Effects of the brain-infecting parasite Pseudoloma neurophilia in laboratory zebrafish (Danio rerio)

Zebrafish (Danio rerio) are increasingly popular model animals in scientific fields ranging from behavioural ecology to neurobiology. It is therefore of increasing concern that half of all zebrafish research facilities are contaminated with the brain-infecting microsporidian parasite Pseudoloma neurophilia. This parasite mainly aggregates in the hindbrain, where it causes chronic yet typically sub-clinical infections. Hence, infected fish often show no obvious disease symptoms and researchers are often unaware of infection status. Previous studies indicate that P. neurophilia reduces growth and alters shoaling behaviour and habituation to fearful stimulus, in zebrafish. These changes in behavioural phenotype suggest that the parasite may affect stress, anxiety and sociability in zebrafish. However, effects of this parasite on host phenotype remains largely uncharted. Thus, in this Thesis, I investigated behavioural, metabolic, neurophysiological and brain transcriptional effects of sub-clinical infections with P. neurophilia in the zebrafish host. The first aim of the Thesis was to identify behavioural effects of P. neurophilia infection in zebrafish across a range of contexts. To this end, infected and uninfected zebrafish were tested in commonly used behavioural paradigms, namely social preference, mirror biting, open field and light/dark preference tests. I found infection to not alter classic behavioural outputs such as sociability and aggression. However, infected individuals displayed reduced activity in all arenas. Furthermore, in accordance with previous studies, infection negatively affected growth, indicating that P. neurophilia is energetically costly for the zebrafish host. This cost is likely related to immune responses mounted by the host. Moreover, behavioural changes may indicate that the parasite has direct effects on the nervous system in zebrafish. The second aim of the Thesis was therefore to study brain transcriptional changes caused by infection. Specifically, I aimed to characterise the immune responses to infection and identify biological processes affected by the parasite. In line with my predictions, RNA-sequencing analysis revealed that the parasite induces a pro-inflammatory response in the zebrafish brain. However, a distinct downregulation of specific immune-related genes also suggests that the parasite takes advantage of specific immune evasion strategies. Surprisingly, P. neurophilia infection had no significant effects on genes related to nervous system function. The initial findings that P. neurophilia reduces growth and activity and induces proinflammatory responses in the brain, indicate that infection constitutes a considerable metabolic cost for the host. In addition, certain neurophysiological (e.g. monoaminergic) responses to infection may not be detectable by RNA-sequencing. Thus, the third aim of the Thesis was to determine metabolic and neurophysiological responses following acute and long-term P. neurophilia infections. In line with my predictions, P. neurophilia infection increased metabolic rate in zebrafish. However, the increase was highest three days after acute exposure (independent of whether the fish had an established infection or not) and mitigated again on day six. Furthermore, acute parasite exposure increased serotonergic and dopaminergic activity, but only in zebrafish with no previous history of infection (naïve). The results suggest that the metabolic and neurophysiological effects of P. neurophilia depends on time post last exposure and previous infection status and that metabolic costs are higher with acute compared to established infection. Since zebrafish frequently encounter infectious spores in their environment, repeated acute infections may represent a substantial metabolic cost to laboratory zebrafish. Taken together, the results obtained in this Thesis indicate that infection is associated with decreased activity and growth, a proinflammatory immune response and elevated metabolism in zebrafish. This phenotype is reminiscent of sickness behaviour (a condition in which acutely infected individuals adopt energy reducing strategies in order to fight infection). This thesis provides evidence that P. neurophilia can affect multiple biological aspects, that potentially have severe consequences for research outcomes. Hence, the findings highlight the importance of proper and standardised health monitoring in animal research facilities, not only for improving animal welfare, but also for ensuring research reproducibility.Sebrafisk (Danio rerio) har i løpet av de siste tiårene blitt en av de mest brukte og populære dyremodellene i biovitenskapelig forskning og brukes i dag i en rekke forskningsfelt, inkludert atferdsbiologi og hjerneforskning. Det er derfor svært bekymringsverdig at halvdelen av alle forskningsfasiliteter, som holder