summer growth. In the 28 day defoliation treatment, FD10 had lower shoot growth rates during the spring-summer period 2016/17 and grew ∼3 kg DM/ha/0Cd lower than the FD2 and FD5 genotypes. The difference in Proot among genotypes was possibly due to different in base photoperiod response. The FD2 genotype showed the most response to Pp direction. This suggests this genotype had a lower base photoperiod response. Therefore this more dormant genotype recharged root reserves at all times of year. In contrast, the winter-active (FD10) genotype had a higher base photoperiod and therefore had less time to recharge root reserves than the FD2 genotype, FD5 was intermediate. This explains the faster decline in root reserves for FD10 growing in colder months in the DF28 regime. Consequently, the root reserves of FD10 declined over time to 1.5 t DM/ha by the end of the experiment in January 2017. It is likely that this progressive reduction in root reserves is the cause of reported decreases in persistence of FD10 genotypes over time. Ongoing monitoring of this experiment will be used to test this hypothesis. Thus this research showed growth differed among FD ratings. Phenological development (phyllochron, branching, canopy structure and leaf senescence) and reproductive development were conservative among FD ratings. In contrast, vegetative growth (leaf area expansion and stem elongation) was most closely correlated with fall dormancy ratings during atumn period.

This project was performed in two parts. The first was focused on light intensity as it affects performance. A randomized complete block design (RCBD) was performed. Broilers, Cobb 500 (n = 1584) were housed in 3 commercial houses (121.9 x 12.2 m). In each house birds were randomized and placed in 72 pens of 121.9 x 121.9 cm (22 bird/pen, males and females). All the treatment groups were provided with 24h light (L) during the first week and then 18L:6Dark (D) and 20 lux from day 7 to 14. The 3 intensity treatments of 5 lux (lx), 10 lx and 20 lx (24 replications) with 18L:6D were started at day 14 and continued until 40 days of age. The second experiment was designed to determine if birds showed a preference for light intensity while eating. A RCBD was performed with 3 different light intensities. Cobb 500 broilers (n= 180), were housed in 1 commercial house. They were placed in 6 pens. Each pen had 3 rooms with a specific light intensity and one feeder so the birds could choose under which intensity to eat after 14d of age. Feed disappearance for each feeder was collected and the lighting program was the same as in trial number one. Also a camera was set to record the feeding behavior of the birds (number of birds per treatment during one hour at a random time during the daylight period, before light turns off and one hour after light turns on). In the first experiment there was no effect of light intensity on the production parameters. In the feed preference experiment there was a significant difference among the treatments (p The results suggest that from a welfare perspective meat-type broiler chickens prefer to eat and drink under 20 lx rather than 5 lx which is the common commercial practice. Results suggest that a greater attention to light intensity, particularly with respect to feeder placement, may not only benefit production performance, but also bird welfare due to their preference for increased light intensity when feeding.

Abstract: Cows fed the same high-grain diet have a large variation in rumen pH and can be categorized as higher or lower risk for developing subacute ruminal acidosis (SARA). The objective of this research was to determine if differences in feeding behaviour existed between the two categories and if these differences could be managed with feeding frequency or photoperiod. In the preliminary studies, the feeding behaviour of 6 higher risk cows and 10 lower risk cows was observed over 24 hours. Cows at a higher risk for SARA ate for a longer period of time (186 vs. 153 min; P = 0.01) soon after feed was delivered once per day in the morning and less time overnight (19 vs. 43 min; P = 0.01) before feed delivery the next day than lower risk cows. In the primary experiment of Experiment 1, 4 higher risk and 4 lower risk cows were fed either once or three times daily and feeding behaviour was observed. Cows that were fed 3 times vs. once daily reduced eating time in the morning (103 vs. 145 min; P < 0.01) and increased eating time overnight (76 vs. 44 min; P < 0.01) which resulted in a decrease in the severity of SARA in higher risk cows (area below a pH of 5.8; 51 vs. 98 pH × min/d; P = 0.05) when they were fed 3 times vs. once daily. In Experiment 2, 30 cows were subjected to a long day or short day photoperiod and behaviour was observed. A long day photoperiod tended to increase eating time between 0300 to 0800 h (53 vs. 39 min; P = 0.06) and reduced overall daily sorting (P = 0.07). These findings suggest that feeding behaviour is a contributing factor to increased risk for SARA, where eating for longer following feed delivery increases the risk. Feeding more often and providing more hours of light increased the daily distribution of eating time, which may reduce the risk for SARA.

Abstract: The goals of this research were to evaluate the interaction effects between photoperiod management and dietary grain allocation in lactating dairy cows and to determine if any relationships exist between photoperiod management and the persistency of lactation in selected dairy herds in Alberta. In study 1, there were no significant interaction effects between photoperiod management and dietary grain allocation on milk production, dry matter intake or body weight gain. Cows that were exposed to long day photoperiod (LP; 16 h of light, 8 h of darkness) increased milk yield by 2. 2 kg/d relative to the animals exposed to short day photoperiod (SP; 8 h of light, 16 h of darkness). However, galactopoietic responses to LP were only detected four weeks after initial light exposure; when cows were fed different diets, after adaptation to light treatment, the effect of LP on milk yield was not detected. Contrarily, cows fed high grain diets increased milk yield and dry matter intake compared with those fed low grain diets. In study 2, we found that animal exposure to light did not differ between summer and winter. Thus, farms that practiced photoperiod management were able to provide long day photoperiod throughout the year whereas cows in farms without photoperiod management were exposed to short photoperiod even in the summer months. The current study showed that persistency of lactation was not different for farms with photoperiod management compared with farms without it. Long day photoperiod can increase milk production in lactating dairy cows but animal responses may be affected to a greater extent by other management practices.

