Percorrer por autor "Guerreiro, Pedro Miguel"
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- Are there differential roles in the parental behaviour of the chameleon cichlid australoheros facetus?Publication . Oliveira, Gonçalo; Saraiva, João; Jesus, Nuno F.; Guerreiro, Pedro MiguelCichlids are social fishes well known for their complex behaviour. The chameleon cichlid, Autraloheros facetus, is native to South American river drainages and is currently established in several Mediterranean-type drainages in southern Portugal as an invasive species. Their high local recruitment, territorially, and parental care activities are possible advantages in competing with native fish and achieving high reproductive success. The main objective of this work was to characterise the behaviours of the males and females of the species A. facetus upon pair formation and their roles during parental behaviour at different stages of offspring development for the purpose of gathering important basic knowledge on fish biology to apply in the control of the species’ populations. To attain this initial goal, we used observation techniques and video recording protocols to characterise the specific activities performed by each individual during reproductive and parental stages (pair formation, eggs, attached larvae, and free-swimming larvae), identifying the main tasks, and assessing the time spent on each task by each member of the reproductive pair. The breeding pairs were obtained as a result of the social hierarchy formed in each social group: groups of six individuals of similar size were placed in individual tanks fitted with a bottom biological filter, in which the temperature was increased to 24 ◦C at an expanded photoperiod. Social behaviours were recorded and characterised, expanded on a previously established ethogram. The results obtained so far allow us to establish a set of aggressive behaviours towards other fish (striking, chasing, biting), nest preparing behaviours (digging, cleaning), caring (caring, fetching), and guarding behaviours towards the offspring (hovering, patrolling). Concerning the rate of occurrence of social behaviours in randomised 5 min periods (frequency of specific behaviours per each 5 min period) show that in the pair formation stage, males present a more aggressive nature with frequent occurrence of striking (rstriking = 1) and biting (rbiting = 0.31). On the other hand, at the egg stage, the rate of occurrence of parental behaviours shows the dominance of females to prepare the nest (rdigging = 0.91) and caring for the eggs (rcaring = 3.64), while males are more vigilant (rparental hovering = 0.95). At the attached larvae stage, this tendency continues with higher occurrence rates: males focus on vigilance (rparental hovering = 1.6) and patrolling (rpatrolling = 2), and females care for the recently hatched larvae (rfetching = 4). Finally, at the free-swimming larvae stage, there is a turn and a small decrease in the occurrence of these activities: males oversee nest maintenance (rdigging = 1.09), and females patrol the tank (rpatrolling = 0.71) and care for the larvae (rfetching = 2.02). These preliminary data suggest differential roles for male and female A. facetus, that evolve during parental behaviour, for which further experimental paradigms will be designed to explore underlying proximate causes.
- Assessing fish welfare in small-scale commercial fixed-net fisheries off the southern portuguese coastPublication . Samel, Vighnesh Nilesh; Costa, Rita; Marçalo, Ana; Frade, Magda; Bentes, Luis; Saraiva, João; Gonçalves, Jorge Manuel Santos; Guerreiro, Pedro Miguel; Claudio D'IglioDespite a growing interest in animal welfare in production systems, research on fish welfare remains limited, particularly in commercial fisheries. Fish caught in fixed-net fisheries experience multiple stressors from the time of capture to mortality on deck considered detrimental to their welfare. We examined the impact of bottom-set gill nets and on-board handling on catch welfare using behavioural and physiological indicators. Vitality assessments were performed on four commercially important fish species on-board fishing vessels through a devised vitality scale that included behaviours, morphological condition and reflexes as indicators of welfare. Physiological stress parameters (Cortisol, Glucose, Lactate and Osmolality) were evaluated in blood collected on deck and analysed in relation to the vitality scores. The vitality at arrival on deck as well as the rate of decrease in vitality differed significantly amongst the tested species. Furthermore, Generalised Linear Models predicted that several biological, operational, and environmental variables significantly affect the extent of time the fish shows activity, and hence, on the welfare. Elevated average cortisol levels were found at all the vitality stages highlighting the stress experienced by fish due to the fishing process. The findings of this study enable us to recommend welfare-friendly methods in set-net fisheries to promote better fishing standards.
