Boldness, activity, and aggression: Insights from a large-scale study in Baltic salmon (Salmo salar L).

Atlantic salmon (Salmo salar) display high levels of agonistic behavior in aquaculture farms, resulting in fin damage and chronic stress. Aggression affects fish growth and performance negatively, and presents a serious welfare problem. Indeed, it would be beneficial to identify, separate or exclude overly aggressive individuals. Research on behavioral syndromes suggests that aggressive behavior may correlate with other behavioral traits, such as boldness and locomotory activity. We aimed to develop a high-throughput method to quantify and predict aggressive behavior of individual parr in hatchery-reared Baltic salmon (Salmo salar L.). We screened approximately 2000 parr in open field (OF) and mirror image stimulation (MIS) tests. We extracted seven variables from video tracking software for each minute of the tests; distance moved and duration moving (activity), the duration in and number of entries to the center of the arena (boldness), the distance moved in and duration spent in the area adjacent to the mirror during the MIS test (aggressiveness) and head direction (lateralization). To investigate the relationship between activity, boldness and aggression we first correlated the first six variables to one another. Second, we assigned individuals to high, medium, low or zero aggression groups based on the MIS test and quantified activity and boldness in each group. Third, we analyzed whether the fish viewed the mirror with the left or right eye. Our results show that medium and low aggressive fish were the most active, while highly aggressive fish showed average activity. Aggressive groups did not differ in boldness. Activity and boldness were positively correlated. Finally, we detected a preference for fish to view the mirror with the left eye. We conclude that aggressiveness cannot be predicted from the results of the OF test alone but that the MIS test can be used for large-scale individual aggression profiling of juvenile salmon.

Improved survival after laparoscopic compared to open gastrectomy for advanced gastric cancer: a Swedish population-based cohort study.

BACKGROUND: Laparoscopic gastrectomy is increasingly used for the treatment of locally advanced gastric cancer but concerns remain whether similar results can be obtained compared to open gastrectomy, especially in Western populations. This study compared the short-term postoperative, oncological and survival outcomes following laparoscopic versus open gastrectomy based on data from the Swedish National Register for Esophageal and Gastric Cancer. METHODS: Patients who underwent surgery with curative intent for adenocarcinoma of the stomach or gastroesophageal junction Siewert type III from 2015 to 2020 were identified, and 622 patients with cT2-4aN0-3M0 tumors were included. The impact of surgical approach on short-term outcomes was assessed using multivariable logistic regression. Long-term survival was compared using multivariable Cox regression. RESULTS: In total, 350 patients underwent open and 272 laparoscopic gastrectomy, of which 12.9% were converted to open surgery. The groups were similar regarding distribution of clinical disease stage (27.6% stage I, 46.0% stage II, and 26.4% stage III). Neoadjuvant chemotherapy was administered to 52.7% of the patients. There was no difference in the rate of postoperative complications, but laparoscopic approach was associated with lower 90 day mortality (1.8 vs 4.9%, p = 0.043). The median number of resected lymph nodes was higher after laparoscopic surgery (32 vs 26, p < 0.001), while no difference was found in the rate of tumor-free resection margins. Better overall survival was observed after laparoscopic gastrectomy (HR 0.63, p < 0.001). CONCLUSIONS: Laparoscopic gastrectomy can be safely preformed for advanced gastric cancer and is associated with improved overall survival compared to open surgery.

Sex-Specific Effects of Acute Ethanol Exposure on Locomotory Activity and Exploratory Behavior in Adult Zebrafish (Danio rerio).