Freezing injury, caused by freezing temperatures in the late fall, midwinter, or early spring, can result in significant loss to grape growers. The damage may range from the partial damage of parts of the plants to the total death of the plant, and may vary between years. Freezing tolerance is a multi-genetic, complex quantitative trait that involves many related traits like dormancy induction, growth cessation, acclimation, deacclimation and bud break. Developing an understanding of the genetics behind the complex trait requires connecting the phenotype with the genotype to enable discovering the underlying genes that can contribute to quantifiable differences between individuals. The objective of this thesis is to detect the genomic location(s) underlying genetic variation in low-temperature response traits like freezing tolerance and bud break using quantitative trait loci analysis (QTL). An F2 population developed by selfing a single F1 from a cross between American species Vitis riparia and a hybrid grapevine Seyval, was used to conduct QTL analysis for subzero temperature tolerance. The low temperature exotherms (LTE) obtained from differential thermal analysis (DTA) were used as phenotype data for the QTL analysis. The phenotyping was conducted in multiple months and dormant seasons. LTE results when the supercooled water inside the bud freezes and identifies the temperature at which buds are killed. Best linear unbiased predictors (BLUP) was also calculated using the LTEs to calculate random genetic effects. Composite interval mapping (CIM) was conducted using either the average LTEs or BLUP values in R/QTL with 1000 permutations and error rate of 5%. The LTEs for individual genotypes varied in the different months. QTLs were identified using either average LTEs or BLUP values for all the months and dormant seasons. QTLs appeared on Chromosome 1, 5, 9, 13 and 16 in different months or dormant seasons. Many potential candidate genes associated with calcium signaling, ethylene signaling, ABA signaling, cellular metabolism and dehydration response were found underlying the 1.5 LOD interval of these QTLs. The bud break phenology was studied at three chilling periods to identify bud break related QTL. Bud break occurs upon the exposure to optimal growth conditions, after the chilling requirement fulfillment transitions the bud from endodormancy to ecodormancy. Three one node cuttings of the grapevines were forced at 13 hours of daylength and 25⁰C/20⁰C thermoperiod in the growth chamber for four weeks. Each week the growth stage of the cuttings was scored using Modified Eichhorn-Lorenz (Modified E-L) phenology scale. This was repeated in multiple dormant seasons and months. The bud break data was ordinal, monotonic and based on repeated measurements, thus similar to data in disease epidemiological studies in which disease severity is scored repeatedly over a time period. Therefore, the concept of area under the curve (AUC) was applied to the bud break data. The area under the bud break progression curve (AUBPC) was calculated for the bud break data of each individual for an individual month. CIM was conducted with the average week 3 phenology score data or AUBPC data in R/qtl using 1000 permutations. Both data processing approaches provided similar QTL results on chromosome 3, 7, 8, 9, 13, 18 and 19. These QTLs encompassed candidate genes involved in calcium signaling, auxin signaling, ethylene signaling, circadian clock signaling, cellular metabolism, primary and secondary metabolism, dehydration stress response, drought stress response, fruit ripening and many transcription factors. The results from this study can be useful in developing low temperature response trait markers that can be applied in marker-assisted breeding and selection to develop the grapevine cultivars suitable for northern continental climates where freezing temperatures are common. This will enhance the sustainability of cold climate grape production and promote expansion of grape growing regions.

Abstract: Photoperiod, or day length, generates a highly accurate biological calendar that allows animals to anticipate and adapt to environmental change throughout the year. Photoperiod coordinates both daily and yearly rhythms associated with sleep/wake cycles, behavior, and reproduction, including mammary development and milk production. In dairy cows, long day (summer-like) photoperiod during lactation, or short day (winter-like) photoperiod during late gestation, enhances milk production. Potential mediators of these effects include changes in mammary cell-turnover and hormonal signaling factors. The effects of photoperiod on the mammary transcriptome and subsequently the molecular mechanisms underlying the milk yield response have not been elucidated. The aim of the work presented here is to identify genes and pathways responsive to photoperiod and associate their differential expression with functional effects of photoperiod in the mammary gland. To address this aim we employed microarray technology in two model systems, cows and mice, with the objectives of: 1. Evaluate the effects of photoperiod on the mammary transcriptome of cows. 2. Identify common effects of photoperiod on mammary function in cows and mice. 3. Evaluate the effects of photoperiod on the mammary transcriptome in mice. 4. Determine if physiological state (gestation or lactation) influences the effects of photoperiod on the mammary transcriptome. 5. Assess common biology between the cow and mouse models. In the bovine mammary gland, we identified 64 photoperiod responsive genes and have interpreted these genes and their associated functions in the context of the mammary gland. Differentially expressed genes were associated with mammary development and immune function consistent with the enhancement of milk production in the ensuing lactation. The transcriptomic signatures across time relative to parturition were not consistent with those in response to photoperiod, suggesting different underlying mechanisms. Furthermore, genes identified in the interaction of photoperiod and time indicate the physiological state of the mammary gland during late gestation influences its response to photoperiod. In the mouse, mammary cell proliferation and gene expression signatures provide substantial evidence that photoperiod can affect the ability of the mouse mammary gland to produce milk, although, we were unable to detect effects of photoperiod on litter weight. Overall, our findings provide several novel insights about the effects of photoperiod on the mammary transcriptome. Firstly, photoperiod manipulation is sufficient stimulation to affect the mouse mammary transcriptome. To that end, we have determined that long day and short day photoperiod affect very different sets of genes that are associated with distinct biological functions. In addition, photoperiod differentially affects gene expression in the mammary gland depending on the physiological state. Lastly, photoperiod can have enduring effects after the cessation of exposure on the mammary transcriptome. Ultimately, this work reveals that photoperiod manipulation induces changes in the mammary transcriptome during both lactation and gestation. The genes and pathways identified here have been grouped into six potential mechanisms that may underlie the effects of photoperiod on mammary development and function in cows and mice.

Abstract: Understanding the flowering gene complex in bread wheat is important to utilize the maximum production potential in a time-limited growing season. Vernalization (Vrn), photoperiod (Ppd) and earliness per se are the three genetic constituents of the flowering gene complex. A series of experiments were conducted to explore the role of each genetic constituent in Canadian spring wheat germplasm. The first experiment revealed vernalization and photoperiod gene frequency and effects in Canadian germplasm released between 1885 and 2008. Vernalization insensitive Vrn-A1a is the most frequent allele in the Canadian hard red spring class and Vrn-B1 is more frequent in Canadian soft white spring class. The photoperiod sensitive allele (Ppd-D1) is being replaced with the insensitive allele, Ppd-D1a, in all modern Canadian germplasm. An overall analysis of the data for the CWRS class revealed an average reduction of 0.04 days/year in maturity and an increase of about 8 kg/ha/year in grain yield. A second study was conducted on the Cutler × AC Barrie population to map and characterize the earliness per se QTL. Three QTLs of earliness per se affecting days to flowering and maturity were mapped on chromosomes 1B (QEps.dms-1B1 and QEps.dms-1B2) and 5B (QEps.dms-5B1). Two grain yield QTLs were mapped on chromosome 5B. The photoperiod insensitive allele Ppd-D1a interacted in an additive fashion with QTLs for flowering and maturity times. The earliness per se QTL QFlt.dms-5B.1 together with Ppd-D1a can induce earlier flowering and could help to elongate grain filling duration for higher grain yields. A comparative study of Vrn alleles suggests that the presence of Vrn-D1 along with Vrn-A1a and Vrn-B1 confers higher grain yield with similar maturity compared to all other Vrn gene groups in soft white spring wheat and we suggested the inclusion of Vrn-D1 allele in elite Canadian germplasm, which has been overlooked in the past. In a final study we found that early flowering and maturity conferred by insensitive vernalization alleles Vrn-A1a and/or Vrn-B1 has not conferred any yield advantage under organic conditions. The results also revealed that there is significant genotype × environment interaction and breeding for organic production should be practiced under organic conditions.

The main aim of this research was to refine best management practices for dryland lucerne (Medicago sativa L.) crops in relation to crop water extraction. To do this, dryland lucerne was established at Lincoln University, New Zealand in two soils which differed in the plant available water content (PAWC). The low PAWC site (Ashely Dene) had a very stony silt loam soil with ~130 mm of water to 2.3 m. The high PAWC site (Iversen 12) is a deep silt loam soil with ~360 mm of water to 2.3 m. The available water for crop extraction in the establishment year was manipulated by imposing the second treatment, sowing date. This resulted in mean annual dry matter (DM) yields which ranged from 0.4 to 21.5 t DM/ha. A detailed examination of lucerne physiology was undertaken to determine how lucerne extracts water from the soil to explain these yield differences.

