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Distinct patterns of gene expression often underlie intra- and intersexual differences, and the study of this set of coregulated genes is essential to understand the emergence of complex behavioural phenotypes. Here, we describe the development of a de novo transcriptome and brain gene expression profiles of wild-caught peacock blenny, Salaria pavo, an intertidal fish with sex-role reversal in courtship behaviour (i.e., females are the courting sex) and sequential alternative reproductive tactics in males (i.e., larger and older nest-holder males and smaller and younger sneaker males occur). Sneakers mimic both female's courtship behaviour and nuptial coloration to get access to nests and sneak fertilizations, and later in life transition into nest-holder males. Thus, this species offers the unique opportunity to study how the regulation of gene expression can contribute to intersex phenotypes and to the sequential expression of male and female behavioural phenotypes by the same individual. We found that at the whole brain level, expression of the sneaker tactic was paralleled by broader and divergent gene expression when compared to either females or nest-holder males, which were more similar between themselves. When looking at sex-biased transcripts, sneaker males are intersex rather than being either nest-holder or female-like, and their transcriptome is simultaneously demasculinized for nest-holder-biased transcripts and feminized for female-biased transcripts. These results indicate that evolutionary changes in reproductive plasticity can be achieved through regulation of gene expression, and in particular by varying the magnitude of expression of sex-biased genes, throughout the lifetime of the same individual.

Air pollution is a major concern issue on Macao since the concentration levels of several of the most common pollutants are frequently above the internationally recommended values. The low air quality episodes impacts on human health paired with highly populated urban areas are important motivations to develop forecast methodologies in order to anticipate pollution episodes, allowing establishing warnings to the local community to take precautionary measures and avoid outdoor activities during this period. Using statistical methods (multiple linear regression (MLR) and classification and regression tree (CART) analysis) we were able to develop forecasting models for the main pollutants (NO2, PM2.5, and O3) enabling us to know the next day concentrations with a good skill, translated by high coefficients of determination (0.82–0.90) on a 95% confidence level. The model development was based on six years of historical data, 2013 to 2018, consisting of surface and upper-air meteorological observations and surface air quality observations. The year of 2019 was used for model validation. From an initially large group of meteorological and air quality variables only a few were identified as significant dependent variables in the model. The selected meteorological variables included geopotential height, relative humidity and air temperature at different altitude levels and atmospheric stability characterization parameters. The air quality predictors used included recent past hourly levels of mean concentrations for NO2 and PM2.5 and maximum concentrations for O3. The application of the obtained models provides the expected daily mean concentrations for NO2 and PM2.5 and maximum hourly concentrations O3 for the next day in Taipa Ambient air quality monitoring stations. The described methodology is now operational, in Macao, since 2020.

Statistical methods such as multiple linear regression (MLR) and classification and regression tree (CART) analysis were used to build prediction models for the levels of pollutant concentrations in Macao using meteorological and air quality historical data to three periods: (i) from 2013 to 2016, (ii) from 2015 to 2018, and (iii) from 2013 to 2018. The variables retained by the models were identical for nitrogen dioxide (NO2), particulate matter (PM10), PM2.5, but not for ozone (O3) Air pollution data from 2019 was used for validation purposes. The model for the 2013 to 2018 period was the one that performed best in prediction of the next-day concentrations levels in 2019, with high coefficient of determination (R2), between predicted and observed daily average concentrations (between 0.78 and 0.89 for all pollutants), and low root mean square error (RMSE), mean absolute error (MAE), and biases (BIAS). To understand if the prediction model was robust to extreme variations in pollutants concentration, a test was performed under the circumstances of a high pollution episode for PM2.5 and O3 during 2019, and the low pollution episode during the period of implementation of the preventive measures for COVID-19 pandemic. 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 exceeding 55 μg/m3 and 400 μg/m3, respectively. The 2013 to 2018 model successfully predicted this high pollution episode with high coefficients of determination (of 0.92 for PM2.5 and 0.82 for O3). The low pollution episode for PM2.5 and O3 was identified during the 2020 COVID-19 pandemic period, with a low record of daily concentration for PM2.5 levels at 2 μg/m3 and O3 levels at 50 μg/m3, respectively. The 2013 to 2018 model successfully predicted the low pollution episode for PM2.5 and O3 with a high coefficient of determination (0.86 and 0.84, 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.

