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"Adult neurogenesis, i.e., the production of new neurons in the adult brain, has been studied intensively in the past years, both in humans and in animal models, as the understanding of this process can have major clinical implications. The study of neurogenesis in fish has been receiving more attention as, unlike mammals, they possess remarkably high levels of adult neurogenesis and a high capability for neuronal regeneration and replacement where neuronal death has occurred. Less is known, however, on the importance of adult neurogenesis for behavioural plasticity, i.e., for the capacity to change behaviour according to context. As a product of the brain, behaviour relies on functional neuronal networks and it may be expected that more permanent changes in behavioural states imply structural reorganization of neuronal circuits, with the integration of new neurons. Interestingly, the high level of brain plasticity of fish is paralleled by a high degree of behavioural plasticity, with many examples of species that change, either reversibly or irreversibly, their behavioural phenotype during their lifetime, as illustrated by species with functional sex-change and alternative reproductive phenotypes. Flexibility in behaviour may thus require a reorganization of neuronal networks underlying these behaviours with recruitment of new neurons. In this thesis, the link between brain and behavioural plasticity was studied in a small marine fish that inhabits the Mediterranean and adjacent Atlantic coasts, the peacock blenny Salaria pavo. In this species, males adopt nests in rock crevices and attract females into the nest for egg laying, with the male taking care of the eggs until hatching. In some populations, a scarcity of nest sites drives smaller and young males to adopt an alternative reproductive tactic to reproduce. These “sneaker” males mimic the females’ morphology and reproductive behaviour in order to illude the larger nesting males and parasitically fertilize eggs during mating events. Sneaker males later transition into the nesting male phenotype, and this major behavioural transformation in the same animal, first courting males and afterwards courting females, may imply significant reorganization of brain areas associated with reproductive behaviour. During the study, a brain atlas for the species was developed and the main cell proliferation regions, i.e. niches of stem cells birth that may differentiate into cells of the nervous system, characterized. Proliferative areas were observed throughout the whole brain and paralleled the pattern described for other teleosts. Proliferative cells were abundant namely in areas like the olfactory bulbs (granular and glomerular), the anterior subdivision of the dorsomedial telencephalon (DMa), the dorsal and ventral part of the ventral subdivision of the dorsomedial telencephalon (DMvd and DMvv), the dorsal part of the dorsal subdivision of the dorsomedial telencephalon (DMdd), the posterior subdivision of the dorsolateral telencephalon (DLp), the posterior zone of the dorsal telencephalic area (DP), the preoptic area (POA), the dorsal, supracommissural and ventral nucleus of the ventral telencephalic area (Vd, Vs and Vv), the optic tectum and its periventricular grey zone (TeO and PGZ), the ventral zone of the periventricular hypothalamus (Hv), the cerebellum, mainly the molecular layer (CCeM) and the caudal lobe (LCa). A study of the brain nuclei activated during female courtship events using immediate early-genes suggested that some of the areas of the social behaviour network (SBN), a set of brain nuclei underlying the expression of social behaviour across vertebrates, are implicated in female courtship, in particular nuclei in the ventral telencephalic regions. This was followed by an experiment to investigate the possible link between cell proliferation and male tactic switch. Nest availability was manipulated to allow a fraction of sneaker males to adopt a nest and start the transition to nesting males. Ten days after the experiment, some of the smaller males had indeed started switching into nesting males, adopting a nest and starting to develop male secondary sexual characters. The pattern of brain proliferation was studied in these fish to try to confirm that the irreversible behavioural transition would be associated with the reorganization of brain nuclei, assuming that cell proliferation relates to neurogenesis and structural reorganization. Transitional males had elevated cell proliferation levels, as compared to males that remained sneakers, in the dorsolateral anterior and posterior telencephalic regions, thought to be homologous to the hippocampus in mammals. Cell proliferation levels were generally elevated in ventral and ventromedial telencephalic nuclei in both sneakers and transitional males, as compared with nesting males and females, areas considered to be homologous to nuclei of the amygdaloid complex of mammals. There was large variation in proliferation levels within transitional males, and in particular one male more advanced in the transition had higher numbers of BrdU-positive cells than the others. This suggests that a longer time-window for detecting the peak in brain cell proliferation associated with tactic transition in some fish may have been needed. Overall, the study supports the hypothesis that behavioural transition in males of this species is paralleled by an increase in cell proliferation in nuclei potentially relevant for the expression of reproductive behaviours, and establishes the peacock blenny as a new relevant model for the study of neuronal plasticity in vertebrates."