This dissertation is divided into two parts. Chapters 1 through 3 are interdisciplinary and focus on legal and scientific perspectives regarding the regulation of genetically engineered crops. Chapters 4 and 5 evaluated the environmental factors that control flowering in Pennsylvania sedge (Carex pensylvanica Lam.). The commercial potential of genetically engineered (GE) crops has not been fully realized in the United States due to environmental litigation that dramatically affected the pace of GE crop development and deregulation. The USDA's Animal and Plant Health Inspection Service (APHIS) regulates GE crops. However, litigation initiated by nongovernmental organizations exposed APHIS's vulnerability to lawsuits under the National Environmental Policy Act. We concluded in chapters 1 and 2 that APHIS did not adequately evaluate the environmental risks of novel crops, and thus left itself open to litigation. In Chapter 3, we described how the biotechnology industry is attempting to avoid regulation of GE crops through the creation of a non-plant pest loophole.Pennsylvania sedge is an upland forest sedge with horticultural potential as a low maintenance groundcover. For large plantings, achenes are preferred, but Pennsylvania sedge typically produces few achenes in its native habitat. As a first step in improving achene production, Chapter 4 evaluated the effect of vernalization and photoperiod on floral initiation and development. We concluded that Pennsylvania sedge is an obligate short day plant that does not require vernalization for flowering. Plants flowered when exposed to daylengths of 6 to 12 hours. Flowering was completely inhibited with 14-hour photoperiods. Chapter 5 examined the environmental factors that control floral gender sequence and inflorescence culm heights. Plants were found to be determined and florally initiated in the fall in the northern United States. A post-floral induction chilling treatment (winter) was necessary to produce protogynous flowering and normal inflorescence culm elongation.

The cardinal temperatures and vernalisation requirements for a selection of vegetables for seed production were studied. Experiment one assessed the rate of germination (1/days) of selected vegetable species over a range of 5-40 °C, in order to calculate the cardinal (base, optimum and maximum) temperatures for germination. The cardinal temperatures for carrot and red beet were 0.1, 30.9 and 40.7 °C, and 4.2, 35.9 and 44.4 °C respectively. Experiment two examined the vernalisation response of imbibed cabbage, carrot and red beet seeds, plus perennial ryegrass as a comparison over duration treatments of 0-12 weeks at 4 °C. Red beet and ryegrass had positive vernalisation responses with anthesis occurring in plants from the 4-12 week durations in red beet, and all durations in ryegrass. The number of days to anthesis and the final number of main stem leaves did not differ significantly (P = 0.143 and P = 0.323 respectively) among vernalisation durations in red beet, but did differ significantly in ryegrass (P

Animals have intrinsic circadian periods of around 24 hours. In order to synchronize to the 24-hour day, circadian rhythms entrain to photic time cues and establish a consistent daily rhythm. The maintenance of a stable phase relationship between circadian rhythms and the light-dark (LD) cycle is necessary for entrainment to occur. It is commonly thought that there is a fixed relationship between the phase angle of entrainment and the intrinsic free-running period, where a smaller phase angle reflects a shorter period. Furthermore, it was thought that individuals with smaller phase angles would adjust faster to advance shift of the LD cycle, and slower to delay shift of the LD cycle. This dissertation reevaluated these relationships in rats using long-term in vivo pineal microdialysis to carefully monitor melatonin secretion rhythm. The studies presented in this dissertation examined both the interindividual differences between outbred rats, as well as differences in melatonin rhythms of inbred rats entrained to different photoperiods. Under long photoperiod (short night), melatonin rhythm has smaller phase angles and shorter durations; the opposite is true for short photoperiods. The phase angle variation, natural as well as manipulated, did not correlate with the free-running period variation in rats. Furthermore, individuals with smaller phase angles reentrained faster in both the advance and delay direction. To gain a greater understanding of the role of photoperiod in melatonin phase angle, the adjustment of the melatonin rhythm to an expansion or compression of dark period was studied by altering the dark period symmetrically or unidirectionally. While reentrainment to an 8h night resulted in very similar melatonin profiles regardless of how the dark period was altered, reentrainment to a 16h night depend significantly on whether the dark period was expanded symmetrically or by morning or evening expansion. These findings demonstrate that the relationship between period, phase, and pattern of reentrainment are quite different than is currently believed, and new analysis may be necessary to understand the underlying biology of the circadian timing system.

Animals inhabiting temperate environments have evolved mechanisms to interpret environmental cues and adjust their behavior and physiology in order to maximize survival and reproductive success. Reproduction is an energetically costly process that is inhibited in the winter in seasonal breeders to conserve energy when the chances of successful reproduction are minimal. Seasonally breeding rodents serve as an ideal model system to investigate the neural control of the reproductive axis in a controlled laboratory setting, as surgical and pharmacological methods of reproductive axis inhibition are not required. The aim of the present series of experiments was to elucidate how the brain processes information about the length of the day in order to communicate time of year to the reproductive axis to coordinate reproductive function and behavior. Specifically, the studies herein aimed to investigate the role that two novel neuropeptides, kisspeptin and gonadotropin-inhibiting hormone (GnIH or RFRP in mammals) serve in processing and communicating day length information to the reproductive axis in several species of seasonally breeding rodents. The results uncover important roles for these two neuropeptides in the interpretation of photoperiodic signals for regulating the process of gonadal regression in response to the short day lengths typical of winter in these species. Additionally, by exploiting natural genetic variability within populations of seasonal breeders, these studies suggest that the phenomenon of nonresponsiveness and phenotypic variation in reproductive photoresponse may result, at least in part, from variation in the morphological features and seasonal response of the RFRP system. As a whole, this body of work provides important insight into the complex neural mechanisms that have evolved for integrating environmental information in order to coordinate reproductive physiology with a changing external environment.

Obesity is America’s fastest growing health threat. Although a primary health risk factor, obesity increases the probability of secondary health consequences such as stroke, diabetes mellitus and heart disease. Siberian hamsters offer a convenient model to study obesity, as they exhibit a photoperiod-driven reversal of obesity during the fall-winter months (i.e., short-days-SD). SD responses in the Siberian hamster, amongst others, include decreased adiposity and gonadal regression. The duration of the dark period is faithfully transmitted into a neuroendocrine melatonin (MEL) signal; that codes seasonal information. The brain communicates with body fat (white adipose tissue- WAT) via the sympathetic nervous system (SNS). The central MEL binding sites, however, necessary for the body fat/lipid mobilization responses during SDs are not precisely known, although melatonin receptor (MEL 1aR) mRNA has been co-localized with sympathetic outflow neurons of WAT in several forebrain areas. This dissertation aims to identify and to characterize the contribution of central sites that are important in seasonal responses in Siberian hamsters. Thus, I asked: Which specific brain sites are both sufficient and necessary to stimulate SD-like decreases in body, WAT and testes mass? Furthermore, I tested if SD-induced decreases in body fat mass are accompanied by increased energy expenditure, specifically brown adipose tissue (BAT) thermogenesis. It is hoped that the identification of brain sites and mechanisms involved in the effortless reversal of obesity in these animals can be applied to treatment opportunities of human obesity.

