Darkness at night during the new moon period alters the expression levels of the clock genes in the brain of a moon-related spawner, the Malabar grouper Epinephelus malabaricus

ABSTRACT Some fish in tropical and subtropical waters exhibit moon-related rhythmicity in their reproduction and migration. We investigated the involvement of the subtypes of cryptochrome (mgCry) in moon-related rhythmicity of the Malabar grouper Epinephelus malabaricus, which spawns around the new moon period. Under natural photoperiodic conditions, the levels of mgCry1 and mgCry2 showed daily variation with peaks at 11:00 and 19:00, respectively, in the diencephalon (including the pituitary), regardless of the moon phase. The weekly changes in mgCry2, but not mgCry1, were moon-dependent, with higher levels around the new moon. When fish were reared under natural moonlight, mgCry2 in the pituitary was higher at 15:00 and 11:00 during the new moon period than the full moon period. Rearing fish under moonlight-exposed or moonlight-blocked conditions resulted in increases in mgCry genes in the pituitary during the night at full moon, suggesting that the pituitary is a target organ of the moonlight-dependent clock system. These findings indicate that mgCry genes in the brain of the Malabar grouper exhibits moon dependency, with changes according to the duration of darkness at night. We conclude that weekly changes in Cry genes in the brain play a role in entraining moon-dependent events in the Malabar grouper.


Introduction
In certain fish inhabiting shallow waters, cues from the moon become a useful zeitgeber and are utilized for entrainment of activity, including reproduction and migration (Takemura et al. 2010).Typical examples of this rhythmic entrainment are found in tropical groupers (Epinephelinae), snappers (Lutjanidae), and spinefoots (Siganidae), in which migration to and aggregation at spawning grounds and subsequent spawning are simultaneously repeated around a specific moon phase.The spiny spinefoot Siganus spinus, the white-spotted spinefoot S. canaliculatus, and the Leopard coral grouper Plectropomus leopardus all show a preference for a new moon for spawning (Samoilys 1997;Hoque et al. 1999;Harahap et al. 2001).The streamlined spinefoot S. argenteus and the goldlined spinefoot S. guttatus spawn during the last quarter moon and the first quarter moon period, respectively (Rahman et al. 2003), and spawning around the full moon has been confirmed in the honeycomb grouper Epinephelus merra (Lee et al. 2002).Such synchrony can lead to increased breeding success for adults and decreased predation risk for offspring (Ikegami et al. 2014).It may also lead to allochronic isolation among different species.
Clock genes may play a role in this phenomenon in scleractinian corals (Levy et al. 2007) and Fishes (Fukushiro et al. 2011;Toda et al. 2014;Takeuchi et al. 2018;Fukunaga et al. 2020).Among the core clock components (period [PER], circadian locomotor output cycles kaput [CLOCK], aryl hydrocarbon receptor nuclear translocator like 1 [BMAL1], and cryptochrome [CRY]), CRY has attracted special attention because it serves as a blue light photoreceptor in the fruit fry Drosophila melanogaster (Lin and Todo 2005) and a light-responsive gene in a light-responsive zebrafish (Danio rerio) cell line (Cermakian et al. 2002).In Levy et al. (2007), Cry2 was upregulated at midnight of the full moon in the stony coral Acropora millepora (Levy et al. 2007).Elevated expression levels of Cry1a, Cry2, and Cry3 have been reported at full moon, the last quarter moon, and the new moon, respectively, in the diencephalon of the goldlined spinefoot, in fish reared under natural moonlight (Takeuchi et al. 2018).In another study, Cry1 and Cry2 were upregulated in the diencephalon and pituitary gland of honeycomb grouper reared under artificial full moon conditions (Fukunaga et al. 2020).Therefore, it is likely that there is an interplay between moonlight and clock genes.
Previous studies on moon cycles in this context have focused on sexually mature fish because reproductive activities show marked moon-related cycles (Takeuchi et al. 2018;Fukunaga et al. 2020).However, it is reasonable to assume that moon-related phenomena can also occur in sexually immature fish.Indeed, spinefoot juveniles migrate synchronously from the open ocean to coastal regions at specific moon phases (Soliman and Yamaoka 2010).This suggests that fish perceive cues from the moon throughout their life history.This may be partially confirmed by examining the levels of clock gene transcripts in the brain of immature fish according to the moon cycle.Hence, in this study, we examined the expression profiles of Cry genes (mgCry1, mgCry2, and mgCry3) in the brain (specifically, the diencephalon, and pituitary gland) of the Malabar grouper Epinephelus malabaricus, a moon-related spawner with a preference for the new moon phase, which has a wide distribution from tropical to temperate regions and is of high economic value in Asian markets.Since aquaculture of the Malabar grouper is expanding, our findings help to develop the artificial and efficient breeding protocol based on the moon-related spawning cycle in this species.Using sexually immature individuals of this species, we compared daily profiles of these genes in four moon phases: new moon, the first quarter moon, full moon, and the last quarter moon.In addition, changes in the transcript levels of these genes according to moonlight conditions were assessed.