A combination of assessment, operational forecast, and future perspective was thoroughly explored to provide an overview of the existing air quality problems in Macao. The levels of air pollution in Macao often exceed those recommended by the World Health Organization (WHO). In order for the population to take precautionary measures and avoid further health risks during high pollution episodes, it is important to develop a reliable air quality forecast. Statistical models based on linear multiple regression (MLR) and classification and regression trees (CART) analysis were successfully developed for Macao, to predict the next day concentrations of NO2, PM10, PM2.5, and O3. Meteorological variables were selected from an extensive list of possible variables, including geopotential height, relative humidity, atmospheric stability, and air temperature at different vertical levels. Air quality variables translate the resilience of the recent past concentrations of each pollutant and usually are maximum and/or the average of latest 24-hour levels. The models were applied in forecasting the next day average daily concentrations for NO2 and PM and maximum hourly O3 levels for five air quality monitoring stations. The results are expected to support an operational air iv quality forecast for Macao. The work involved two phases. On a first phase, the models utilized meteorological and air quality variables based on five years of historical data, from 2013 to 2017. Data from 2013 to 2016 were used to develop the statistical models and data from 2017 was used for validation purposes. All the developed models were statistically significantly valid with a 95% confidence level with high coefficients of determination (from 0.78 to 0.93) for all pollutants. On a second phase, these models were used with 2019 validation data, while a new set of models based on a more extended historical data series, from 2013 to 2018, were also validated with 2019 data. There were no significant differences in the coefficients of determination (R2) and minor improvements in root mean square errors (RMSE), mean absolute errors (MAE) and biases (BIAS) between the 2013 to 2016 and the 2013 to 2018 data models. In addition, for one air quality monitoring station (Taipa Ambient), the 2013 to 2018 model was applied for two days ahead (D2) forecast and the coefficient of determination (R2) was considerably less accurate to the one day ahead (D1) forecast, but still able to provide a reliable air quality forecast for Macao. To understand if the prediction model was robust to extreme variations in v pollutants concentration, a test was performed under the circumstances of a high pollution episode for PM2.5 and O3 during 2019, and a low pollution episode during 2020. Regarding the high pollution episode, the period of the Chinese National Holiday of 2019 was selected, in which high concentration levels were identified for PM2.5 and O3, with peaks of daily concentration for PM2.5 levels exceeding 55 μg/m3 and the maximum hourly concentration for O3 levels exceeding 400 μg/m3. For the low pollution episode, the 2020 period of implementation of the preventive measures for COVID-19 pandemic was selected, with a low record of daily concentration for PM2.5 levels at 2 μg/m3 and maximum hourly concentration for O3 levels at 50 μg/m3. The 2013 to 2018 model successfully predicted the high pollution episode with high coefficients of determination (0.92 for PM2.5 and 0.82 for O3). Likewise, the low pollution episode was also correctly predicted with high coefficients of determination (0.86 and 0.84 for PM2.5 and O3, respectively). Overall, the results demonstrate that the statistical forecast model is robust and able to correctly reproduce extreme air pollution events of both high and low concentration levels. Machine learning methods maybe adopted to provide significant improvements in combination of multiple linear regression (MLR) and classification and regression vi tree (CART) to further improve the accuracy of the statistical forecast. The developed air pollution forecasting model may be combined with other measures to mitigate the impact of air pollution in Macao. These may include the establishment of low emission zones (LEZ), as enforced in some European cities, license plate restrictions and lottery policy, as used in some Asian, tax exemptions on electric vehicles (EVs) and exclusive corridors for public transportations. Keywords: Air pollution; Particulate Matter; Ozone; Macao; Statistical air quality forecast; Pollution episodes; Chinese national holiday; COVID-19

Aggression clearly has an adaptive value as it is necessary to secure resources for survival, growth, and reproduction. The Siamese fighting fish, Betta splendens, have endured a prolonged process of artificial selection for winning paired-fight contests across Southeast Asia, resulting in strains of short-fin aggressive “fighters”. Across centuries, Thai breeders have been selecting these strains by discarding loser batches and allowing winner batches to breed, claiming that they are significantly more aggressive than wild-types. This natural experiment provides a powerful context to investigate the biological basis of aggressive behaviour in fish, the topic of this thesis. To study aggression, it is important to validate and standardize behavioural assays appropriate for the species under study. Further, different aggression-eliciting stimuli, such as live opponents, 3D models, video playback, or mirror