Aggression is a feature of many clinical disorders including autism, Alzheimer’s disease, bipolar disorder, and schizophrenia. The available treatment options act to prevent impulsive aggression through modulation of GABAergic and dopaminergic pathways which come with metabolic and dyskinetic side effects. The mechanism underlying aggressive motivation, however, has not been elucidated. In addition, previous studies have been heavily biased towards males of various species. Mimicking changes in day length, or photoperiod, in the laboratory is a natural manipulation used to examine seasonal changes in aggressive behavior in many species. In response to the reduction in the duration of light exposure, animals undergo gonadal regression and become reproductively quiescent. During this non-breeding season in male photoperiod-responsive animals, aggressive behavior increases significantly. Although studies have shown offensive aggression remains elevated in female rodents, seasonal regulation of this behavior in females has not been thoroughly studied. The neuropeptide arginine-vasopressin (AVP) has been implicated in the facilitation of aggressive behavior in male rodents and fishes; therefore, it is useful to examine AVP as a modulator of seasonal aggression in females. Because the actions of AVP in female social behavior may be hormonally-dependent, we investigated the hormonal mechanisms that regulate the expression of AVP receptors and the behavioral actions of AVP on aggression. In addition to changes in gonadal steroid hormones during the non-breeding season, we identified photoperiod-dependent alterations in adrenal hormone secretion as AVP plays a role in regulation of hypothalamic-pituitary-adrenal axis (HPA) activity and anxiety-like behaviors in animal models.

The genetic diversity of elite temperate maize germplasm is very narrow in comparison to the genetic diversity available across of the entire species. Tropical maize germplasm has frequently been cited as a potential source of enhanced genetic diversity that could be used to increase corn productivity. One obstacle to utilizing tropical maize germplasm in temperate breeding programs is photoperiod sensitivity, which is very common in tropical adapted maize lines. An investigation of the quantitative trait loci (QTL) contributing to maize photoperiod sensitivity may increase the facility and ability with which maize breeders can adapt tropical maize germplasm to temperate latitudes. The inductive phase of maize photoperiod sensitivity was studied in a diverse set of temperate and tropical lines. From a factorial mating of two temperate and two tropical inbreds, four populations of recombinant inbred lines (RIL) were developed for the purpose of mapping the QTL underlying photoperiod sensitivity in tropical maize. Plants were grown in both long- and short-day environments and a number of traits were measured in each environment. These traits include flowering time, plant height, leaf number, and ear structure traits. The trait differences between long- and short-day environments were reported as the photoperiodic responses of the RILs. Utilizing the data of both individual and combined mapping populations, QTL were identified using Iterative QTL Mapping (iQTLm). The positions and effects of these QTL were compared between populations and with flowering time and plant height QTL from other mapping studies. We detected four regions in the genome that produced large photoperiod effects and named these ZmPR1, ZmPR2, ZmPR3, and ZmPR4. These four QTL accounted for 75% of the phenotypic diversity for photoperiod measured by our analysis. Similar QTL positions have been detected by other researchers studying photoperiod sensitivity and flowering time in maize. In addition to a major QTL on chromosome 3, QTL affecting the tasseled ear phenotype of maize were found in the ZmPR3 and ZmPR4 QTL regions, implicating these QTL as also having effects on floral morphology. The four ZmPR loci are the most promising as targets of marker-assisted selection against photoperiod sensitivity in maize. Verification of the four ZmPR QTL was undertaken in four BC2F3:4 mapping populations. The B73 × CML254 had the same parentage as one of the RIL populations originally used to identify the ZmPR loci. We verified the presence of three of the four ZmPR loci in this population. We also found that the other three mapping populations, derived from crosses of B73 × Ki3, B73 × CML247, and B73 × Ki11 showed significant flowering time and plant height associations at some of the ZmPR loci. Winter nurseries were used to verify that the ZmPR4 QTL, which was the strongest photoperiodic flowering time QTL detected in previous mapping studies, was indeed a photoperiodic and not flowering time per se QTL. An F2 population of Ki14 × CML254 was used to identify allelism at the ZmPR loci. Only ZmPR4 and ZmPR2 showed allelism between these two tropical lines for flowering time and height traits, respectively. The utility of QTL mapping for applied breeding programs is often limited by the transferability of QTL across populations and by the lack of precision in QTL mapping. One solution to these obstacles is to fine-map and clone the gene or genes underlying the QTL. We developed several heterogeneous inbred families (HIFs) from four RIL populations in order to facilitate fine-mapping. We observed several traits in these HIFs in phytotron, greenhouse, and field environments. We report the manner with which the HIFs were derived, as well as some observations and notes about future fine-mapping directions with these HIFs.

Northern red oak (NRO), Quercus rubra L., is a valuable tree species for timber, wildlife, restoration, and urban forestry. Seed production and growth responses from different maternal sources can be widely variable. Rooting choice stem cuttings of NRO could prove to be a time-saving nursery practice, yielding prime candidates for outplanting. This study sought to develop effective protocols to root NRO stem cuttings at high frequencies and to examine the effects of several factors during rooting and subsequent containerized growth. Supplemental light and heat were administered to determine the effects of artificially extending the growing season of successfully rooted cuttings. Different container sizes were used to determine which size encouraged optimum containerized growth and survival. In addition, shoot production enhancement was studied through pruning of field grown stock seedlings so that stem cutting material could be readily available throughout the growing season; different seedling sources were used to examine provenance variability for shoot production. Also, early (ES) and late season (LS) cuttings were taken to determine the viability of rooting at different times during the growing season. After two seasons of containerized growth, final measurements of NRO rooted cuttings were recorded to determine efficacy of prior treatments. Geographic source and prune height treatments were both found to have significant impact on the number of new shoots generated by NRO stock plants. We found large stem caliper may enhance donor plant ability to generate new shoots for rooting as compared to small stem caliper. Geographic source and prune height treatment significantly affected rooting ability of ES cuttings, which rooted at 64% overall. Rooting percentages were lower for LS stem cuttings (53% overall), but showed the possibility of LS rooting for nursery operations; prune height alone was statistically significant for rooting ability of LS cuttings. During the first growing season, both growing environment and container size were significant in encouraging shoot growth post-rooting. After the first growing season and subsequent overwintering, survival rates of rooted cuttings were 75% overall. In this study, the smallest container size used during the first growing season was the most beneficial to rooted cutting growth based on final attribute measurements of height, root collar diameter (RCD), root mass, shoot mass, and root-to-shoot ratio, all of which have been proposed to be good indicators of field performance. Extending the growing season immediately after rooting significantly affected RCD and root mass measurements as well.