Animals and experimental design
All experiments were conducted in compliance with the guidelines of the Animal Care and Use Committee of the University of the Ryukyus and the regulations for the care and use of laboratory animals in Japan.
Juvenile Malabar grouper (19.1 ± 1.5 cm in total length) were obtained from Okinawa Prefectural Sea Farming Center, Motobu, Okinawa, Japan, and transferred to Sesoko Station, Tropical Biosphere Research Center (TBRC), University of the Ryukyus, Motobu, Okinawa, Japan.They were housed in outdoor stock tanks (capacity 3 metric tons) with aerated running seawater and fed daily (Higashimaru, Kagoshima, Japan) at 10:00 under conditions of natural photoperiod and ambient water temperature until the experiments.
As mgCry2 has already been cloned (Yamashina et al. 2019), we cloned and characterized only mgCry1 and mgCry3 (Experiment 1).Fish (n = 8) were taken from the outdoor stock tanks, anesthetized on ice, and then sacrificed by decapitation.The whole brain was dissected from four individuals and used for molecular cloning.The brains from another four individuals were dissected and then separated into various specific regions, including the diencephalon, telencephalon, optic tectum, pituitary, cerebellum, and medulla oblongata.In addition, the retina and several peripheral tissues (heart, liver, spleen, gill, intestine, and ovary) were sampled from four individuals.These samples were immediately frozen in liquid nitrogen and then stored at −80°C until RNA extraction.
To examine the moon-related expression profiles of the mgCry gene (Experiment 2), fish were kept in outdoor tanks (capacity 3 metric tons) with aerated running seawater for 1 month under a natural photoperiod in June (sunrise and sunset times, 06:00 and 20:00, respectively) and water temperature at Sesoko Station.After the acclimatization period, six individuals were taken from the tanks every 4 h from 12:00 in each moon phase.After anesthetization on ice, the fish were sacrificed by decapitation, the whole brain was removed, and the diencephalon and pituitary were separated.These samples were immediately frozen in liquid nitrogen and then stored at −80°C until RNA extraction.
The effects of blocking moonlight on the expression profiles of mgCry genes in the brain were examined during the full moon period (Experiment 3) in November (sunrise and sunset times, 06:30 and 17:47, respectively).This was carried out based on the experimental design by Takemura et al. (2004).Briefly, fish were housed in four floating cages (90 cm × 50 cm × 50 cm) in outdoor tanks with natural photoperiod and water temperature.After a 1-month acclimatization period, two floating cages were covered with a black sheet from sunset to sunrise during the full moon period to prevent exposure of the fish to moonlight (moonlight-blocked group).Fish in the residual cages were exposed to moonlight (moonlight-exposed group).Six individuals in both groups were taken every 4 h from 15:00.After anesthetization on ice, the pituitary gland was dissected out, frozen in liquid nitrogen, and stored at −80°C until RNA extraction.

Cloning
Total RNA was extracted from tissues using Tripure Isolation Reagent (Roche Diagnostics, Indianapolis, IN, U.S.A.) according to the manufacturer's instructions.First-strand cDNA was reverse transcribed from 1 µg total RNA using the reverse transcriptase supplied with the Prime Script RT reagent in a gDNA eraser kit (Takara Bio, Kusatsu, Japan) according to the manufacturer's protocol.
The predicted amino acid sequences encoded by mgCry1 and mgCry3 cDNAs were deduced using the ORF Finder program (NCBI: http://www.ncbi.nlm.nih.gov/projects/gorf/). Sequence identity was verified by searching the NCBI database using BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi).ClustalX2 was used to generate a neighbor-joining phylogenetic tree with bootstrap confidence values based on 1000 replicate (Felsenstein 1985).

