Effects of two genes (DRD4 and SERT) on great tit (Parus major) behaviour and reproductive traits
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2018-09-19
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Abstract
Loomadel esinevaid püsivaid käitumistunnuseid nimetatakse käitumistüüpideks või ka isiksusteks. Siia kuuluvad näiteks julgus võtta riske, agressiivsus, sotsiaalsus ning ekstravertsus. Isiksuse avaldumist kujundavad lisaks keskkonnast tulenevatele teguritele ka geenid. Teades geene, mis kujundavad käitumist, on võimalik paremini mõista loomade kohanemisedukust erinevates keskkondades. Lisaks mõjutavad mitmed geenipiirkonnad inimesel ärevushäirete tekkimist, depressiooni ja ka skisofreenia avaldumist. Selleks, et uurida erinevaid käitumisega seotud aspekte ning geene, kasutatakse sageli mudelliike. Võrreldes inimesega on mudelina kasutatavatel liikidel enamasti oluliselt lühem elutsükkel, mis võimaldab lihtsamini uurida muu hulgas ka käitumise mõju paljunemisedukusele ning jälgida tunnuste päritavust ehk säilimist põlvkonnast põlvkonda.
Käitumistunnuste avaldumine toimub läbi keerulise mehhanismi, kus oluline osa on virgatsainetel, mis mõjutavad otseselt närvisüsteemi tööd. Peamised sellised ühendid on 1) dopamiin, mis mõjutab uurivat käitumist, 2) serotoniin, mis reguleerib ärevust ja agressiivsust ning 3) noradrenaliin, mis seostub motivatsiooniga. Nii on käitumise uurimisel peamised kandidaatgeenid need, mille puhul on näidatud olulist mõju virgatsainete toimimisele. Et aga varasemates uurimustes on saadud käitumisgeenide mõjude osas vastukäivaid tulemusi, siis on oluline teha uuringuid erinevates keskkondades ja populatsioonides.
Praeguses teadustöös sooviti vastust küsimusele, kas varasemalt uuritud kaks geenipiirkonda, mis mõjutavad inimesel ning loomadel ärevust (serotoniini transporteri geen/SERT geen) ning uurivat käitumist või riskijulgust (dopamiini retseptori geen D4/DRD4 geen) omavad olulist mõju ka lindude käitumisele. Varasemad uuringud on näidanud, et DRD4 geen mõjutab rasvatihaste ja primaatide uurivat käitumist, koerte impulsiivsust ning hiirte hüperaktiivsust. Rasvatihasel, kes on üks paremini uuritud liike, pole aga kõigis populatsioonides seoseid DRD4 geeni ja käitumise vahel leitud. Seega uuriti praeguses teadustöös DRD4 geeni ja käitumise vahelisi seoseid nii välikatses kui ka aviaariumis (katseruumis), et mõista geeni mõju erinevates keskkondades. Teiseks, SERT geeni on kõige rohkem uuritud inimestel seoses erinevate käitumishäiretega (näiteks depressioon, ärevus ning suitsiid). Ka teistel imetajatel mõjutavad SERT geeni piirkonnad just ärevusega seotud käitumist. Lindudel on aga seda geeni veel võrdlemisi vähe uuritud, ehkki on näidatud, et mitmed SERT geeni piirkonnad mõjutavad lindudel impulsiivsust ning reaktsiooni uudsele objektile või olukorrale. Seega, käesoleva töö teine uurimisküsimus keskendus SERT geeni ja käitumise vahelistele seostele lindudele loomuomases keskkonnas. Kolmandaks, kuna DRD4 ja SERT geenid on seotud ka paljunemisedukuse ja kaaslase valikuga, uuriti teadustöös nende geenide mõju lindude pesitsusedukusele.