Seasonal adaptations have evolved in animals and are believed to promote survival and reproduction through yearly changes in the environment. These seasonal responses are physiological, morphological, and behavioral and are coordinated by day length (photoperiod) information in non-tropical rodents. In the laboratory, short days promote a winter phenotype, whereas long days promote a summer phenotype. This dissertation was designed to examine the effects of photoperiod on adult plasticity of physiology, brain, and behavior in male white-footed mice (Peromyscus leucopus) that may represent seasonal adaptations to the changing environment. Recent evidence suggests that significant plasticity occurs in adult systems including the central nervous system. The first studies examined the effects of photoperiod on angiogenesis in the periphery and the brain. Short days altered expression of angiogenesis genes in testes and brain and decreased cerebral blood flow compared with long days. The next set of studies was based on the finding that short days decreased brain mass and impaired spatial learning and memory compared with long days. The hippocampus is involved in spatial learning and memory and is one of the few brain regions associated with significant adult plasticity. Short days decreased hippocampal volume, altered hippocampal dendritic spine density, modified the effects of learning experience on neurogenesis, and dampened long-term potentiation compared with long days. Photoperiod alters concentrations of various hormones, some of which affect the hippocampal morphology and function. Testosterone reversed the short-day impairment of spatial learning and memory indirectly of hippocampal steroid receptors, but did not affect long-day performance. Corticosterone altered spatial learning and memory depending on photoperiod and duration of exposure and short days altered the hypothalamic-pituitary-adrenal axis compared with long days. The effects of photoperiod on the other brain region characterized by significant adult plasticity, the olfactory bulbs, and olfaction was examined. Neurogenesis increased in the caudal olfactory bulbs of short-day mice. Finally, I tested the effects of photoperiod and testosterone on depressive- or anxiety-like behaviors and observed few differences. Taken together, photoperiod appears to coordinate adult plasticity within the endocrine and nervous system that result in behavioral changes hypothesized to be adaptive to a seasonally-changing environment.

The success of the yellow perch Perca flavescens aquaculture industry depends largely on increasing growth efficiency to reduce production costs, and increasing fingerling availability for year-round production. Two characteristics of yellow perch biology pose challenges toward these goals. 1) Yellow perch exhibit sexually dimorphic growth, detected concurrently as the fish enter puberty, with many slower-growing males that may never achieve market size. These growth differences between genders also promote a situation in which cannibalism can quickly deplete a perch population reared under intensive conditions. 2) Yellow perch spawn once a year in the spring, and most producers are limited to a production schedule centered on this event. Two sets of studies were conducted to evaluate culture methods for the inhibiting maturation for promotion of somatic growth, and evaluate the practice of 'cold-banking' for off-season fingerling availability. Four combinations of constant and natural photothermal regimes were used to impose environmental conditions on juvenile fish that inhibited maturation and promoted somatic growth. Immature perch were expected to demonstrate reduced dimorphic growth pattern and overall growth enhancement. Results indicate that a) constant temperature is the strongest promoter of overall growth regardless of photoperiod; b) constant photoperiod also promotes overall growth regardless of temperature; c) natural (decreasing) photoperiod initiates maturation in yellow perch regardless of temperature; d) natural temperature is required for late maturational processes; e) constant temperature and constant photoperiod together confer the best overall growth performance, fully inhibit maturation in both genders, and suppress a sexually dimorphic growth pattern up to 53g average weight. Two studies were conducted evaluating the growth of cold-banked fish and costs associated with commercial application of this culture technique. Perch banked at 10 C on a reduced ration for 60 and 180 days were reared up to 240 and 120 days post-banking respectively. Post-banking culture conditions (and 0 days banked control conditions) were satiation feeding at 22 C. Growth, SGR, and feed conversion did not differ among the 60-day, 180-day treatments and controls up to 120 days post-banking. Growth, specific growth rate (SGR), and feed conversion did not differ between controls and 60-day treatment up to 240 days post-banking. The additional cost of cold banking yellow perch on a commercial scale was evaluated at Deca-J Farms (Clinton, NC) for 4 groups of fish cold banked (11 C, restricted ration) for four different durations (98, 169, 238, and 308 days). Growth was compared to controls (0 days banked, 24 C, satiation feeding) over 60 days post-banking. Growth observations indicated that perch grew during banking and post-banking SGR was negatively correlated with banking duration. Cold banking at Deca-J farms cost an estimated additional $0.15 per fish, with $0.09 per fish attributed to maintenance (variable) costs.

Japanese stiltgrass is a nonnative invasive grass that occurs in many habitats and is a management concern throughout the eastern United States. Experiments were conducted to address biological and ecological considerations for effective management of Japanese stiltgrass and restoration of native plant communities. Conventional management recommendations emphasize Japanese stiltgrass removal in autumn prior to flowering. Investigations of Japanese stiltgrass reproductive biology were conducted to assess biological considerations that impact such management strategies. Japanese stiltgrass was grown in long day growth chambers (26/22 C) and plants were moved 2, 6, or 10 wk after germination to short day growth chambers (26/22 or 22/18 C). All plants exposed to short days flowered, while under long days no plants flowered. No difference in inflorescence number or shoot dry weight was observed between the two temperature regimes. Japanese stiltgrass populations from North Carolina, Pennsylvania, and Virginia flowered similarly in response short day conditions. Furthermore, inflorescences from natural stands of Japanese stiltgrass were harvested four times; one raceme branch beginning to emerge through the leaf sheath, one raceme branch fully elongated and a second branch visible, fully expanded inflorescence, and in concert with natural dispersal. Following > 90 d storage, seed germination from each harvest averaged 13, 51, 95, and 100%, respectively, suggesting management designed to prevent seed production should be implemented before flowering. In forested areas, herbicides that selectively control Japanese stiltgrass while preserving native vegetation may be desired. The efficacy of three selective postemergence herbicides (fenoxaprop-P, imazapic, and sethoxydim) applied early, mid-, or late season were compared. The herbicides, averaged across application timings, controlled Japanese stiltgrass 83 to 89% and reduced seedhead production 79 to 94%. Seedling emergence was reduced 89, 70, and 78% in spring 2004 by fenoxaprop-P, imazapic, and sethoxydim, respectively, applied in 2003. Additionally, fenoxaprop-P or sethoxydim applied twice (4 wk interval) at half-label or full labeled rates controlled Japanese stiltgrass, providing 92% reduction in biomass and 97 to 98% seedhead reduction. Experiments were conducted to determine if Japanese stiltgrass seeds possess dormancy and if so, to determine conditions required to overcome dormancy and for successful germination. Mature Japanese stiltgrass seeds were collected in autumn 2002 and 2003, cold stratified moist at 4 C or stored dry at 21 C, 0 to 90 d. After storage, seeds were incubated in petri dishes in alternating or constant temperatures and exposed to 14 h or 0 h (total dark) photoperiod. Less than 1% of seeds stored 0 or 15 d germinated while > 95% of seeds stored 90 d germinated, suggesting primary innate dormancy upon natural dispersal. A three-year experiment was conducted in two forest sites to document the ecological impacts of conventional and alternative selective management on Japanese stiltgrass populations and native flora recruitment and establishment. Conventional management treatments included hand-pulling, mowing, or glyphosate (1.1 kg ai/ha) applied in autumn compared to hand-pulling or fenoxaprop-P (0.19 kg ai/ha) applied as needed throughout the season. All management treatments significantly reduced Japanese stiltgrass cover and seed bank over time compared to no management and decreases in relative Japanese stiltgrass cover and seed bank populations were greater in 2004 than 2003, after two seasons of management. However, selective management was more suitable than nonselective management or no management for recruitment and re-establishment of native plants and increasing overall species richness. Additionally, relative cover of other exotic plants decreased 6% over time, suggesting that removal of Japanese stiltgrass did not increase invasion of other exotic plant species.

