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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 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.

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.

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.

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 multiple regression (MR) analysis were developed successfully for Macao to predict the next day concentrations of PM10, PM2.5, and NO2. All the developed models were statistically significantly valid with a 95% confidence level with high coefficients of determination (from 0.89 to 0.92) for all pollutants. The models utilized meteorological and air quality variables based on five years of historical data, from 2013 to 2017. The data from 2013 to 2016 were used to develop the statistical models and data from 2017 were used for validation purposes. A wide range of meteorological and air quality variables were identified, and only some were selected as significant dependent 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-hour levels. The models were applied in forecasting the next day average daily concentrations for PM10, PM2.5, and NO2 for the air quality monitoring stations. The results are expected to be an operational air quality forecast for Macao.

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.

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.

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.

In Southeast Asia, males of the Siamese fighting fish Betta splendens have been selected across centuries for paired-staged fights. During the selection process, matched for size males fight in a small tank until the contest is resolved. Breeders discard losing batches and reproduce winner batches with the aim of increasing fight performance. We assessed the results of this long-term selection process by comparing under standard laboratory conditions male and female aggressive behaviour of one strain selected for staged fights (“fighters”) and one strain of wild-types. The aggressive response of adult fish was tested against their mirror image or a size-matched conspecific. Fighter males were more aggressive than wild-type males for all measured behaviours. Differences were not only quantitative but the pattern of fight display was also divergent. Fighter males had an overall higher swimming activity, performing frequent fast strikes in the direction of the intruder and displaying from a distance. Wild-type males were less active and exhibited aggressive displays mostly in close proximity to the stimuli. Females of the fighter strain, which are not used for fights, were also more aggressive than wild-type females. Aggressive behaviours were correlated across male and female fighter siblings, suggesting common genetic and physiological mechanisms to male and female aggression in this species. The study further shows that results were largely independent of the stimulus type, with the mirror test inducing similar and less variable responses than the live conspecific presentation. These results suggest that selection for male winners co-selected for high-frequency and metabolic demanding aggressive display in males and also enhanced female aggression, opening a wide range of testable hypothesis about the ultimate and proximate mechanisms of male and female aggression in B. splendens.

In southeast Asia, males of the Siamese fighting fish, Betta splendens, have been selected across centuries for winning paired staged fights and previous work has shown that males from fighter strains are more aggressive than wild-types. This strong directional selection for winners is likely to have targeted aggression-related endocrine systems, and a comparison between fighter and wild-type strains can bring into evidence the key hormones implicated in aggression. Here, we compared the plasma levels of the androgen 11-ketotestosterone (KT) and of the corticosteroid cortisol (F) in F2 males of a fighter and a wild-type strain raised under similar laboratory conditions. We show that F was generally lower in fighter as compared with wild-type males, while no overall differences in KT levels were detected between strains. When presented with a mirror-induced aggressive challenge, post-fight levels of F increased but more significantly so in wild-type males, while KT increased in males of both strains. After the challenge, fighter males had higher levels of KT as compared with wild-type males, while the pattern for F was opposite. As compared with animals in social groups, wild-type males placed under social isolation had lower F levels, while KT decreased for fighters. Taken together, this data suggests that while wild-type males responded to aggression with an increase in circulating levels of both androgens and corticosteroids, males selected for winning fights maintained a blunt F response, increasing only KT levels. These data agree with the hypothesis that a combination of high levels of androgens and low levels of corticosteroids is associated with high aggression. Overall, these results seem to indicate that selection for winning had a stronger impact in the hypothalamus-pituitary-interrenal axis than in the hypothalamus-pituitary–gonadal axis 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.