Kui eelnevad käitumiskatsed ja geeniuuringud on tehtud enamasti laboritingimustes, siis praegune töö viidi läbi loodusliku rasvatihaste populatsiooniga ning lindudele omases keskkonnas. See võimaldab genotüübi ja käitumise vahelisi seoseid laiemalt testida, sest laboris ei pruugi kõik käitumistunnused alati avalduda. Lisaks tehti suur osa katsetest pesitsusperioodil, et hinnata käitumisgeenide mõju paljunemisedukusele. Läbiviidud käitumiskatsete eesmärgiks oli jälgida isendite hakkamasaamist uudse või stressirohke olukorraga. Põhikatses filmiti vanalindude toitmiskäitumist (pesapoegade toitmist) Edela-Eestis Kilingi-Nõmme katsealal. Katsetes vaadeldi nii tavalist toitmissagedust, kui hinnati uudsest objektist tingitud toitmispausi pikkust. Sarnane korduskatse viidi läbi ka kontrollitud tingimustes ja enne pesitsusaega, et välistada keskkonnast tulenevad mõjutusi. Lisaks hinnati emaslindude reaktsiooni võimalikule sissetungijatele ning vanalindude käitumist lõksuminekul (tugev stressiolukord). Lisaks käitumiskatsetele määrati lindude genotüüp nii DRD4 kui SERT geeni osas.
Töö tulemusena selgus, et mõlemad geenid (DRD4 ja SERT) on Eesti rasvatihaste populatsioonis polümorfsed, st geenipiirkonnad varieeruvad isendite vahel. Mõlemad geenid seostusid ka lindude käitumisega. DRD4 geen mõjutas isaslindude uurivat käitumist nii pesitsusajal vabas looduses kui ka laborikatses (aviaariumis). Emaslindudel ilmnes DRD4 geeni mõju käitumisele aviaariumikatses enne pesitsusaega, aga mitte vabas looduses poegade toitmise ajal. Küll mõjutas emaslindude käitumist vabas looduses pesakonna suurus, mis tõenäoliselt võis varjutada geeni mõju käitumisele. Lisaks uuriti selles katses ka lindude stressitaset. Kõige madalam stressihormooni tase oli nendel isenditel, kellel olid olemas DRD4 geeni erinevad alleelid (heterosügoodid). Uudsele objektile lähenedes olid need linnud kõige altimad ja kiiremad lähenejad ning vabastamisel tegid nad ka kõige sagedamini hoiatushüüde. Sarnaselt on varasemalt rasvatihastel leitud, et hoiatushüüdude suurem hulk näitab tõepoolest julgemat käitumist. Ka madal stressihormooni tase viitab, et need indiviidid võiksid stressiolukorras paremini hakkama saada. Seega, vaatamata väikestele erinevustele aviaariumi ja katsete vahel, mida tehti vabas looduses, on tõenäoline, et DRD4 mõjutab oluliselt lindude käitumisotsustuste kujunemist ning võib olla seotud ka stressitaseme kujunemisega.
Teiseks uuriti SERT geeni mõju tihaste käitumisele. Selgus, et rasvatihastel esineb selles geenis mitmeid polümorfseid piirkondi (SNPd), mis võivad käitumist mõjutada. Uuriva käitumisega tundmatu objekti suhtes oli seotud polümorfism, mis paikneb geeni kolmandas eksonis. Et katses kasutati sama käitumistunnust, mida DRD4 geeni puhulgi, kontrolliti ühtlasi seoseid ka DRD4 geeni ja SERT geeni vahel. Ehkki sellised geenid võivad olla aheldatud ja koos päranduda, siis uuritud tihasepopulatsioonis sellist seost ei leitud. SERT geeni uuringud näitasid ka, et geen mõjutab enam neid olukordi, mis on tihaste jaoks stressirohkemad ja ohustavamad kui DRD4 geeni puhul. Näiteks leiti seos SERT geeni ekson 8 SNP197 ja lõksumineku vahel. Stressirohke olukord loodi ka katses emaslindudega haudeperioodil, kus SERT geeni mõju käitumisele ilmnes siis, kui pesa ohustas võimalik sissetungija. Katses imiteeriti rähnitopisega rünnakut pesakastile ning leiti, et polümorfism SERT geeni eksonis 1 mõjutas seda, kas emaste reageering on aktiivne (susin ning rünnak topise suunas) või passiivne (ei tee midagi). Kokkuvõtvalt, serotoniini transporteri geen on oluline tegur lindude käitumise kujundamisel erinevates olukordades. Geen mõjutas nii reageeringut uudsele objektile, lõksule kui ka sissetungijale ning omas märkimisväärselt suuremat mõju käitumisele olukordades, mis põhjustavad rohkem ärevust. Nii DRD4 kui SERT geeni puhul uuriti nende mõju lindude pesitsusedukusele. Seega vaadeldi lisaks erinevaid pesitsusega seotud tunnuseid nagu näitkes munade arvu, munemise algusaega, koorunud poegade arvu, pesakonna suurust ning poegade arvu lennuvõimestumisel. Enamik neist tunnustest polnud kummaski kandidaatgeenis käitumist mõjutavate polümorfismidega otseselt seotud. Küll aga leiti seos SERT eksonis 8 paikneva SNP197 ja pesakonna suuruse vahel. Suuremate pesakondadega olid heterosügoodid, kes ühtlasi näitasid käitumiskatses julgemat käitumist uudse objekti suhtes. Teine polümorfism (SNP457) eksonis 8 seostus koorunud poegade arvuga. See SNP pole küll käitumisega otseselt seotud, kuid mõjutas tõenäoliselt kuidagi otsust, millal munemist alustada. Nii võib arvata, et pesitsusedukus tihastel küll olla mõjutatud SERT poolt käitumise kaudu, kuid on vähe või pole seotud DRD4 geeniga.