Lucerne (Medicago sativa L.) was found to be a more productive dryland forage option than chicory (Cichorium intybus L.) or red clover (Trifolium pratense L.). This was concluded from superior annual dryland yields of 20 t DM/ha from lucerne compared with 14-16 t DM/ha for chicory and red clover. This yield advantage was achieved by higher growth rates during both cool spring/autumn periods and dry summer periods. Lucerne was also the most persistent species maintaining a botanical composition of 94% six seasons after establishment, compared with 65% for chicory and 0% for red clover. All three species had similar herbage quality (25% crude protein, 11.5 MJ ME/kg DM) and grazing stock consumed 30% more protein and energy from lucerne than chicory or red clover crops. The superior lucerne production during dry periods was due to increased water extraction up to 2.8 m depth, compared with ~1.9 m for chicory and red clover. All three crops displayed a top-down perennial water extraction pattern with an extraction front velocity of ~15 mm/day. Depletion of available water capacity in each layer of the soil profile was exponential following the arrival of the extraction front. A detailed examination of lucerne physiology was conducted to understand seasonal variation, and the effects of water shortages on forage yield. Total DM production under non-water and non-temperature limiting conditions was related to total intercepted radiation. The total radiation use efficiency (RUE) was found to be 1.6 g/MJ. However, there was a seasonal change in DM partitioning between shoot and perennial organs (roots and crowns) and its influence on forage yield was quantified by converting total RUE to shoot RUE. The shoot RUE was 1.3 g/MJ in September, gradually decreased to a constant 1.0 g/MJ from mid-December late-January and then abruptly decreased to 0.6 g/MJ in March/April. Temperature also influenced shoot production and this was quantified by multiplying RUE by a linear factor that declined from unity at a mean regrowth cycle air temperature of 18°C to zero at 0°C. Seasonal changes in radiation interception were quantified by studying the influence of temperature and photoperiod (Pp) on the components of leaf area index (LAI) expansion. Specifically, main-stem node appearance was linear in response to Tt and the phyllochron was 37±7°Cd for from August-January. However, phyllochron increased to 60 °Cd when the Pp on the day 150 °Cd before the first node decreased to 16 h (24 January). Continued decrease in Pp gave a 5.6 °Cd/h Pp reduction in phyllochron returning, it to 37±7 °Cd at a Pp of 13.5 h (15 March). There was a poor relationship between main-stem node appearance and LAI expansion, suggesting branching and leaf expansion have different seasonal responses to environment. Water shortages were quantified by crop transpiration (ET) relative to the crops ET demand. Crop ET was calculated from water balance by removing evaporation losses from the soil and outer canopy. Crop ET demand (EPT) was calculated from Penman evapotranspiration potential (EP) multiplied by crop cover and a calibration coefficient (0.86), determined by regressing the ET of irrigated crops against EP. The RUE and LAI of dryland crops was expressed as a fraction of irrigated crops (fD/I) to quantify the effects of water stress. The LAI expansion of lucerne was the most sensitive process with fD/I of 1.0 at an ETIEPT of 0.97 decreasing to 0.1 at an ET/EPT of 0.22. There was a 1: 1 relationship between the fD/I of RUE and ET/EPT. It is concluded that the improved understanding of lucerne environmental responses presented in this thesis must be considered when examining yield variability of lucerne.

To develop reproductive control technologies for wild stoat (Mustela erminea) populations there is a requirement for year-round breeding in captive animals. This study tested the hypothesis that a long-day photoperiod applied to stoats during winter months would stimulate reproduction in these animals. Adult stoats (12 males and 12 females) were captured from the wild during summer and autumn. From 14 May half of the animals were subjected to artificial lighting, which reached 16 h/day on 30 June and continued at this daily duration until November. Controls experienced natural changes in daylight. Faecal samples were collected for hormone analysis. Vaginal cytology and physical changes associated with oestrus were monitored in females and scrotal size was monitored in males. In early spring months there was a high incidence of keratinised vaginal epithelial cells and signs of oestrus in light-treated females that were absent in the controls. Mean faecal oestradiol concentration was highly variable (1- 20 ng/g dry weight), and was not different between treated and control groups of females. Light-treated males had higher (P < 0.05) mean faecal testosterone concentrations than controls ( e.g. 272 ±. 32 versus 163 ±. 31 ng/g dry weight respectively, on 4 September) and larger (P < 0.05) scrotal dimensions in August. These results show that stoats are amenable to photoperiodic stimulation of breeding activity and provide some of the first reproductive endocrinology data for this species in New Zealand.

Grown for its distinctive foliage, Strobilanthes dyerianus is a popular bedding and container plant. A problem in production is that over-wintered stock plants often flower. Once the plant becomes reproductive, stem elongation slows and floral buds arise from every node, rendering the plants useless for propagation. The objectives of this research were to examine the effectiveness of manipulating environmental factors and the application of ethephon on preventing floral bud initiation, as well as determine optimal nitrogen rate for stock plant culture. The first experiment was performed in a glass greenhouse and ran 11 weeks, utilizing 8 h, 10 h, 12 h and a 4 h night interruption photoperiod treatments to determine critical photoperiod. None of the photoperiod treatments were significant for inhibiting flowering and there was a positive correlation between plant size and flowering. A second experiment was performed in growth chambers to create three photoperiods (8 h, 12 h and 16 h) with two temperature regimes (24C day/ 21C night and 17C day/ 14C night) for a total of six treatments. Neither photoperiod nor temperature inhibited bud initiation, and there was no correlation between plant size and flowering. The third experiment examined potential use of ethephon to maintain vegetative plants. Six rates of ethephon (0, 150, 300, 450, 600 or 750 mg·Lˉ¹) were applied at three frequencies (weekly, biweekly and monthly) over an eleven week period. Floral initiation was not totally inhibited, but flowering was highly correlated to plant size. Additionally, plant growth response to nitrogen was examined to determine the optimal rate for stock plant production. Plants were treated with 0, 100, 200, 300 or 400 mg·Lˉ¹ N from a 15 N – 2.2 P – 12.4 K fertilizer at each irrigation for eight weeks. There were no differences among plant quality ratings for plants receiving 100, 200, 300 or 400 mg·Lˉ¹ N, and plants grown with 200 mg·Lˉ¹ N had the largest leaf area and shoot dry weight.