Tissue distributions of mgCry1 and mgCry3 expression
The tissue distributions of mgCry1 and mgCry3 expression were examined using a CFX96 Real Time System (Bio-Rad, Hercules, CA, U.S.A.) and Go Taq qPCR Master mix (Promega).Total RNA extracted from each tissue (1 µg) was treated with gDNA Eraser at 37°C for 15 min to avoid contamination with genomic DNA and then used for cDNA synthesis with a PrimeScript RT Reagent Kit and gDNA Eraser (Takara Bio).The obtained cDNAs were subjected to PCR amplification with primer pairs specific for mgCry1 and mgCry3.For the detection of mgCry1 and mgCry3 mRNAs, PCR was carried out in a final volume of 10 µL containing 5 µL 2× Go Taq qPCR Master mix, 0.3 µL forward and reverse primers, 2.4 µL nuclease-free water, and 2 µL cDNA template.The cycling conditions for qPCR were denaturation at 95°C for 2 min followed by 40 cycles of 95°C for 15 s, and 60°C for 1 min.A standard curve was created using 10-fold dilutions of plasmid DNA standard and qPCR with specific primers in accordance with the protocol provided by Applied Biosystems (Foster City, CA, U.S.A.).The housekeeping gene Elongation factor 1 alpha (mgEf1α) was used to normalize the transcript levels of reproduction-related genes.Measurements of mgCry1, mgCry3, and mgEf1α expression were made in duplicate.The relative mRNA expression levels of mgCry1 and mgCry3 were calculated using the ΔΔ Ct method.Verification of mgEf1α as an internal control gene was previously checked by comparing transcript levels among three candidate genes, i.e. mgActin, mgEf1α, and mgRpl8 (Yamashina et al. 2019).

Statistical analysis
All data are expressed as the mean ± standard error of the mean (SEM).Data regarding daily variation in mgCrys around the four moon phases were analyzed by one-way analysis of variance (ANOVA) with the Tukey -Kramer multiple comparison test.The transcript levels of mgCrys were compared between two moon phases using Student's t test.In all analyses, P < .05 was taken to indicate statistical significance.

Molecular cloning and characterization of mgCry1 and mgCry3
The partial sequences of the cloned mgCry1 (512 bp) and mgCry3 (1171 bp) had open reading frames encoding protein products of 161 and 390 amino acids, respectively, with high levels of amino acid sequence identity to homologs from other species: 98% for mgCRY1 with those of other vertebrates (Supplementary Figure S1) and 85-95% for mgCRY3 with those of other teleosts (Supplementary Figure S2).Phylogenetic analysis of CRY was carried out (see Figure 1) using mgCRY1 (BBJ06076.1),mgCRY3 (BBJ06077.1),and mgCRY2 (BBJ06078.1;Supplementary Figure S3).These exclusively clustered with the CRY group, where mgCRY2 and mgCRY3 belonged to the teleost CRY1 and CRY3 groups, respectively (Figure 1).Both mgCry1 and mgCry3 were expressed in all tissues examined in the present study, with neural tissues showing particularly high levels of both gene transcripts (Supplementary Figure S4).

Daily expression of mgCry genes according to the moon cycle
The levels of mgCry gene transcripts in the diencephalon, including the pituitary gland, were measured at 4 h intervals by qPCR (Figure 2).Each mgCry showed a similar daily profile across the four moon phases.The transcript levels of mgCry1 and mgCry2 increased during the photophase, peaking at 11:00 and 19:00, respectively.By contrast, that of mgCry3 showed minimal daily fluctuation, but increased at 11:00 and 03:00.The daily fluctuations of mgCry1 and mgCry2 were statistically significant for all phases.However, a significant difference was only seen during the last quarter moon for mgCry3.
Next, the transcript levels of mgCry1 and mgCry2 were compared between the peaks and troughs of each moon phase (Figure 3).This figure is the subset of data replotted from Figure 2. Since we became aware of the difference between the peaks and troughs of each moon phase, we reanalyzed and replotted the data to account for these variations.No significant differences were observed for mgCry1 between the peak time (11:00) and trough time (23:00).On the other hand, the transcript level of mgCry2 was significantly higher at the peak time (19:00) than at the trough time (03:00).
Next, daily variation in the expression of the mgCry gene in the pituitary gland was evaluated between the full and new moon periods (Figure 4).There were significant day-high and night-low variations in the transcript levels of mgCry1 and mgCry2 but not mgCry3.A significant difference between the two moon phases was found at 15:00 and 11:00 specifically for mgCry2, with higher levels observed around the new moon.