Kokkuvõtvalt, töö tulemusena selgus nii DRD4 kui ka SERT geeni mõju tihaste uurivale käitumisele ning riskijulgusele. Oluline on märkida, et vabas looduses võib pesitsusaeg mõjutada DRD4 geeni ja käitumise vahelisi seoseid. Seega tuleks edasistes uuringutes omavahel võrrelda erinevaid keskkondi ning perioode, mõistmaks olulisi tegureid, mis käitumist võivad kujundada. Mõõtmised peamiselt laboritingimustes võivad olla ka ühtlasi põhjuseks, miks varasemates töödes pole seoseid DRD4 geeni ja käitumise vahel alati leitud. SERT geenis kirjeldatud polümorfismid osutavad geenipiirkondadele, mida pole varasemalt uuritud, kuid, millega tasub tulevikus edasi töödata, kuna need SNPd omavad mõju käitumisele. Seejuures on oluline silmas pidada neid polümorfisme SERT geenis, mis kujundavad pesitsusedukust ja suunavad seeläbi looduslikku valikut. Teades käitumist mõjutavaid geene, on võimalik keskenduda põhjuslikele seostele, mis kujundavad käitumisvastuseid. See omakorda aitab mõista kohanemisedukust nii tihastel, inimestel kui ka muudel loomaliikidel.
Genetic studies give the opportunity to understand potential mechanisms underlying individual behavioural variation. Behaviour varies between individuals but the individual response over the situations and environments stays often persistent. Adapting the reaction to situation or being behaviourally more plastic in changing environment is costly and often limited. Limits of behavioural plasticity are affected by the environment itself, other behavioural traits and genes. Therefore, knowing these genes that affect behaviour and the neurotransmitter networks behind behavioural responses, enables to understand individual variation in behaviour. Studies of behavioural genetics also include behavioural disorders in humans, such as depression, anxiety and schizophrenia; adaptation in animals (eg reintroduction and colonization) and finally domestication and selective breeding in farming. Behavioural traits are a part of complex system that involves nerve system, hormones, neurotransmitters and genes. Two main candidate genes that are widely studied have been associated with anxiety and novelty-seeking and are related to dopamine and/or serotonin neurotransmission. These are the serotonin transporter gene (SERT) and the dopamine receptor gene D4 (DRD4). Given that dopamine and serotonin regulate behavioural responses and are related to the variation in individual genotypes, the main focus of the thesis was to study the effect of these candidate genes on behavioural traits in a natural population. As most of the experiments were conducted during the breeding season, reproductive traits were also included to analysis. To study this, different behavioural experiments were conducted both in the aviary and in the wild. The great tit was used as a model species that allowed to conduct experiments both in the wild and in the captivity. Individual behavioural variation was measured at the presence of novel object, the nest box trap and during the simulated predator attack. Most of the behavioural experiments were conducted during the breeding season that enabled to include reproductive traits to the analysis. For gene analyses, blood samples were collected for DNA separation and sequencing. The results show that candidate genes (DRD4 and SERT) are polymorphic among Estonian great tits. Moreover, individual behavioural response to novel object was affected by both candidate genes. The DRD4 gene is related to male behavioural response in the natural environment and in the aviary. In female great tits, there was no effect of the DRD4 gene on behaviour in the experiment conducted in the wild. However, while conducting the experiment in the aviary and before the start of the breeding season, a similar correlation between the DRD4 gene and behaviour was found in females as well. Therefore, potential effect of the breeding season on female