images, may elicit non-equivalent behavioural and physiological responses. For B. splendens, in particular, quantifying aggression from live fights is not ethically acceptable as the high levels of aggression of this species usually result in injuries or even death of the opponent. In Chapter II, it was shown that mirror images elicit very similar aggressive displays and endocrine responses to an interacting opponent behind a transparent partition, validating the use of this test to measure aggression in this model species. Further, it was shown that circulating levels of both androgens (11-ketotestosterone and testosterone) and corticosteroids (cortisol) increased in response to the aggression challenge, even in the absence of conflict resolution, questioning the role of these hormones during present and future aggressive contests. Using the previously validated mirror assay and also tests with live conspecifics, we assessed the impact of selection for winning by comparing, in Chapter III, male and female aggressive behaviour of lab-raised fighter and wild-type strains. The hypothesis that selection for male winners enhanced aggressive displays was confirmed, suggesting that the duration and frequency of threat and attack behaviour correlates with winning probability. However, females of the fighter strain, which are not selected for fights, were also more aggressive than wild-type females. This suggests that male and female aggression share common genetic pathways and physiological mechanisms and raises the possibility that selection for alleles that favour male aggression may have promoted intersexual genetic conflict in this species. After confirming the expected differences in aggressive behaviour between fighter and wild-type fish, the following question was whether endocrine systems, in particular those previously shown to respond to aggression, could have been targeted by the selection process. From previous studies in fish and other vertebrates, it was hypothesised that selection for winners could have increased constitutive levels of androgens or led to an enhanced androgen response to a social challenge. However, in chapter IV, it was shown that levels of 11-ketotestosterone and its response to aggression was similar in males of both strains, questioning the role of androgens in the modulation of aggression in B. splendens. On the contrary, constitutive levels of cortisol and the response of this hormone to an aggression challenge were higher in wild-type compared with fighter fish, supporting previous findings that associated high aggression with a blunted cortisol response. Overall, results from Chapter IV suggest that selection for winning had a stronger impact on the hypothalamus-pituitary-interrenal axis than in the hypothalamus-pituitary-gonadal axis. My results support the assumption of the “Challenge Hypothesis” proposed by John Wingfield and collaborators in 1990 to explain the relationship between androgens and aggression, according to which androgen levels above a reproductive baseline are a consequence of the frequency and intensity of social interactions, in particular of male-male agonistic encounters. It is becoming clear that androgens increase rapidly after an aggressive contest, independently of fight outcome. However, the function of this increase remains unclear as the frequency of aggressive displays was unrelated with post-fight androgen levels and constitutive levels of androgens, and androgen responsiveness, were similar between fighter and wild-type males. Results obtained for cortisol agree with a “corticosteroid-mediated dominance hypothesis” whereby low baseline levels and a blunted response of corticosteroids would be associated with a dominant status and high aggression. The work advances our knowledge about the endocrine regulation of aggressive behaviour in B. splendens and opens several testable hypotheses about the role of androgens and corticosteroids in the regulation of fish aggressive behaviour

According to the United Nations (UN) World Urbanization Prospects 2018, cities are growing in both size and number, posing challenges for sustainable development. With urbanization, the increase of impermeabilization and landscape fragmentation urges for territorial planning and resources management. To promote resilient and resourceful cites, strategic planning where nature-based solutions (NBS) are included into the built environment, counteracting the negative effects of urbanization through the provision of ecosystem services, should be considered. Green roofs (GR) are an example of NBS that provide a panoply of ecosystem services at the level of the building and city, besides contributing for climate change mitigation and adaptation. This NBS can be included in the urban planning agenda in the new building or as a retrofit solution in the existing built environment. Macao SAR (here mentioned as Macao) is the second more densely populated city in the world, according to the UN Department of Economic and Social Affairs Population Division. The present study aims to assess the inclusion of GR in Macao and assess the ecosystem services that they may provide in a holistic approach aligned with the city’s sustainable development. For this purpose, an extensive GR implemented in Taipa Island of Macao was monitored in terms of associated biodiversity and carbon cycle assessment. Further on, it was carried out a comparison between this GR and a conventional roof (CR) in terms of surface thermal