Alternative reproductive tactics (ARTs) evolve when there is strong intra-sexual competition between conspecifics for access to mates. Typically, larger “bourgeois” males reproduce by securing the access to reproductive resources while smaller “parasitic” males reproduce by stealing fertilizations from larger males. A number of factors can influence the reproductive success of each tactic, including intrinsic (e.g. size) and extrinsic (e.g. tactic relative frequency) variables. An example where plastic ARTs occur is the peacock blenny Salaria pavo, with large males reproducing by defending nests and attracting females (bourgeois tactic) and small males reproducing by achieving sneaked fertilizations (parasitic tactic). In this study, we conducted field observations on individually tagged animals to determine their social network and collected eggs from 11 nests to determine the fertilization success of each male tactic. Paternity estimates for 550 offspring indicated an average fertilization success for nest-holder males of 95%. Nest-holder male morphological traits and social network parameters were tested as predictors of fertilization success, but only the number of sneakers present in the nest-holder’s social networks was found to be a predictor of paternity loss. Although male morphological traits had been previously found to be strongly correlated with reproductive success of nest-holder males, as measured by the number of eggs collected in the male’s nest, no correlation was found between any of the measured morphological traits and fertilization success for these males. The results suggest a stronger influence of the social environment than of morphological variables in the proportion of lost fertilizations by nest-holder males of this species.

The occurrence of endocrine disrupting chemicals (EDCs) is a major issue for marine and coastal environments in the proximity of urban areas. The occurrence of EDCs in the Pearl River Delta region is well documented but specific data related to Macao is unavailable. The levels of bisphenol-A (BPA), estrone (E1), 17α-estradiol (αE2), 17β-estradiol (E2), estriol (E3), and 17α-ethynylestradiol (EE2) were measured in sediment samples collected along the coastline of Macao. BPA was found in all 45 collected samples with lower BPA concentrations associated to the presence of mangrove trees. Biodegradation assays were performed to evaluate the capacity of the microbial communities of the surveyed ecosystems to degrade BPA and its analogue BPS. Using sediments collected at a WWTP discharge point as inoculum, at a concentration of 2 mg l−1 complete removal of BPA was observed within 6 days, whereas for the same concentration BPS removal was of 95% after 10 days, which is particularly interesting since this compound is considered recalcitrant to biodegradation and likely to accumulate in the environment. Supplementation with BPA improved the degradation of bisphenol-S (BPS). Aiming at the isolation of EDCs-degrading bacteria, enrichments were established with sediments supplied with BPA, BPS, E2 and EE2, which led to the isolation of a bacterial strain, identified as Rhodoccoccus sp. ED55, able to degrade the four compounds at different extents. The isolated strain represents a valuable candidate for bioremediation of contaminated soils and waters.

Fishes show remarkably diverse aggressive behaviour. Aggression is expressed to secure resources; adjusting aggression levels according to context is key to avoid negative consequences for fitness and survival. Nonetheless, despite its importance, the physiological basis of aggression in fishes is still poorly understood. Several reports suggest hormonal modulation of aggression, particularly by androgens, but contradictory studies have been published. Studies exploring the role of chemical communication in aggressive behaviour are also scant, and the pheromones involved remain to be unequivocally characterized. This is surprising as chemical communication is the most ancient form of information exchange and plays a variety of other roles in fishes. Furthermore, the study of chemical communication and aggression is relevant at the evolutionary, ecological and economic levels. A few pioneering studies support the hypothesis that aggressive behaviour, at least in some teleosts, is modulated by “dominance pheromones” that reflect the social status of the sender, but there is little information on the identity of the compounds involved. This review aims to provide a global view of aggressive behaviour in fishes and its underlying physiological mechanisms including the involvement of chemical communication, and discusses the potential use of dominance pheromones to improve fish welfare. Methodological considerations and future research directions are also outlined.

The prevalence of microplastics in the environment has become a major global conservation issue. One primary source of environmental microplastics is personal care and cosmetic products (PCCPs) containing microbeads. The market availability of PCCPs containing microbeads and the level of contamination of coastal sediments by microplastics was studied in one of the most densely populated cities in the world, Macao in China. We found that PCCPs containing microbeads are still widely available for sale in the region, with over 70% of surveyed PCCPs containing at least one type of microbeads as an ingredient, with polyethylene (PE) being the most common one. In an estimate, the use of PCCPs in the territory may release over 37 billion microbeads per year into the environment via wastewater treatment plants. The density of microplastics in coastal sediments varied between 259 and 1,743 items/L of sediment, amongst the highest reported in the world. The fraction of < 1 mm was the most abundant, representing an average of 98.6% of the total, and correlated positively with the abundance of larger sized fragments. The results show that although environmental pollution with microplastics released from PCCPs usage is significant, other sources, namely fragmentation of larger plastic debris, likely contribute more to the issue. The study highlights the magnitude of the problem at a local level and suggests possible mitigating strategies.