sebrafisk, er kontaminert med den hjerneinfiserende mikrosporidia-parasitten Pseudoloma neurophilia. Denne parasitten angriper primært hjernen, der den etablerer kroniske, dog oftest subkliniske infeksjoner. Det betyr at infiserte fisk ofte ikke viser tegn på sykdom, og at forskere derfor ofte jobber med infisert fisk uten at de vet det. Tidligere studier har indikert at P. neurophilia reduserer vekst, påvirker fiskegruppedynamikk og hvordan sebrafisk responderer på faretruende stimuli. Disse funnene indikerer at denne hjerneparasitten påvirker atferdsparametre som stressrespons, angst og sosiabilitet hos sebrafisk, men stort sett er effektene av denne parasitten på vertens fenotype ukjente. I denne avhandlingen undersøker jeg effekter av subklinisk P. neurophilia-infeksjon på atferd, metabolisme, nevrofysiologi og genuttrykk i hjernen hos sebrafisk. Det første delmålet i denne avhandlingen var å identifisere og kartlegge effekter av P. neurophilia-infeksjon på atferd hos sebrafisk på tvers av flere kontekster. Til dette formålet ble både infiserte og ikke-infiserte sebrafisk testet i atferdstester som er mye benyttet av sebrafiskforskere. Disse testene brukes til å måle atferdsparametre, som for eksempel sosial preferanse, aggressivitet, angstatferd og dristighet. Til tross for parasittens privilegerte plassering i hjernen, fant jeg ingen effekter av infeksjon på disse klassiske atferdsparametrene. På den annen side fant jeg at infiserte individer viste nedsatt aktivitet i alle atferdsarenaene de ble testet i. I tråd med tidligere studier, fant jeg også at P. neurophilia-infeksjon reduserte vekst. Samlet sett tyder redusert aktivitet og vekst på at P. neurophilia utgjør en betydelig kostnad for sebrafiskens energiressurser. Antageligvis er denne kostnaden relatert til sebrafiskens immunrespons til infeksjonen. Atferdsendringene kan også skyldes at parasitten påvirker nervesystemet og hjernen mer direkte. Det andre delmålet i denne avhandlingen var derfor å karakterisere transkripsjonelle responser på P. neurophilia-infeksjon i hjernen. Mer spesifikt ville vi karakterisere immunrespons på infeksjon og identifisere biologiske prosesser påvirket av parasitten. RNA-sekvenseringsanalyse av hjernevev avslørte, ikke helt overraskende, at parasitten induserer en pro-inflammatorisk respons i sebrafiskehjernen. På den annen side tyder nedregulering av spesifikke immunrelaterte gener på at parasitten også utnytter spesifikke unnvikelsesstrategier for å unnslippe immunsystemet. En slik unnvikelsesstrategi kan være avgjørende for parasittens evne til å etablere kroniske infeksjoner. Til tross for at parasitten invaderer nerveceller i hjernen, fant vi ingen effekter på gener involvert i nervesystemets funksjon. Disse foreløpige funnene som viser at P. neurophilia reduserer aktivitet og vekst, og induserer en kraftig immunrespons i sebrafisk, peker på at infeksjonen er kostbar og kan øke sebrafiskens metabolske krav. Noen potensielle effekter på nervesystemet kan man heller ikke detektere ved hjelp av RNA-sekvensering (f.eks. monoaminresponser). Det tredje delmålet i avhandlingen var derfor å bestemme metabolske og nevrofysiologiske responser på akutt versus etablert infeksjon. I samsvar med våre forventninger fant jeg at P. neurophilia øker metabolsk rate hos sebrafisk. Økningen var riktignok størst tre dager etter akutt eksponering for P. neurophilia-sporer og uavhengig av tidligere infeksjonsstatus (ikke tidligere infisert versus etablert infeksjon). Interessant nok ble denne økningen i metabolisme reversert seks dager etter eksponering. Videre så jeg at akutt men ikke etablert parasitteksponering, økte monoaminerg (serotonerg og dopaminerg) aktivitet i hjernen til sebrafisken. Resultatene tyder på at metabolske og nevrofysiologiske effekter av P. neurophilia avhenger av tid etter siste eksponering og at akutte infeksjoner er mer kostbare enn etablerte infeksjoner. I og med at sebrafisk stadig utsettes for infeksiøse parasittsporer i akvariet, kan gjentatte akutte infeksjoner utgjøre en betydelig metabolsk kostnad for sebrafisk i forskningslaboratorier. Samlet viser forsøkene i denne avhandlingen at P. neurophilia-infeksjon reduserer aktivitet og vekst, induserer en kraftig immunrepons og øker fiskens metabolske omkostninger. Denne fenotypen kan minne om det man ser ved sykdomsatferd, en velkjent tilstand der dyr og mennesker tillegger seg energireduserende strategier for å bekjempe akutte infeksjoner. Resultatene viser også at P. neurophilia kan påvirke mange biologiske aspekter hos sebrafisk, som potentielt kan ha alvorlige konsekvenser for forskningsresultater, der man bruker sebrafisk med subklinisk P. neurophilia-infeksjon. Derfor fremhever resultatene viktigheten av ordentlig og standardisert helseovervåkning i forsøksfasiliteter som holder sebrafisk. Dette vil ikke bare forbedre dyrevelferd, men vil også være nødvendig for reproduserbarhet av forskning som benytter sebrafisk som forsøksdyr.publishedVersio

