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Effect of elevated pCO2 and environmental oxazepam on the behavior and physiology of teleost fish
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience. (Behavioral neuroendocrinology (Winberg))
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Description
Abstract [en]

This doctoral thesis investigated the effect of two aquatic pollutants on the behavior and physiology of teleost fish: i) elevated concentrations of carbon dioxide (CO2) in the context of ocean acidification and ii) low concentrations of the anxiolytic pharmaceutical oxazepam in the context of pharmaceutical pollution.

Anthropogenic emissions of CO2 are lowering the pH of the oceans. Studies on coral reef fish exposed to CO2 concentrations projected for the year 2100 (~1000 μatm) reported alarming behavioral effects, of which attraction to predator odor was the most surprising. To explain this behavioral reversal, it was hypothesized that ion-regulatory adjustments to compensate for the decrease in blood pH would result in altered transmembrane gradients of chloride ions, rendering the major inhibitory neurotransmitter in the brain, γ-aminobutyric acid (GABA), excitatory. We investigated whether zebrafish (Danio rerio), an often used model species, showed similar behavioral disruptions in elevated CO2. Zebrafish behavior was however largely unaffected by an approximately month long exposure to ~1600 μatm CO2. We continued by investigating the reproductive, anxiety-related behavior and aggression in another model species, the three-spined stickleback (Gasterosteus aculeatus). However, also stickleback behavior and responses to social subordination were not affected by CO2, in contrast to earlier findings. We concluded that CO2 had no major effect on the behavior of zebrafish and three-spined stickleback.

In the second part of this thesis, I investigated behavioral effects of oxazepam, an anxiolytic pharmaceutical (benzodiazepine) acting on the GABA system. Studies on perch (Perca fluviatilis) have shown that exposure to dilute concentrations of oxazepam (1.8 μg L-1), close to those found outside the municipal sewage treatment plant in Uppsala (0.58 μg L-1), can increase activity, decrease sociality and increase feeding rates. I show that similar oxazepam concentrations can also affect zebrafish. Moreover, I show that females are more sensitive to oxazepam showing reduced anti-predator responses at 0.57 μg L-1 while in males this effect was observed first at 60 μg L-1. Furthermore, and in contrast to wild-caught zebrafish, laboratory zebrafish did not show any effect of the oxazepam exposure. This finding has implications for the use of laboratory zebrafish in ecotoxicological and pharmacological studies, as results might not translate to wild fish. Finally, I show that zebrafish can develop tolerance to the anxiolytic effects of oxazepam during chronic (28 days) exposure. This is an important discovery that could mitigate the effects of this form of pharmaceutical pollution on wild fish.

Abstract [sv]

Doktorsavhandlingen undersöker effekter av två olika vattenföroreningar på beteende och fysiologi hos benfisk, nämligen: i) förhöjda koncentrationer av koldioxid (CO2) i relation till havsförsurning och ii) låga koncentrationer av den ångestdämpande farmakologiska substansen oxazepam i samband med förorening av vatten med läkemedel.

Utsläpp av CO2 sänker havets pH-värde och studier där korallrevsfiskar exponerats för CO2-koncentrationer i nivå med vad som beräknats för år 2100 (~1000 μatm) har rapporterat om alarmerande beteendestörningar, såsom att försöksfiskar inte längre undvek lukt av rovfisk utan istället attraherades till den. En hypotes är att detta omvända beteende orsakas av att de mekanismer som är nödvändiga för att återställa fiskens syra/bas-balans resulterar i att hjärnans främsta inhibitoriska signalsubstans, γ-aminobuturatsyra (GABA), istället får en excitatorisk effekt. Vi har undersökt om sebrafisk (Danio rerio), som är en vanlig modellorganism, visar liknande beteendeförändringar vid förhöjd CO2. Sebrafiskens beteende var emellertid i stort sett opåverkat efter ungefär en månads exponering för ~1600 μatm CO2. Vi fortsatte med att undersöka beteendeeffekter av förhöjda halter av CO2 hos storspigg (Gasterosteus aculeatus), en annan vanlig modellorganism. Inte heller hos denna art kunde vi påvisa några tydliga beteendeeffekter av CO2 varför vi drog slutsatsen att CO2 inte har några påtaglig beteendeeffekter, vare sig hos sebrafisk eller storspigg.

I den andra delen av doktorsavhandlingen undersökte vi hur miljörelevanta koncentrationer av det ångesthämmande läkemedlet oxazepam påverkar GABA systemet och fiskars beteende. Tidigare studier på abborre (Perca fluviatilis) har visat att 1.8 μg L-1oxazepam, vilket är i nivå med vad som påträffas utanför Uppsala avloppsreningsverk (0.58 μg L-1), ger ökad simaktivitet, minskad attraktion till stimmet och ökat födointag. I doktorsavhandlingen visar jag att liknande koncentrationer av oxazepam också påverkar sebrafisk och att honor är mer känsliga för oxazepam då dessa visar minskat anti-predator beteende redan vid 0.57 μg L-1 medan denna effekt inte uppträder hos hanar förrän vid koncentrationen 60 μg L-1. Dessutom fann vi att laboratoriestammar i motsats till vildfångade sebrafiskar inte visade några av dessa oxazepam-inducerade beteendeeffekter, en upptäckt som kan få konsekvenser för användandet av sebrafisk i ekotoxikologiska och farmakologiska studier, eftersom resultat som erhålls på labstammar av sebrafisk inte är direkt överförbara till vild fisk. Slutligen visar min studie att sebrafisk kan utveckla tolerans för de ångesthämmande effekterna av oxazepam vid kronisk (28 dygn) exponering. Detta är ett viktigt resultat som kan tyda på att effekterna av läkemedelsföreningar på vild fisk, åtminstone i detta fall, kan vara mindre än befarat.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2019. , p. 63
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1614
Keywords [en]
fish, GABA, CO2, ocean acidification, pharmaceutical pollution, oxazepam
National Category
Behavioral Sciences Biology Physiology Pharmaceutical Sciences
Identifiers
URN: urn:nbn:se:uu:diva-395993ISBN: 978-91-513-0807-4 (print)OAI: oai:DiVA.org:uu-395993DiVA, id: diva2:1366189
Public defence
2019-12-13, Room B21, Biomedical Center, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2019-11-20 Created: 2019-10-28 Last updated: 2019-11-20
List of papers
1. Zebrafish (Danio rerio) behaviour is largely unaffected by elevated pCO2
Open this publication in new window or tab >>Zebrafish (Danio rerio) behaviour is largely unaffected by elevated pCO2
2016 (English)In: Conservation Physiology, E-ISSN 2051-1434, Vol. 4, no 1Article in journal (Refereed) Published
Abstract [en]