"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."

The levels of air pollution in Macao often exceeded the levels recommended by WHO. In order for the population to take precautionary measures and avoid further health risks under high pollutant exposure, it is important to develop a reliable air quality forecast. Statistical models based on linear multiple regression (MR) and classification and regression trees (CART) analysis were developed successfully, for Macao, to predict the next day concentrations of NO2, PM10, PM2.5, and O3. 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. The models utilized meteorological and air quality variables based on 5 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. A wide range of meteorological and air quality variables was identified, and only some were selected as significant independent variables. Meteorological variables were selected from an extensive list of 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-h 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 be an operational air quality forecast for Macao.

The demand for plastic has led to enormous plastic waste in the environment, which persist and negatively impact the ecosystems. Polyethylene terephthalate (PET) is one of the most common thermoplastic polymers available on the market. The concerns about plastic waste generated an interest in strategies to enhance its biodegradation and finding alternative polymers. In this work was investigated the possibility of using bacteria to degrade PET and to produce bioplastics (Polyhydroxyalkanoates, PHAs). Finally, the integration of the two processes was tested. Overall, the work aimed to investigate the potential to recycle PET into bioplastic using bacteria. The potential of bacterial consortia from various environmental samples to degrade PET granules in liquid matrix was investigated. . The results revealed maximum PET granules degradation of 1.1 % by one of the tested consortia. PET degradation intermediate terephthalic acid (TPA) was not detected at the end of 55 days. Fourier-transform infrared spectroscopy (FTIR) results showed major spectral peak shifts and bends on PET chemical structure compared to non-inoculated control. The biodegradation of PET films buried in the soil (A), with mangrove plants (B), and bioaugmented with a bacterial consortium (C) was also investigated. The experiments were conducted for 270 days at ambient conditions. The results revealed no difference between treatments in the degradation, with a maximum weight loss of 0.118 % in the bioaugmented treatment. Nevertheless, Scanning Electron Microscope (SEM) and FTIR results indicated significant surface changes, spectral peak shifts, and stretches in PET chemical structures. Bacterial consortia isolated from the soil of the experimental treatments were assessed for degradation of PET monomers, TPA and monoethylene glycol (MEG), and intermediate Bis(2-hydroxyethyl) terephthalate (BHET). The consortia were inoculated in flasks containing minimal media with 1000 mg/L TPA or BHET or1113 mg/L MEG as the sole carbon source. Results showed complete degradation of TPA and significant degradation of BHET (96.09%), and MEG (83.65%) by the consortia. In the second part of the study, bacteria were isolated from various environmental samples and screened for PHA production using Sudan Black B staining on colonies and smeared glass slides. Transmission Electron Microscope images were captured to confirm the intracellular PHA inclusions. A total of 35 isolates were screened for PHA, and 22 showed positive staining. The isolate showing higher levels of PHA synthesis (EC2-30-3) was identified based on 16S rRNA gene sequence as Bacillus sp. and selected for PET monomers degradation and fermentation cultures for PHA production. It was cultured in minimal (Moreira et al., 2013) media with 1000 mg/L TPA and 1113 mg/L MEG as the carbon source for eight days. The isolate grew better in media containing MEG, which was selected as a substrate model for PHA fermentation. To integrate PET monomers biodegradation and production of PHA, the isolate was cultured in 0.2 % MEG. A control with 0.2 % of glucose was prepared, and the cultures were incubated for 96 hours. Bacillus sp. EC2-30-3 showed higher PHA accumulation in media supplied with MEG (40.31%) than glucose (25.53%). This is the first report showing that Bacillus sp. uses PET monomer as carbon source to produce a biopolymer. FTIR results of the extracted PHA identified its functional units as C–H, CH3, C=O, and C–O groups. The absorption bands obtained are closely related to the structure of PHB. The study thus confirmed the ability of the isolated bacteria to degrade PET monomers and produce biopolymers. The results of this work open the possibility for upscaling the use of bacteria to mitigate the impact of PET on the environment while producing environmentally friendly bioplastics