behaviour play role as the brood size was related to female behavioural response. As novel situations could be stressful, individual corticosterone (CORT) levels were included to the studies. Stress hormone levels were correlated with individual vocal response during release after the aviary experiment. Moreover, the CORT levels were related to DRD4 gene. The lowest levels of acute CORT were observed in CT heterozygotes suggesting that these individuals are less influenced by handling stress. Indeed, when facing the novel object, CT heterozygotes were bolder to approach. Also, when birds were released, CT heterozygotes conducted most of the alarm calls that could refer to bolder behaviour. The SERT gene polymorphisms were also related to the behavioural reaction to the novel object as a SNP in exonic region 3 correlates with behavioural reaction at the presence of different types of novelty: a camera (inducing the shortest delay), an object on the nest box and the nest box trap (inducing the longest delay). Moreover, the SERT gene seems to play significant role when the situation is more stressful. A SNP in the SERT gene exon 8 is only related to behavioural response towards the trap on the nest box as the carriers of the T allele were bolder than CC homozygotes. Also, a SNP in the exonic region 1 is associated with individual risk taking behaviour during simulated predator attack during incubation period. Behavioural adaptation could also affect the frequencies of the SERT gene polymorphisms. A pilot sample of urban and rural birds showed that one particular SNP (SNP234) that is present in former studies and relates to explorative behaviour is missing in Estonian rural population. In urban birds (especially nestlings), the SNP234 was present. Most of the behavioural traits in the current thesis were conducted during the breeding season that enabled to study several reproductive traits toghether with behavioural decisions that might shape breeding success in birds. Therefore, as DRD4 and SERT genes are related to behavioural decisions at the presence of novelty, it was assumed that these genes could play role in breeding success as well. However, the DRD4 gene was not directly related to any reproductive trait studied. The SERT gene polymorphism in exon 8 SNP457 was related to the start of egg-laying even though this particular SNP had no effect on behaviour. Also, the second polymorphism in exon 8, SNP197 was significantly related with the number of hatched young where heterozygotes had larger broods compared to homozygotes. This SNP also affected individual behavioural response at the presence of the trap where heterozygotes were bolder to enter than homozygotes. Therefore, the SERT gene polymorphisms could be either directly or indirectly related to individual fitness as several gene regions in different tests seem to have some effect on breeding success. In conclusion, the aim of this thesis was to study the effect of two candidate genes on bird behaviour and reproductive traits. The results show that DRD4 and SERT genes are important in shaping behavioural decisions at the presence of different stressors and novelty. Moreover, the stress hormone levels could affect the behavioural response related to the DRD4 gene. The SERT gene might be either directly or indirectly related to reproductive traits. The study with DRD4 gene also indicates that conducting experiments in different environments and seasons could have significant effect on individual behaviour. Therefore, in subsequent studies, picking the study site and timing of the experiment should be considered carefully. Differences in the environment could be also the reason why no correlation between the candidate genes and behaviour was found in some populations. By knowing the effects of candidate genes on behaviour, it is possible to expand studies to causal mechanisms that shape behavioural traits.