Behavioural effects of the common brain-infecting parasite Pseudoloma neurophilia in laboratory zebrafish (Danio rerio)

Research conducted on model organisms may be biased due to undetected pathogen infections. Recently, screening studies discovered high prevalence of the microsporidium Pseudoloma neurophilia in zebrafish (Danio rerio) facilities. This spore-forming unicellular parasite aggregates in brain regions associated with motor function and anxiety, and despite its high occurrence little is known about how sub-clinical infection affects behaviour. Here, we assessed how P. neurophilia infection alters the zebrafish´s response to four commonly used neurobehavioral tests, namely: mirror biting, open field, light/dark preference and social preference, used to quantify aggression, exploration, anxiety, and sociability. Although sociability and aggression remained unaltered, infected hosts exhibited reduced activity, elevated rates of freezing behaviour, and sex-specific effects on exploration. These results indicate that caution is warranted in the interpretation of zebrafish behaviour, particularly since in most cases infection status is unknown. This highlights the importance of comprehensive monitoring procedures to detect sub-clinical infections in laboratory animals.publishedVersio

Effects of the brain-infecting parasite Pseudoloma neurophilia in laboratory zebrafish (Danio rerio)

Zebrafish (Danio rerio) are increasingly popular model animals in scientific fields ranging from behavioural ecology to neurobiology. It is therefore of increasing concern that half of all zebrafish research facilities are contaminated with the brain-infecting microsporidian parasite Pseudoloma neurophilia. This parasite mainly aggregates in the hindbrain, where it causes chronic yet typically sub-clinical infections. Hence, infected fish often show no obvious disease symptoms and researchers are often unaware of infection status. Previous studies indicate that P. neurophilia reduces growth and alters shoaling behaviour and habituation to fearful stimulus, in zebrafish. These changes in behavioural phenotype suggest that the parasite may affect stress, anxiety and sociability in zebrafish. However, effects of this parasite on host phenotype remains largely uncharted. Thus, in this Thesis, I investigated behavioural, metabolic, neurophysiological and brain transcriptional effects of sub-clinical infections with P. neurophilia in the zebrafish host. The first aim of the Thesis was to identify behavioural effects of P. neurophilia infection in zebrafish across a range of contexts. To this end, infected and uninfected zebrafish were tested in commonly used behavioural paradigms, namely social preference, mirror biting, open field and light/dark preference tests. I found infection to not alter classic behavioural outputs such as sociability and aggression. However, infected individuals displayed reduced activity in all arenas. Furthermore, in accordance with previous studies, infection negatively affected growth, indicating that P. neurophilia is energetically costly for the zebrafish host. This cost is likely related to immune responses mounted by the host. Moreover, behavioural changes may indicate that the parasite has direct effects on the nervous system in zebrafish. The second aim of the Thesis was therefore to study brain transcriptional changes caused by infection. Specifically, I aimed to characterise the immune responses to infection and identify biological processes affected by the parasite. In line with my predictions, RNA-sequencing analysis revealed that the parasite induces a pro-inflammatory response in the zebrafish brain. However, a distinct downregulation of specific immune-related genes also suggests that the parasite takes advantage of specific immune evasion strategies. Surprisingly, P. neurophilia infection had no significant effects on genes related to nervous system function. The initial findings that P. neurophilia reduces growth and activity and induces proinflammatory responses in the brain, indicate that infection constitutes a considerable metabolic cost for the host. In addition, certain neurophysiological (e.g. monoaminergic) responses to infection may not be detectable by RNA-sequencing. Thus, the third aim of the Thesis was to determine metabolic and neurophysiological responses following acute and long-term