buffering. The results of this study have shown to be very promising in terms of the applicability of GR in Macao and on the relevant ecosystem services that they can provide. First, data loggers were installed in the GR and CR to assess the impact on temperature mitigation. The GR had a significant effect in decreasing and buffering temperature at the surface, with temperatures of 35.6 ºC in the GR substrate comparing with temperatures of 57.7 ºC in the CR surface. The results show that GR can be a cost-efficiency strategy to reduce heating of buildings and their associated environmental and economic costs with cooling. Second, the biodiversity of plants and animals was assessed using a combination of methods. It was observed a high level of biodiversity under a low maintenance and low disturbance of GR vegetation, with 23 species of plants and 34 species of animals identified. This shows that GR can contribute to increase biodiversity in cities. Third, an experiment was run with leaf litter bags to investigate the role of macrofauna (e.g., insects) in the decomposition of organic matter. With macrofauna activity, the decomposition rate increased 17 % when compared to decomposition rates without macrofauna, suggesting that the animals harboured in GR, in particular insects, play a key role in organic matter decomposition and thus on the carbon cycle in cities. This work is, to our knowledge, the first empirical GR study in Macao. The results support the application of GR in Macao for temperature mitigation, biodiversity increase and carbon sequestration, promoting the integration of this NBS in the future urban planning and policy of the city. More generally, the work supports the use of GR as an important NBS to mitigate the impact of climate change in urban settings

The physiological mechanisms underlying variation in aggression in fish remain poorly understood. One possibly confounding variable is the lack of standardization in the type of stimuli used to elicit aggression. The presentation of controlled stimuli in videos, a.k.a. video playback, can provide better control of the fight components. However, this technique has produced conflicting results in animal behaviour studies and needs to be carefully validated. For this, a similar response to the video and an equivalent live stimulus needs to be demonstrated. Further, different physiological responses may be triggered by live and video stimuli and it is important to demonstrate that video images elicit appropriate physiological reactions. Here, the behavioural and endocrine response of male Siamese fighting fish Betta splendens to a matched for size conspecific fighting behind a one-way mirror, presented live or through video playback, was compared. The video playback and live stimulus elicited a strong and similar aggressive response by the focal fish, with a fight structure that started with stereotypical threat displays and progressed to overt attacks. Post-fight plasma levels of the androgen 11-ketotestosterone were elevated as compared to controls, regardless of the type of stimuli. Cortisol also increased in response to the video images, as previously described for live fights in this species. These results show that the interactive component of a fight, and its resolution, are not needed to trigger an endocrine response to aggression in this species. The study also demonstrates for the first time in a fish a robust endocrine response to video stimuli and supports the use of this technique for researching aggressive behaviour in B. splendens.

The role of hormones as modulators of aggressive behavior in fish remains poorly understood. Androgens and corticosteroids, in particular, have been associated with aggressive behavior in fish but it is still not clear if animals adjust the secretion of these hormones to regulate behavior during ongoing fights, in response to fight outcomes in order to adjust aggressive behavior in subsequent fights, or both. With its stereotyped displays and high aggression levels, the Siamese fighting fish Betta splendens is an excellent model to investigate this question. Here, we compared the behavioral and endocrine response of male B. splendens to fights where there is no winner or loser by presenting them with a size-matched live interacting conspecific behind a transparent partition or with a mirror image. The aggressive response started with threat displays that were overall similar in frequency and duration towards both types of stimuli. Fights transitioned to overt attacks and interacting with a live conspecific elicited a higher frequency of attempted bites and head hits, as compared with the mirror image. There was a pronounced increase in plasma androgens (11-ketotestosterone and testosterone) and corticosteroids (cortisol) levels in response to the aggression challenge, independent of stimulus type. Post-fight intra-group levels of these hormones did not correlate with measures of physical activity or aggressive behavior. A linear discriminant analysis including all behavioral and endocrine data was a poor classifier of fish from the conspecific and mirror trials, showing that overall the behavioral and endocrine response to mirror images and conspecifics was similar. The results show that fight resolution is not necessary to induce an evident increase in peripheral levels of androgens and corticosteroids in B. splendens. However, the function of these hormones during present and future aggressive contests remains to be clarified.