This study tested the hypothesis that a split night photoperiod would induce spring-like growth rates during winter like that seen in sheep. The study was run from 6 May until 18 September, 2002, where weaner red deer stags were housed inside up until the last 2 weeks of this period when they were returned to pasture. Whilst inside the stags (n=10) were subjected to either a long day photoperiod (16L:8D, where L = h of light, D = h of darkness), natural daily photoperiods (short days experiencing the normal seasonal pattern of daylight) or natural daily photoperiods with a 1 h period of light during the dark phase (split night). During the first 12 weeks of the study live weight gain was comparable in all groups (approx 150g/day). However growth of long day treated animals began to surpass that of the other treatments so that by 6 August mean live weight of the long-day treated group was significantly (P

Continuous organogenesis in plants is possible because of the specification of stem cells at the meristems. Genetic studies using Arabidopsis thaliana have isolated a multitude of mutants showing defects in meristem development. Identifying the mutated genes and elucidating how their products interact will be the key to understand how meristems function. Mutations at the CLAVATA (CLV) 1, CLV2 and CLV3 loci lead to the accumulation of stem cells in the shoot and flower meristems. CLV1 and CLV3 genes are specifically expressed in these meristems and encode a receptor-like kinase (RLK) and a putative ligand, respectively, indicating that these genes are involved in signal transduction events regulating stem cells. The CLV2 gene regulates both meristem and organ development. Genetic studies suggested that CLV1, CLV2 and CLV3 genes function in the same pathway for meristem development. I have isolated the CLV2 gene and found it encodes a receptor-like protein (RLP), with a presumed extracellular domain composed of leucine-rich repeats (LRRs), but with a very short predicted cytoplasmic tail. In addition, the chromosomal region in which CLV2 is located contains an extremely high rate of polymorphism. CLV2 is the first RLP shown to regulate development in plants. CLV2 mRNA is expressed in many tissues including the shoot apex and flowers. These results are consistent with a model in which CLV2 forms a signaling complex with CLV1 and CLV3. Analysis of predicted sequences show that plant RLPs and RLKs have several structural features for potential protein-protein interactions. I have shown that clv1, clv2, and clv3 flower defects are suppressed under short day (SD) growth condition, and that this is a true photoperiodic response regulated by genes in the long day (LD)-dependent flowering promotion pathway in Arabidopsis. Inactivation of the LD pathway is sufficient to suppress clv2 under LD and activation of the LD pathway restores the clv phenotypes under SD. These results reveal a novel role of photoperiod in flower meristem development in Arabidopsis. Analysis of the dominant negative alleles of clv1 under SD suggest that the SD suppression may be mediated by a CLV1-like protein specific to the flower meristem.

Argentine stem weevil (ASW) Listronotus bonariensis (Kuschel) is an introduced species that attacks improved graminaceous pasture and crop throughout much of New Zealand. It is recognised as the most important arthropod pest of ryegrass in this country. While most research over the last 10-15 years has focused on plant resistance and biological control, little consistent information is available on aspects of the biology of the weevil. The main aim of this research was to investigate, in detail, the biology of ASW considering particularly preimaginal and imaginal development. A method for ASW rearing under laboratory conditions using natural food sources was developed. The effect of adult population density on egg laying and other reproductive variables was assessed in the laboratory. A weak trend with adult density was shown for the variables recorded, with females laying fewer eggs, having shorter mean ovariole length, fewer oocytes in their ovarioles and fewer eggs in their calyces at higher densities. Eggs were laid in ryegrass sheaths and leaves; the percentage of eggs laid in the leaves was not significantly different between adult population densities. The fecundity, fertility of females and the viability of eggs laid in a "mass" rearing situation was also assessed. A mean of 384 eggs were laid per female with a mean of 4 eggs per day. Mean fertility was 97% and 95% of the eggs were viable. The time to peak oviposition was 19 days and 50% of the eggs had been laid 35 days after emergence. A method for egg incubation, larval rearing and prepupal and pupal extraction under laboratory conditions was established. A mean of 75% of prepupae plus pupae were recovered from the rearing pots and on average, 92% developed into adults. The fecundity, fertility, longevity and other development parameters of oligopausing and non-oligopausing adults were assessed at 20°C. The average duration of the preoligopause, oligopause and postoligopause periods was 76.2, 538.0 and 96.3 days, respectively. Longevity averaged 590.7 days for females and 617.2 days for males, with no significant difference between the means. An average of 150.8 eggs were laid per female. The average number of eggs laid per female, per day was 1.3 during preoligopause and 1.1 during postoligopause. On average, 56.4% of the eggs were laid during the preoligopause and 43.6% during the postoligopause period. Fertility and viability of eggs averaged 96.2% and 94.2% during preoligopause, and 99.4% and 96.9% during postoligopause. Couples were reared at various temperatures to determine any effect on fecundity and other reproductive and development parameters. The mean total number of eggs laid per female ranged from 150-263 at 35 and 25°C, respectively. The time to peak oviposition after emergence was 24-25 days at 20°C, 20-21 days at 25/10°C (alternating temperatures), 14-15 days at 25°C and 10-11 days at 30 and 35°C. Fifty percent of the eggs had been laid 56-67 days, 44-45 days, 32-33 days, 20-21 days and 14-15 days after emergence at 20, 25/10, 25, 30 and 35°C, respectively. No oviposition was observed at 40°C and only 18% of the females laid eggs at 15°C. The mean percentage of fertile and viable eggs laid at 35°C (66.9 and 61.6%, respectively) was significantly lower (P < 0.05) than at the other temperatures (> 95%). Mean longevity for non-oligopausing females ranged from 41.4-144 days at 35 and 20°C, respectively. The development time tended to be longer at a constant temperature of 20°C than at alternating temperatures of 25/10°C with the same means, except for the postoviposition period. There was a significant effect (P < 0.05) of temperature on the occurrence of oligopause in the laboratory; the higher the temperature, the lesser incidence of oligopause was observed. Oligopausing females and males were dissected to check their reproductive condition. A regression was fitted to the development rate (1/day) of the preoviposition period versus temperature and was well described by a linear regression model (r²= 0.99). The lower threshold temperature for the preoviposition period was 12.1°C and 141 day-degrees were required before oviposition. The effect of photoperiod and its interaction with temperature on oviposition was investigated using laboratory-reared adults and overwintering field-collected adults. The mean duration of the preoviposition period of laboratory-reared females was not significantly different (P> 0.05) between photoperiods within the same temperature, but was significantly different (P < 0.05) between temperatures. There was a significant effect (P < 0.05) of photoperiod on the mean number of eggs laid per day by laboratory-reared females, but there was no significant effect (P> 0.05) for overwintering field-collected females. The effect of temperature on the development and survival of ASW preimaginal stages was assessed under laboratory conditions. No hatching occurred at 10 and at 40°C and no survival was observed after the second instar for larvae maintained at those temperatures from the beginning of the first instar. Between 15 and 35°C the only significant mortality observed was in the prepupal stage at 35°C. The development time for the preimaginal stage was significantly shorter (P < 0.001) as temperature was increased and ranged from an average of 19.3 days at 35°C to 115.8 days at 15°C. The development time of preimaginal stages was significantly longer (P < 0.05), except for the prepupal stage, at a constant temperature of 20°C than at an alternating temperature of 25/10°C, both with the same mean temperature. ASW female pupae were significantly heavier (P < 0.05) than male pupae at all temperatures, whereas there was no significant influence (P > 0.05) of temperature on the average weight of female or on the average weight of male pupae, except that at 35°C female pupae were significantly heavier (P < 0.05) than at 30°C. The sex ratio at each temperature was not significantly different (P > 0.05) from 1:1. Based on the development time at constant temperatures, the lower threshold temperatures (T₀) and the day-degrees (DD) requirements were calculated for all preimaginal stages and validated at varying temperatures in the glasshouse and in the laboratory. Regressions were fitted to the development rates (1/days) versus temperature for each preimaginal stage and period, and were well described by a linear regression model as indicated by high r² values (> 0.97). There was no significant difference (P> 0.05) between T₀ of the preimaginal stages, larval period and preimaginal period, which ranged from 10.7°C for the 3rd instar to 13.3°C for the 1st instar. The average DD requirements for most of the stages at varying temperatures in the glasshouse and in the laboratory were lower than the DD requirements estimated at constant temperatures, however, most of them fell within the confidence intervals given for the DD requirements at constant temperature. The T₀ and DD requirements to complete a generation were calculated and the number of generations possible at Lincoln (Canterbury) was estimated to be 1.7. The implications of these findings are discussed.