The zebrafish (Danio rerio) is an established model organism in pharmacology and biomedicine, including in research on alcohol use disorders and alcohol-related disease. In the past 2 decades, zebrafish has been used to study the complex effects of ethanol on the vertebrate brain and behavior in both acute, chronic and developmental exposure paradigms. Sex differences in the neurobehavioral response to ethanol are well documented for humans and rodents, yet no consensus has been reached for zebrafish. Here, we show for the first time that male zebrafish of the AB strain display more severe behavioral impairments than females for equal exposure concentrations. Adult zebrafish were immersed in 0, 1 or 2% (v/v) ethanol for 30 min, after which behavior was individually assessed in the zebrafish Multivariate Concentric Square Field™ (zMCSF) arena. Males exposed to 2% ethanol showed clear signs of sedation, including reduced activity, increased shelter seeking and reduced exploration of shallow zones. The 1% male group displayed effects in the same direction but of smaller magnitude; this group also explored the shallow areas less, but did not show a general reduction in activity nor an increase in shelter seeking. By contrast, 1 and 2% exposed females showed no alterations in explorative behavior. Females exposed to 2% ethanol did not display a general reduction in activity, rather activity gradually increased from hypoactivity to hyperactivity over the course of the test. This mixed stimulatory/depressant effect was only quantifiable when locomotory variables were analyzed over time and was not apparent from averages of the whole 30-min test, which may explain why previous studies failed to detect sex-specific effects on locomotion. Our results emphasize the importance of explicitly including sex and time as factors in pharmacological studies of zebrafish behavior. We hypothesize that the lower sensitivity of female zebrafish to ethanol may be explained by their greater body weight and associated larger distribution volume for ethanol, which may render lower brain ethanol concentrations in females.

The zebrafish Multivariate Concentric Square Field: A Standardized Test for Behavioral Profiling of Zebrafish (Danio rerio).

The zebrafish (Danio rerio) is an important model organism in the study of the neurobiological basis of human mental disorders. Yet the utility of this species is limited by the quality of the phenotypical characterization tools available. Here, we present a complex testing environment for the quantification of explorative behavior in adult zebrafish, the zebrafish Multivariate Concentric Square Field™ (zMCSF), adapted from the rodent equivalent that has been used in > 40 studies. The apparatus consists of a central open area which is surrounded by a dark corner with a roof (DCR), corridors, and an inclined ramp. These areas differ in illumination, water depth, and are sheltered or exposed to different degrees. We quantified behavior of male and female wild-caught and AB strain zebrafish in the zMCSF (day 1) and cross-validated these results using the novel tank diving test (NTDT) (day 2). To assess the effect of repeated testing, AB zebrafish we tested a second time in both tests 1 week later (on days 7 and 8). We detected strong differences between the strains, with wild zebrafish swimming faster and spending more time in the corridors and on the ramp, while they avoided the open area in the center. AB zebrafish were less hesitant to enter the center but avoided the ramp, and often left one or more zones unexplored. No major sex differences in exploratory behavior were detected in either strain, except for a slightly higher velocity of AB males which has been reported before. Importantly, the zMCSF was largely resilient to repeated testing. The diving test revealed only one difference confined to one sex; wild females paid more visits to the top third than AB females. In isolation, this finding could lead to the conclusion that wild zebrafish are more risk-taking, which is incorrect given this strain's avoidance of open areas. To conclude, our results suggest that the zMCSF presents a sophisticated behavioral tool that can distinguish between different magnitudes and types of risk, allowing the user to create an intricate behavioral profile of individual adult zebrafish.

Chronic Exposure to Oxazepam Pollution Produces Tolerance to Anxiolytic Effects in Zebrafish (Danio rerio).

Environmental concentrations of the anxiolytic drug oxazepam have been found to disrupt antipredator behaviors of wild fish. Most experiments exposed fish for a week, while evidence from mammals suggests that chronic exposure to therapeutic concentrations of benzodiazepines (such as oxazepam) results in the development of tolerance to the anxiolytic effects. If tolerance can also develop in response to the low concentrations found in the aquatic environment, it could mitigate the negative effects of oxazepam pollution. In the current study, we exposed wild-caught zebrafish to oxazepam (∼7 µg L-1) for 7 or 28 days and evaluated behavioral and physiological parameters at both time points. Females showed reduced diving responses to conspecific alarm pheromone after 7 days, but not after 28 days, indicating that they had developed tolerance to the anxiolytic effects of the drug. Zebrafish males were not affected by this oxazepam concentration, in line with earlier results. Serotonin turnover (ratio 5-HIAA/5-HT) was reduced in exposed females and males after 28 days, indicating that brain neurochemistry had not normalized. Post-confinement cortisol concentrations and gene expression of corticotropin-releasing hormone (CRH) were not affected by oxazepam. We did not find evidence that chronically exposed fish had altered relative expression of GABAA receptor subunits, suggesting that some other still unknown mechanism caused the developed tolerance.