Genetic studies give the opportunity to understand potential mechanisms underlying individual behavioural variation. Behaviour varies between individuals but the individual response over the situations and environments stays often persistent. Adapting the reaction to situation or being behaviourally more plastic in changing environment is costly and often limited. Limits of behavioural plasticity are affected by the environment itself, other behavioural traits and genes. Therefore, knowing these genes that affect behaviour and the neurotransmitter networks behind behavioural responses, enables to understand individual variation in behaviour. Studies of behavioural genetics also include behavioural disorders in humans, such as depression, anxiety and schizophrenia; adaptation in animals (eg reintroduction and colonization) and finally domestication and selective breeding in farming. Behavioural traits are a part of complex system that involves nerve system, hormones, neurotransmitters and genes. Two main candidate genes that are widely studied have been associated with anxiety and novelty-seeking and are related to dopamine and/or serotonin neurotransmission. These are the serotonin transporter gene (SERT) and the dopamine receptor gene D4 (DRD4). Given that dopamine and serotonin regulate behavioural responses and are related to the variation in individual genotypes, the main focus of the thesis was to study the effect of these candidate genes on behavioural traits in a natural population. As most of the experiments were conducted during the breeding season, reproductive traits were also included to analysis. To study this, different behavioural experiments were conducted both in the aviary and in the wild. The great tit was used as a model species that allowed to conduct experiments both in the wild and in the captivity. Individual behavioural variation was measured at the presence of novel object, the nest box trap and during the simulated predator attack. Most of the behavioural experiments were conducted during the breeding season that enabled to include reproductive traits to the analysis. For gene analyses, blood samples were collected for DNA separation and sequencing. The results show that candidate genes (DRD4 and SERT) are polymorphic among Estonian great tits. Moreover, individual behavioural response to novel object was affected by both candidate genes. The DRD4 gene is related to male behavioural response in the natural environment and in the aviary. In female great tits, there was no effect of the DRD4 gene on behaviour in the experiment conducted in the wild. However, while conducting the experiment in the aviary and before the start of the breeding season, a similar correlation between the DRD4 gene and behaviour was found in females as well. Therefore, potential effect of the breeding season on female behaviour play role as the brood size was related to female behavioural response. As novel situations could be stressful, individual corticosterone (CORT) levels were included to the studies. Stress hormone levels were correlated with individual vocal response during release after the aviary experiment. Moreover, the CORT levels were related to DRD4 gene. The lowest levels of acute CORT were observed in CT heterozygotes suggesting that these individuals are less influenced by handling stress. Indeed, when facing the novel object, CT heterozygotes were bolder to approach. Also, when birds were released, CT heterozygotes conducted most of the alarm calls that could refer to bolder behaviour. The SERT gene polymorphisms were also related to the behavioural reaction to the novel object as a SNP in exonic region 3 correlates with behavioural reaction at the presence of different types of novelty: a camera (inducing the shortest delay), an object on the nest box and the nest box trap (inducing the longest delay). Moreover, the SERT gene seems to play significant role when the situation is more stressful. A SNP in the SERT gene exon 8 is only related to behavioural response towards the trap on the nest box as the carriers of the T allele were bolder than CC homozygotes. Also, a SNP in the exonic region 1 is associated with individual risk taking behaviour during simulated predator attack during incubation period. Behavioural adaptation could also affect the frequencies of the SERT gene polymorphisms. A pilot sample of urban and rural birds showed that one particular SNP (SNP234) that is present in former studies and relates to explorative behaviour is missing in Estonian rural population. In urban birds (especially nestlings), the SNP234 was present. Most of the behavioural traits in the current thesis were conducted during the breeding season that enabled to study several reproductive traits toghether with behavioural decisions that might shape breeding success in birds. Therefore, as DRD4 and SERT genes are related to behavioural decisions at the presence of novelty, it was assumed that these genes could play role in breeding success as well. However, the DRD4 gene was not directly related to any reproductive trait studied. The SERT gene polymorphism in exon 8 SNP457 was related to the start of egg-laying even though this particular SNP had no effect on behaviour. Also, the second polymorphism in exon 8, SNP197 was significantly related with the number of hatched young where heterozygotes had larger broods compared to homozygotes. This SNP also affected individual behavioural response at the presence of the trap where heterozygotes were bolder to enter than homozygotes. Therefore, the SERT gene polymorphisms could be either directly or indirectly related to individual fitness as several gene regions in different tests seem to have some effect on breeding success. In conclusion, the aim of this thesis was to study the effect of two candidate genes on bird behaviour and reproductive traits. The results show that DRD4 and SERT genes are important in shaping behavioural decisions at the presence of different stressors and novelty. Moreover, the stress hormone levels could affect the behavioural response related to the DRD4 gene. The SERT gene might be either directly or indirectly related to reproductive traits. The study with DRD4 gene also indicates that conducting experiments in different environments and seasons could have significant effect on individual behaviour. Therefore, in subsequent studies, picking the study site and timing of the experiment should be considered carefully. Differences in the environment could be also the reason why no correlation between the candidate genes and behaviour was found in some populations. By knowing the effects of candidate genes on behaviour, it is possible to expand studies to causal mechanisms that shape behavioural traits.
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Keywords
great tit, behavior, reproduction (biol.), animal experiments, dopamine, serotonin, genes, neurotransmitters, behaviour modifications, nest