- Assessment of the reproductive status of captive populations of endangered leuciscid species from the iberian peninsula: a. hispanica, I. lusitanicum and a. occidentalePublication . Hernandez, Ana; Gil, Fátima; Sousa-Santos, Carla; Cabrita, Elsa; Guerreiro, Pedro Miguel; Gallego, VictorPopulations of freshwater fish species endemic to the Iberian Peninsula have been declining since the mid-20th century, and the captive breeding of highly endangered species is considered to be a useful tool to restock declining populations. A pioneer project of supportive breeding of critically endangered fish started in 2007 at the Aquário Vasco da Gama (AVG), and this work aims to show the reproductive status of the breeders which make up the current captive broodstoks. Populations of different endangered leuciscid species (Anaecypris hispanica, Iberochondrostoma lusitanicum and Achondrostoma occidentale) were sampled at AVG during the spring of 2022. Breeders were counted and sexed, and males were stripped to check for the presence of spermatozoa. The sperm volume was assessed visually, and spermatozoa motility was assessed by a CASA system. Sperm samples were classified into four classes based on the percentage of motile cells: C-I ≤ 25%, C-II = 25–50%, C-III = 50–75%; and C-IV > 75%. The captive population of A. hispanica consisted of 63 individuals and showed a 40% of spermiating males, with an average volume of 5–10 µL. The histogram of sperm quality reported that 15% males had sperm motility of C-II, 50% of males had sperm motility of C-III and, finally, 35% of males had sperm with the high-quality class (C-IV). The population of I. lusitanicum consisted of 599 individuals and showed 93% of spermiating males, with an average volume of 15–20 µL. The histogram of sperm quality reported that most part of the males had good sperm quality belonging to C-III and C-IV class (26% and 71%, respectively), while just 1 male showed bad quality sperm (C-II). The captive population of A. occidentale consisted of 193 individuals, showing a 62% of spermiating males with an average volume of 20–25 µL. The histogram showed that 6% males had sperm motility of C-I, 26% of males had sperm motility of C-II, the most part of the males (45%) showed a sperm quality of C-III and, finally, 23% of males had sperm with the high-quality class (C-IV). Since the project began in 2007, more than 12,000 fish of these three critically endangered species have been released to restock the populations from which the respective wild breeders were caught. All captive fish were released after a maximum of three consecutive generations in captivity, and new stocks were established with wild adults from the target populations, to avoid the negative effects of inbreeding and genetic drift on the original genetic pool.
- Combined effects of PVC microplastics and thermal rise alter the oxidative stress response in Antarctic fish Harpagifer antarcticus and Sub-Antarctic Harpagifer bispinisPublication . Nualart, Daniela P.; Paschke, Kurt; Guerreiro, Pedro Miguel; McCormick, Stephen D.; González-Wevar, Claudio; Cheng, Chi-Hing Christina; Chacoff, Luis VargasThis study evaluated the oxidative stress response in two cold-water fish species, the Antarctic Harpagifer antarcticus and the sub-Antarctic H. bispinis, following exposure to single and combined stressors: polyvinyl chloride (PVC) microplastics and thermal increase. Fish were exposed for 24 h to two temperature regimes (2 degrees C and 5 degrees C for H. antarcticus; 8 degrees C and 12 degrees C for H. bispinis) and were orally administered a PVC microplastic solution (200 mg/L). Oxidative stress was assessed through transcriptional and enzymatic activity analyses of key antioxidant markers: catalase, glutathione peroxidase, superoxide dismutase, and glutathione reductase. In H. antarcticus, gill antioxidant gene expression decreased in response to both stressors when applied individually, while H. bispinis exhibited transcriptional upregulation under the same conditions. In H. antarcticus, enzymatic activity in gill tissues increased for all four enzymes following exposure to both microplastics and elevated temperature. In contrast, H. bispinis showed a differential enzymatic response: thermal stress induced CAT activity, whereas exposure to microplastics specifically increased GR activity. At the hepatic level, H. antarcticus displayed increased transcription of antioxidant genes following exposure to both stressors. In H. bispinis, transcriptional upregulation was limited to GR and SOD in response to microplastics. However, under the combined exposure of multiple stressors, an inactivation of the antioxidant enzyme response was observed in the gills. This could indicate a functional collapse of the antioxidant system under conditions of exacerbated acute stress. The observed responses raise concerns about the potential vulnerability of polar and subpolar fishes, considering their ecological importance in trophic networks and the increasing pressure from multiple anthropogenic stressors in a rapidly changing climate.