Low concentrations of the benzodiazepine drug oxazepam induce anxiolytic effects in wild-caught but not in laboratory zebrafish.

Pollution by psychoactive pharmaceuticals has been found to disrupt anti-predator behaviors of wild fish. The challenge is now to identify which of the many psychoactive drugs pose the greatest threat. One strategy is to screen for behavioral effects of selected pharmaceuticals using a single, widely available fish species such as zebrafish. Here, we show that although such high-throughput behavioral screening might facilitate comparisons between pharmaceuticals, the choice of strain is essential. While wild-caught zebrafish exposed to concentrations of the anxiolytic drug oxazepam as low as 0.57 µg L-1 showed a reduction in the response to conspecific alarm pheromone, laboratory strain AB did not respond to the alarm cue, and consequently, the anxiolytic effect of oxazepam could not be measured. Adaptation to the laboratory environment may have rendered laboratory strains unfit for use in some ecotoxicological and pharmacological studies, since the results might not translate to wild fish populations.

Characterization of the γ-aminobutyric acid signaling system in the zebrafish (Danio rerio Hamilton) central nervous system by reverse transcription-quantitative polymerase chain reaction.

In the vertebrate brain, inhibition is largely mediated by γ-aminobutyric acid (GABA). This neurotransmitter comprises a signaling machinery of GABAA, GABAB receptors, transporters, glutamate decarboxylases (gads) and 4-aminobutyrate aminotransferase (abat), and associated proteins. Chloride is intimately related to GABAA receptor conductance, GABA uptake, and GADs activity. The response of target neurons to GABA stimuli is shaped by chloride-cation co-transporters (CCCs), which strictly control Cl- gradient across plasma membranes. This research profiled the expression of forty genes involved in GABA signaling in the zebrafish (Danio rerio) brain, grouped brain regions and retinas. Primer pairs were developed for reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The mRNA levels of the zebrafish GABA system share similarities with that of mammals, and confirm previous studies in non-mammalian species. Proposed GABAA receptors are α1ß2γ2, α1ß2δ, α2bß3γ2, α2bß3δ, α4ß2γ2, α4ß2δ, α6bß2γ2 and α6bß2δ. Regional brain differences were documented. Retinal hetero- or homomeric ρ-composed GABAA receptors could exist, accompanying α1ßyγ2, α1ßyδ, α6aßyγ2, α6aßyδ. Expression patterns of α6a and α6b were opposite, with the former being more abundant in retinas, the latter in brains. Given the stoichiometry α6wßyγz, α6a- or α6b-containing receptors likely have different regulatory mechanisms. Different gene isoforms could originate after the rounds of genome duplication during teleost evolution. This research depicts that one isoform is generally more abundantly expressed than the other. Such observations also apply to GABAB receptors, GABA transporters, GABA-related enzymes, CCCs and GABAA receptor-associated proteins, whose presence further strengthens the proof of a GABA system in zebrafish.

Zebrafish (Danio rerio) behaviour is largely unaffected by elevated pCO2.

Ocean acidification, the decrease in ocean pH caused by anthropogenic emission of carbon dioxide, can cause behavioural disturbances in marine teleost species. We investigated whether AB-strain zebrafish (Danio rerio) show similar behavioural disturbances in the presence of elevated CO2, because this model species could open up a toolbox to investigate the physiological and neurological mechanisms of CO2 exposure. We found no effect of elevated CO2 (~1600 µatm) on the behaviour of zebrafish in the open field test, indicating that zebrafish are largely insensitive to this elevated CO2 level. In the detour test of lateralization, however, zebrafish exposed to elevated CO2 swam more often to the right, whereas individuals exposed to control CO2 (~400 µatm) had no preference for left or right. This may indicate that some behaviours of some freshwater fishes can be altered by elevated CO2 levels. Given that elevated CO2 levels often occur in recirculating aquaculture and aquarium systems, we recommend that dissolved CO2 levels are measured and, if necessary, the aquarium water should be aerated, in order to exclude CO2 level as a confounding factor in experiments.