The Tasmanian lacewing (Micromus tasmaniae Walker) is a common aphid predator and is known from Australia and New Zealand. It is a cold-adapted insect and there is no diapause in any life cycle stage. A review of the literature on Tasmanian lacewing and cabbage aphid, Brevicoryne brassicae (L.) reveals that there is a need to determine the rate of predation by lacewing larvae under different combinations of physical environmental factors, e.g., temperature, humidity and photoperiod before this predator can be exploited as a biocontrol agent to control cabbage aphids in cabbage crops. The rate of predation, (e.g., prey consumption per larva I day) and the total number of prey consumed per larva during the period of larval development was determined under different constant temperatures, fluctuating daily temperatures and different constant relative humidities. The influence of temperature and humidity at constant photoperiod (16L : SD) on larval mortality, duration of larval development and physiological time required to complete larval development has been determined in this study. In addition the total mean number of prey consumed by Tasmanian lacewing larvae during the period of larval development using a range of arenas with different internal surface areas was also investigated. The main findings of this study were that the rate of predation and the rate of larval development was linear in the temperature range 10°C - 25°C. The rate of predation under fluctuating daily temperature was higher than that of the corresponding mean temperature. Humidity also influenced the larval development period and the physiological time required to complete development. Larvae took the least time to develop at 60% RH but this increased with increasing relative humidity from 60% RH to 90% RH. A rise and fall of predation rate was noticed with increasing humidity. Arenas with different internal surface areas showed an impact on the rate of predation of cabbage aphids by Tasmanian lacewing larvae. The mean total predation increased with increasing surface area up to a certain level (e.g., 300 ±25 cm² ) and thereafter remained unchanged. Discussion is provided on the number of Tasmanian lacewing larvae required to be released in the field to achieve successful biological control of cabbage aphids as well as future research required commercialise this biological control agent.

This thesis is aimed at improving techniques for induced maturation and artificial fertilization of Morone. Chapter 1 is a study of hybrid bass (n = 73) exposed to 6- or 9-month photothermal cycles which were formed by excising summer or summer and fall photothermal conditions from an annual regime. Shortened cycles abbreviated the interval between spawning and recrudescence, and advanced maturation by five and four months, respectively. All females developed functionally mature oocytes and ovulated after hormone injection. Maximum testicular sizes were similar and occurred after similar periods (153-199 d). Milt was produced in most (> 90%) males. Some milt characteristics differed among males in the three cycles, but fertility was similar. Findings suggest that excised cycles can advance maturation in hybrid bass, permitting off-season spawning. In Chapter 2, effects of stripping frequency (3 times in 7 days) on semen quality was determined by measuring concentration, motility, osmolality, pH and sugars of milt. Male and female effects on fertility variation were measured by crossing all males and females. Sperm concentration was estimated by spermatocrit, ultraviolet and fluorometric absorbance. Motility (58 sec) and milt pH (8.44) were lowest (p < 0.05) after the third stripping. Concentration (1.76 x 10⁹ cells mL⁻¹) and osmolality also decreased with stripping frequency, but inconsistently among males. Fertility was highest during the second stripping and was correlated with milt pH (r = 0.762). Fertility variation was caused by females (p = 0.001). Results provide the first characterization of Morone semen quality. They suggest that frequent stripping alters milt parameters, that females account for most fertility variation, and that sperm concentration can be estimated reliably by spermatocrit.

Thirty Dorset Down x (Border Leicester x Corriedale) female lambs were used to determine the effect of two fixed photoperiods (16L:8D and 8L:16D) on growth rate and body composition. Pair-feeding and controlled conditions were maintained for 91 days. Comparative slaughter procedures were used to determine changes in body composition. No significant treatment differences were detected (P > 0.05). However trends observed indicate the existence of a photoperiod effect on growth. Sheep in the long photoperiod (16L:8D) gained bodyweight slightly faster and had a higher protein content of gain.

A questionnaire on St. John's wort was presented to all South Island Noxious Plants Officers. Responses show that St. John's wort biocontrol is perceived as only partially effective. Areas of serious infestation remain, but no alternative to biocontrol is practical. By means of insecticide interference with biocontrol at an experimental site near Lake Tekapo the findings of Syrett and Hancox (1985) were confirmed, namely that biocontrol by Chlysolina hyperici reduced St. John's wort shoot numbers during the important spring stock grazing period. After spring the beetles had no measurable effect. Food consumption of laboratory-reared C. hyperici larvae was measured by weighing food sprigs before and after feeding periods. The fourth (last) instar larvae consumed 82% of the total food consumed during the larval period and showed the highest rate of consumption. Laboratory experiments using controlled daylength cabinets showed that whereas moistening of aestivally diapausing C. hyperici does not break their diapause, a change in day length from sixteen to eight hours of daylight does break diapauses, initiating heavier feeding, reproductive maturation, mating and oviposition. The necessity for "photoperiod matching" of biocontrol agents is emphasized. Measurements of field-collected insects' flight muscles showed that after peaking in late summer (early in aestivation) the muscles shrank during autumn as the insects became reproductive. It is argued that the benefit of such shrinkage lies in the mobilization of nutrients rather than in vacation of body space or enforcement of winter flightlessness. Quarantining of the host plant's oil-soluble toxin hypericin in fat or oil globules may have led to a reliance by C. hyperici on muscle tissue as a "negotiable" nutrient store.

The effects of a shortened daily photoperiod or daily, afternoon injection of melatonin on the the onset of the breeding season and other seasonal changes associated with the transition from summer to winter were examined in pubertal red deer hinds. Eleven pubertal hinds and four non lactating adult hinds were randomly allocated to three treatment groups. Control hinds (Group 1) were maintained under natural photoperiodic conditions. From 8 January until 31 March one group of hinds (Group 2) was subjected to a daily photoperiodic regime of eight hours light to sixteen hours dark (8L:16D) by keeping them in a darkened room from 1600h until 0800h the following day and another group of hinds (Group 3) was given an intramuscular injection of 3 mg melatonin in safflower oil daily at 1600h. Silent ovulations seemed to occur in many of the hinds, but this study did not establish whether the treatments utilised here had any effects on these silent ovulations. Earlier calving may give rise to calves with a body weight advantage over those born later, at least until their first winter. Mating and calving data from the adult hinds was too limited to indicate whether the advancement of these events achieved in the yearlings could readily translated to older animals.