P. neurophilia infections. In line with my predictions, P. neurophilia infection increased metabolic rate in zebrafish. However, the increase was highest three days after acute exposure (independent of whether the fish had an established infection or not) and mitigated again on day six. Furthermore, acute parasite exposure increased serotonergic and dopaminergic activity, but only in zebrafish with no previous history of infection (naïve). The results suggest that the metabolic and neurophysiological effects of P. neurophilia depends on time post last exposure and previous infection status and that metabolic costs are higher with acute compared to established infection. Since zebrafish frequently encounter infectious spores in their environment, repeated acute infections may represent a substantial metabolic cost to laboratory zebrafish. Taken together, the results obtained in this Thesis indicate that infection is associated with decreased activity and growth, a proinflammatory immune response and elevated metabolism in zebrafish. This phenotype is reminiscent of sickness behaviour (a condition in which acutely infected individuals adopt energy reducing strategies in order to fight infection). This thesis provides evidence that P. neurophilia can affect multiple biological aspects, that potentially have severe consequences for research outcomes. Hence, the findings highlight the importance of proper and standardised health monitoring in animal research facilities, not only for improving animal welfare, but also for ensuring research reproducibility

Behavioural effects of the common brain-infecting parasite Pseudoloma neurophilia in laboratory zebrafish (Danio rerio)

Research conducted on model organisms may be biased due to undetected pathogen infections. Recently, screening studies discovered high prevalence of the microsporidium Pseudoloma neurophilia in zebrafish (Danio rerio) facilities. This spore-forming unicellular parasite aggregates in brain regions associated with motor function and anxiety, and despite its high occurrence little is known about how sub-clinical infection affects behaviour. Here, we assessed how P. neurophilia infection alters the zebrafish´s response to four commonly used neurobehavioral tests, namely: mirror biting, open field, light/dark preference and social preference, used to quantify aggression, exploration, anxiety, and sociability. Although sociability and aggression remained unaltered, infected hosts exhibited reduced activity, elevated rates of freezing behaviour, and sex-specific effects on exploration. These results indicate that caution is warranted in the interpretation of zebrafish behaviour, particularly since in most cases infection status is unknown. This highlights the importance of comprehensive monitoring procedures to detect sub-clinical infections in laboratory animals

Effects of Pseudoloma neurophilia infection on the brain transcriptome in zebrafish (Danio rerio)

Laboratory zebrafish are commonly infected with the intracellular, brain‐infecting microsporidian parasite Pseudoloma neurophilia . Chronic P. neurophilia infections induce inflammation in meninges, brain and spinal cord, and have been suggested to affect neural functions since parasite clusters reside inside neurons. However, underlying neural and immunological mechanisms associated with infection have not been explored. Utilizing RNA‐sequencing analysis, we found that P. neurophilia infection upregulated 175 and downregulated 45 genes in the zebrafish brain, compared to uninfected controls. Four biological pathways were enriched by the parasite, all of which were associated with immune function. In addition, 14 gene ontology (GO) terms were enriched, eight of which were associated with immune responses and five with circadian rhythm. Surprisingly, no differentially expressed genes or enriched pathways were specific for nervous system function. Upregulated immune‐related genes indicate that the host generally show a pro‐inflammatory immune response to infection. On the other hand, we found a general downregulation of immune response genes associated with anti‐pathogen functions, suggesting an immune evasion strategy by the parasite. The results reported here provide important information on host–parasite interaction and highlight possible pathways for complex effects of parasite infections on zebrafish phenotypes