- A comparison of olfactory sensitivity in seawater- and freshwater-adapted bass, dicentrarchus iabraxPublication . Velez, Zélia; Hubbard, Peter; Guerreiro, Pedro MiguelFish rely heavily on olfaction for many aspects of their lives including foraging, defense, migration, and reproduction. Olfactory receptor neurons in the olfactory epithelium are in direct contact with the water, and are, therefore, exposed to changes in water chemistry. The European seabass, Dicentrarchus labrax, uses estuaries as feeding grounds and migrates between seawater and brackish water; but some can be found in 100% freshwater. However, little is known about how the olfactory system adjusts to waters of such different ionic composition and whether this affects its function and ability to discriminate between odorants. The aim of this study was, therefore, to compare olfactory sensitivity in seabass adapted to either seawater (SW) or freshwater (5 ppt; FW), to odorants conveyed at different salinities, using multi-unit recording from the olfactory nerve. In SW-adapted fish, olfactory sensitivity to amino acids (AA) was consistently higher when AA were presented in seawater (SW-AA) than when presented in freshwater (FW-AA), whereas in FW-adapted fish, olfactory sensitivity to FW-AA was either equal or slightly lower to SW-AA. SW-adapted fish responded to decreases in external [Ca2+] and to increases in external [Na+ ]. FWadapted fish responded to increases of both ions. In SW-adapted fish, Ca2+-free artificial seawater (ASW) completely inhibited olfactory responses to amino acids, whereas Na+ -free ASW had no effect. However, in FW-adapted fish, lack of either ion in the water had no effect. Taken together, these results suggest that, as a primarily marine species, the olfactory system of the seabass is more sensitive in seawater; however, it can still function in freshwater, albeit with reduced sensitivity. Furthermore, in seawater, the olfactory transduction process is likely mediated by influx of external Ca2+, but not Na+ . In FW-adapted fish, the transduction process relies on neither external Ca2+ nor Na+ , suggesting that the process of hyperosmoregulatory ability to adjust to life in ion-poor water. Further work is needed to clarify how changes in salinity affect olfactory sensitivity, and the mechanisms by which euryhaline species are able to adapt to such changes when moving between media of different ionic composition and variable pH.
- Evaluating repetitive mucus extraction effects on mucus biomarkers, mucus cells and skin-barrier status in a marine fish modelPublication . Sanahuja, Ignasi; Guerreiro, Pedro Miguel; Girons, Albert; Fernández-Alacid, Laura; Ibarz, AntoniAmong all the mucosal barriers, the skin and its surrounding mucus are possibly the main defensive tools used by fish against the environment. Due to its less-invasive extraction, the study of its production and functions has gained high interest in the last years. However, there are still many gaps in research, such as the possible alteration of mucus composition or the skin integrity resulting from the sampling process. In the current study, skin mucus extraction impacts were determined by comparing the effects of one-single extraction (SEG; single extraction group) and three successive extractions (REG; repetitive extractions group, separated by 4 days) on mucus properties and on skin epithelial integrity. In terms of analytical evaluation, plasma biomarkers and plasma antibacterial capacity were also determined. With regard skin histology and skin barrier properties, both SEG and REG did not evidence differences with respect to intact skin (ØEG). Interestingly, the repetitive mucus extraction protocol seemed to activate skin mucus turnover, significantly increasing the number of low-size mucus cells (cell area < 100 µm2 ) and reducing the number of high-size mucus cells (cell area > 150 µm2 ). Repetitive extraction of skin mucus diminished the amounts of soluble protein and glucose in mucus with regard to one-single extraction and increased cortisol exudation. These metabolites remained unaltered in plasma, indicating the different response among both sampling targets. Despite mucus biomarkers modification, the antibacterial capacity against the pathogenic bacterial (P. anguilliseptica and V. anguillarum) was maintained in both plasma and mucus irrespective of the number of mucus extractions. Overall, the mucus sampling protocol scarcely affected skin integrity and mucus antibacterial properties and only modified metabolites exudation, evidencing a feasible and minimally invasive method for studying fish health and welfare as an alternative or as a complement to plasma. The knolwdege provided here highlighted that this methodology is putatively transferable to farm culture conditions and showed that it is very useful for the study of threatened species aimed at preserving fish welfare.
