Fig 1.
Experimental setup and photoperiod regimes used in this study.
Sea bass were purchased and maintained from February until March under natural photoperiod (black line). On the 9th of March the fish were separated into three groups, one exposed to simulated natural photoperiod (NP, broken line), a second to continuous light (LL, spotted line) and a third to 9L: 15D (AP, continuous line). On the 2nd of April the AP group was switched to a long day light regime (15L: 9D) and on the 4th of May a short day regime (9L: 15D). Fish were sampled on the 5th, 6th and 8th of May. Horizontal diamonds indicate the spermiation period for the AP and NP groups. The LL group did not spermiate.
Table 1.
List of primers used in RT-qPCR and accession numbers of corresponding genes.
Fig 2.
Reproduction related gene network predicted by promoter framework analysis.
The genes presented in this network were predicted to be co-regulated with the circadian clock-gnrh2-kiss/kissr genes. Solid lines indicate direct interactions between network partners (nodes), broken lines indicate indirect actions between nodes. A black arrow with a solid line indicates that one node acts on the other and a black arrow with a broken line indicates the node acts indirectly on another node, while a white arrow with a solid line indicates interaction between nodes involving translocation.
Fig 3.
Transcript levels of clock genes in the brain of sea bass exposed to different photoperiods.
qPCR analysis was used to measure the transcript levels of clock1 and npas2 genes in whole brains of fish exposed to simulated normal (NP), accelerating (AP) and continuous light (LL). Each value is the mean ± SEM (n = 6 fish per group at each sampling time) of the relative expression. Asterisk (*) indicates significant differences of AP and LL fish relative to control fish (NP) (P<0.05).
Fig 4.
Transcript levels of reproductive axis genes in the brain of sea bass exposed to different photoperiods.
The transcript levels of the predicted reproductive axis-related genes (gnrh1, gnrh2, kiss2 and kiss1rb) was measured by qPCR in whole brains of sea bass exposed to simulated normal (NP), accelerating (AP) and continuous light (LL). Each value is the mean ± SEM (n = 6 individual fish per group at each sampling time) of the relative expression. Asterisk (*) indicates significant differences of AP and LL fish relative to control fish (NP) (P<0.05).
Fig 5.
Transcript levels of galanin 1 and estrogen 1 receptors in the brain of sea bass exposed to different photoperiods.
The transcript levels of galr1a, galr1b and esr1 were measured in whole brains of sea bass exposed to normal (NP), accelerating (AP) and continuous light (LL) using qPCR. Each value is the mean ± SEM (n = 6 fish per group at each sampling time) of the relative expression. Asterisk (*) indicates significant differences of AP and LL fish relative to control fish (NP) (P<0.05).
Fig 6.
Diagram summarizing the results obtained in this study.
The key genes involved in gonadotropin stimulation and activation of the BPG axis are highly responsive soon after photoperiodic shift and prior to pubertal activation. Accelerating photoperiod regimes, AP (red arrows), increased npas2, gnrh1, gnrh2, kiss2 and kiss1rb, demonstrating that light not only affects the circadian clock but also key genes involved in gonadotropin stimulation. Continuous light, LL (blue arrows), down-regulated gnrh1, kiss2 and esr1 but had no effect on kiss1rb. Several candidate genes involved in signalling the metabolic/nutritional status of the individual were identified and galr1b gene expression was upregulated by AP and down-regulated by LL. sst-somatostatin, gal- galanin, gcgr- glucagon receptor, lep- leptin, ghr- growth hormone receptor, clock- circadian locomotor output cycles kaput, npas2-neuronal PAS domain protein 2, bmal1 (artnl)- Aryl hydrocarbon receptor nuclear translocator-like, galr1a- galanin receptor 1a, galr1b- galanin receptor b, esr1- estrogen receptor 1, gnrh1- gonadotropin releasing hormone 1, gnrh2- gonadotropin releasing hormone 2, kiss1- kisspeptin 1, kiss2- kisspeptin 2, kiss1rb- kisspeptin 1 receptor b, kiss2r- kisspeptin 2 receptor, FSHb-follicle stimulating hormone beta, LHb- luteinizing hormone beta, SCN-suprachiasmatic nucleus.