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.

Keywords
Behavioural lateralization, detour, gabazine, ocean acidification, open field, zebrafish
National Category
Climate Research Behavioral Sciences Biology
Identifiers
urn:nbn:se:uu:diva-319061 (URN)10.1093/conphys/cow065 (DOI)28066551 (PubMedID)
Available from: 2017-03-31 Created: 2017-03-31 Last updated: 2019-10-28Bibliographically approved
2. No effect of elevated carbon dioxide on reproductive behaviors in the three-spined stickleback
Open this publication in new window or tab >>No effect of elevated carbon dioxide on reproductive behaviors in the three-spined stickleback
2017 (English)In: Behavioral Ecology, ISSN 1045-2249, E-ISSN 1465-7279, Vol. 28, no 6, p. 1482-1491Article in journal (Refereed) Published
Abstract [en]

Ocean acidification, the reduction in ocean pH resulting from anthropogenic emissions of carbon dioxide (CO2), has been predicted to alter the behavior of fishes. During experimental exposure to CO2 concentrations projected for the year 2100 (~1000 μatm), fish have been reported to display disturbances in activity, learning, behavioral lateralization, and even attraction to predator cues. Reproductive behaviors have received far less attention, despite an intensive research effort on ocean acidification and its ecological importance. Here, we investigate whether elevated levels of CO2 affect reproduction in breeding pairs of the three-spined stickleback, Gasterosteus aculeatus, a model species in behavioral, evolutionary ecology, and environmental toxicology. We found that males under both present day levels (400 μatm) and future levels (1000 μatm) of CO2 developed normal sexual ornaments, pursued normal nest building activi-ties, exhibited similar levels of courtship behaviors and displacement fanning, and had the same mating probability. Moreover, fanning behavior during the paternal care period followed what is expected for the species for males from both treatments, and there was no effect of treatment on the numbers of offspring produced. This study is the first to investigate the effect of elevated CO2 on the com-plete breeding cycle in detail, studying an array of highly fitness-relevant traits. Our study showing surprising resilience of fish repro-duction is an important contribution in order to realistically predict the impacts of future ocean acidification.

Place, publisher, year, edition, pages
Oxford University Press, 2017
National Category
Behavioral Sciences Biology
Identifiers
urn:nbn:se:uu:diva-331063 (URN)10.1093/beheco/arx112 (DOI)000415675000010 ()
Funder
Swedish Research Council Formas, 2013–947Swedish Research Council Formas, 2009–596Swedish Research Council, 621-2012-4679The Royal Swedish Academy of Sciences, (FOA14SLC027The Royal Swedish Academy of Sciences, FOA14SLC016
Available from: 2017-10-10 Created: 2017-10-10 Last updated: 2019-10-28Bibliographically approved
3. No evidence that exposure to elevated pCO2 affects behaviour or monoamine neurotransmitters of three-spined stickleback
Open this publication in new window or tab >>No evidence that exposure to elevated pCO2 affects behaviour or monoamine neurotransmitters of three-spined stickleback
Show others...
(English)Manuscript (preprint) (Other academic)
National Category
Behavioral Sciences Biology
Identifiers
urn:nbn:se:uu:diva-395988 (URN)
Available from: 2019-10-28 Created: 2019-10-28 Last updated: 2019-10-28
4. Low concentrations of the benzodiazepine drug oxazepam induce anxiolytic effects in wild-caught but not in laboratory zebrafish
Open this publication in new window or tab >>Low concentrations of the benzodiazepine drug oxazepam induce anxiolytic effects in wild-caught but not in laboratory zebrafish
Show others...
2020 (English)In: Science of the Total Environment, ISSN 0048-9697, E-ISSN 1879-1026, Vol. 703, article id 134701Article in journal (Refereed) Epub ahead of print
Abstract [en]

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.

National Category
Behavioral Sciences Biology
Identifiers
urn:nbn:se:uu:diva-395990 (URN)10.1016/j.scitotenv.2019.134701 (DOI)
Available from: 2019-10-28 Created: 2019-10-28 Last updated: 2019-11-25Bibliographically approved
5. Chronic exposure to oxazepam pollution produces tolerance to anxiolytic effects in zebrafish (Danio rerio)
Open this publication in new window or tab >>Chronic exposure to oxazepam pollution produces tolerance to anxiolytic effects in zebrafish (Danio rerio)
Show others...
(English)In: Article in journal (Other academic) Submitted
National Category
Behavioral Sciences Biology
Identifiers
urn:nbn:se:uu:diva-395991 (URN)
Available from: 2019-10-28 Created: 2019-10-28 Last updated: 2019-10-28

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