- Evaluation of an acute osmotic stress in European Sea bass via skin mucus biomarkersPublication . Ordóñez-Grande, Borja; Guerreiro, Pedro Miguel; Sanahuja, Ignasi; Fernández-Alacid, Laura; Ibarz, AntoniEuropean sea bass is a marine teleost which can inhabit a broad range of environmental salinities. So far, no research has studied the physiological response of this fish to salinity challenges using modifications in skin mucus as a potential biological matrix. Here, we used a skin mucus sampling technique to evaluate the response of sea bass to several acute osmotic challenges (for 3 h) from seawater (35%_) to two hypoosmotic environments, diluted brackish water (3%_) and estuarine waters (12%_), and to one hyperosmotic condition (50%_). For this, we recorded the volume of mucus exuded and compared the main stress-related biomarkers and osmosis-related parameters in skin mucus and plasma. Sea bass exuded the greatest volume of skin mucus with the highest total contents of cortisol, glucose, and protein under hyper salinity. This indicates an exacerbated acute stress response with possible energy losses if the condition is sustained over time. Under hypo salinity, the response depended on the magnitude of the osmotic change: shifting to 3%_ was an extreme salinity change, which affected fish aerobic metabolism by acutely modifying lactate exudation. All these data enhance the current scarce knowledge of skin mucus as a target through which to study environmental changes and fish status.
- The immune system in antarctic and subantarctic fish of the genus harpagifer is affected by the effects of combined microplastics and thermal increasePublication . Nualart, Daniela P.; Guerreiro, Pedro Miguel; Paschke, Kurt; McCormick, Stephen D.; Cheng, Chi-Hing Christina; Vargas-Chacoff, LuisRising ocean temperatures due to climate change, combined with the intensification of anthropogenic activity, may lead to changes in the physiology and distribution of native species. Compounding climate stress, microplastic particles (MPs) enter the oceans through wastewater and the breakdown of macroplastics. Depending on their composition, they can be harmful and act as a vehicle for toxic substances, although their effects on native Antarctic and subantarctic species are unknown. Notothenioid fish are members of this group and are found inside and outside Antarctica, such as the Harpagifer, which has adapted to the cold and is particularly sensitive to thermal increases. Here, we aimed to evaluate the innate immune response in the head kidney, spleen, and foregut of two notothenoid fish, Harpagifer antarcticus and Harpagifer bispinis, exposed to elevated temperatures and PVC (polyvinyl chloride) microplastics. Adults from both species were collected on King George Island (Antarctica) and Punta Arenas (Chile), respectively. Specimens were assigned to a control group or exposed to a temperature increase (TI) or PVC microplastics (MPs), separately or in combination (MPs + TI). MP exposures were oral (gavage) for 24 h or aqueous (in a bath) for 24 and 48 h. Using real-time qPCR, we evaluated the relative gene expression of markers involved in the innate immune response, including tlr2 (toll-like receptor 2), tlr4 (toll-like receptor 4), myd88 (myeloid differentiation factor 88), nfkb (nuclear factor kb), il6 (interleukin 6), and il8 (irterleukin 8). We found differences between treatments when H. antarcticus and H. bispinis were exposed independently to MPs or thermal increase (TI) in the experiment with a cannula, showing an up-regulation in transcripts. In contrast, a down-regulation was observed when exposed in combination to MP + TI, which looked to be tissue-dependent. However, transcripts related to innate immunity in the bath experiment increased when exposure to both stressors was combined, mostly at 48 h. These results highlight the importance of evaluating the effects of multiple stressors, both independently and in combination, and whether these species will have the capacity to adapt or survive under these conditions, especially in waters where temperature is increasing and pollution is also rising, primarily from MP-PVC, a plastic widely used in various industries and among the population.
- Metabolic responses and resilience to environmental challenges in the sedentary batrachoid halobatrachus didactylusPublication . Molina, Juan M.; Kunzmann, Andreas; Reis, João; Guerreiro, Pedro MiguelThe Lusitanian toadfish, Halobatrachus didactylus is a marine teleost found in coastal lagoons and river estuaries, often exposed to important changes in salinity, temperature and reduced oxygen. Sedentary species, with strong site fidelity and low migratory ability along the temperature gradient such as this may be especially impacted by climate change. We aimed at establishing the tolerance limits to acute temperature and oxygen changes, and evaluate respiratory and metabolic responses in chronic control, warm and hypoxic (35% O2 ) conditions. Critical temperature maximum (CTmax) was determined in 12 individuals exposed to a temperature ramp of 3 ◦C per hour starting at 18 ◦C, and was found to be 34.8 ± 0.66 ◦C. Critical oxygen level (PO2crit) was determined in 8 fish at 18 ◦C while performing intermittent respirometry and oxygen depletion was created by nitrogen injection in the tank. PO2crit was calculated as the inflexion point between oxyregulation and oxyconformation, which was found to be around 1.2 mgO2/L, but fish survived down to 3% O2 , recovering from 0.2 mgO2/L but showing increased hematocrit (Hct), red blood cell (RBC) counts and blood pH. We also quantified routine aerobic scope and daily activity patterns, finding this fish to be extremely sedentary. H. didactylus showed one of the lowest daytime basal metabolic rates (MR) found in the literature but activity increased significantly at night (over two-fold when closed inside the metabolic chambers). The effect of temperature on metabolic rate (MR) was evaluated using a temperature ramp ranging from 8 to 32 ◦C (1 ◦C/h). Acute temperature changes resulted in a steady increase in MR up to circa 29 ◦C, beyond which MR become increasingly variable, especially among smaller individuals. Indeed, small fish appear to show high- and low-MR groups, and were more susceptible to heat and hypoxia than larger individuals. In chronic acclimation, the MR was increased by 3- and 4-fold (hypoxia vs. normoxia) in fish at 28 ◦C in relation to those at 12 ◦C. Standard MR were not statistically different between normoxia and hypoxia at 12 ◦C, but maximum MR in hypoxia was only about 2/3 of that in normoxia. Fish in high temperature lost weight (mean −3.1%) and had higher metabolism, while in low temperature, weight increased (mean +9.3%) and metabolism was low, and HIS was significantly lower in high temperature groups. Fish in hypoxic conditions showed consistently high Hct but not RBC or hemoglobin (Hb). Overall this study indicates that H. didactylus is highly tolerant to hypoxia and temperature variations. It remains to be seen if other populations along the Atlantic coast show similar metrics. The measured CTmax is close to the actual maximum temperature possible to experience in Ria Formosa ponds during summer, and it would not be unexpected to find this species establishing stable populations in other regions if climate change forces it out of its actual distribution.
- Sperm characterization of endangered leuciscids endemic from the iberian peninsula: gamete storage as a tool for helping ex-situ breeding programsPublication . Hernandez, Ana; Sousa-Santos, Carla; Gil, Fátima; Guerreiro, Pedro Miguel; Cabrita, Elsa; Gallego, VictorPopulations of freshwater fish species endemic to the Iberian Peninsula have been declining since the mid-20th century, and several types of actions (from in situ to ex situ measurements) have been applied over the past decades for preserving these species. However, limited knowledge about their reproductive biology makes it necessary to investigate different aspects of the reproductive cycle for improving breeding programs. The main objectives of this work were to advance knowledge concerning sperm kinetics and spermatozoa morphology and to develop protocols for the short- and long- term storage of gametes. Populations of different endangered leuciscid species (Anaecypris hispanica, Iberochondrostoma lusitanicum, Achondrostoma occidentale, and Squalius aradensis) were sampled during the spring of 2022 both in captive populations kept at Aquário Vasco da Gama (AVG) and in wild populations from different Portuguese rivers. Sperm samples were collected and sperm motion parameters were assessed, for the first time, for these four species. Sperm kinetics differed between species in motility and velocity traits, also showing a different number of sperm subpopulations. The longevity of sperm (swimming period) was also different among species: the shortest period was obtained for the wild population of S. aradensis (values close to zero at 40 s), and the longest swimming period for the captive population of I. lusitanicum (values close to zero at 120 s). Furthermore, different storage trials were carried out diluting the sperm in a extender solution (75 mM NaC1, 70 mM KC1, 2 mM CaC12, 1 mM, MgSO4, l0 mM Hepes, pH 8) at a ratio 1:20 (sperm:extender). Sperm quality (>40% of motile cells) was kept for a maximum of four days of storage, depending on the species. In addition, new cryopreservation protocols (using DMSO, Methanol and/or egg yolk) were tested for cryobanking the sperm of these threatened species. Cryopreserved samples showed significantly lower motility when compared with fresh samples, and the best results were obtained for I. lusitanicum, reaching 20% of motile cells after thawing using 10% of DMSO supplemented with 10% of egg yolk. This study is the first of its kind to successfully achieve gamete cryopreservation of Iberian endemic and endangered freshwater fish species, developing new and useful tools to complement the management and conservation programs.