Effects of blocking moonlight on cry gene transcripts
The groups were divided and housed as described in the Methods.The moonlightblocked group showed significant differences in mgCry gene transcript levels in the pituitary gland (Figure 5).This group showed significantly higher transcript levels at 19:00 and 11:00 for mgCry1; at 19:00, 11:00, and 15:00 for mgCry2; and at various time points except 15:00 and 19:00 for mgCry3.However, there were no significant differences in the transcript levels of these three mgCry genes at different time points in the diencephalon between the moonlight-exposed and moonlight-blocked groups (Figure 5).
previously (Yamashina et al. 2019), we focused on these three genes.Based on the phylogenetic analysis, it is possible that mgCry genes have similar function to Cry genes, which are molecular components of circadian clock and play a key role in the transcriptional and translational feedback loops (Alifu et al. 2021;Iida et al. 2022).In addition, mgCry genes may be involved in light-dependent behavioral rhythms because PER2, CRY1a and CRY1b in zebrafish helps the synchronization of cellular clock in a lightdependent manner and contribute to behavioral rhythm formation (Hirayama et al. 2019).
When fish were reared under a natural day -night cycle, the transcript levels of mgCry1 (peak at 11:00) and mgCry2 (peak at 19:00) in the diencephalon (with the pituitary gland) showed clear daily variation with an increase during photophase around all moon phases.These findings are in good agreement with the daily profiles of Cry1 and Cry2 transcript levels reported previously in the brain and pituitary gland of the European seabass Dicentrarchus labrax (Del Pozo et al. 2012;Herrero and Lepesant 2014), the diencephalon of the goldlined spinefoot S. guttatus (Takeuchi et al. 2018), and the diencephalon and pituitary gland of the honeycomb grouper E. merra (Fukunaga et al. 2020).Taken together, these findings suggest that Cry genes are light-responsive in fishes and play primary roles in their circadian systems, although they show different responses among species under the light -dark cycle.Experiments involving light manipulation have supported this notion, where Cry genes are upregulated in various organs and cultured cell lines of certain fish species; in one study, exposure to a 1 h light pulse induced increases in the levels of Cry1a and Cry2a expression in PAC2 cells and larvae of zebrafish (Weger et al. 2011).In another, rearing under conditions of constant light and constant darkness resulted in upregulation of Cry1 and Cry2, respectively, in the brain of the tropical damselfish Chrysiptera cyanea (Takeuchi et al. 2015).In still another, transcriptomic analyses identified 13 light-responsive genes, including Cry1 and Cry2, in the skin, brain, and liver of the Southern platyfish Xiphophorus maculatus (Lu et al. 2018).As we observed no daily variation in the transcript levels of mgCry3 in the diencephalon (with pituitary gland) around the new moon, first quarter moon, or full moon periods, this gene The peak times (11:00 for mgCry1 and 19:00 for mgCry2) and the bottom times (23:00 for mgCry1 and 03:00 for mgCry2) were selected from Figure 2 and reorganized according to the moon phase: the new moon (NM), first quarter moon (FQM), full moon (FM), and last quarter moon (LQM).Each value is expressed as mean ± standard error of the mean (SEM).Different letters indicate a significant difference in the transcript levels of each gene within a day at a significant level of P < .05.  and c), diencephalon (d, e, and f) of the Malabar grouper.After acclimatization, fish (six individuals per each sampling point) in a cage were exposed to moonlight throughout the night during the full moon (the moonlight-exposed group), while the other group were experienced disrupted moonlight during the same period (the moonlight-disrupted group).After the brain was taken from fish at 4-hour intervals, the transcript levels of mgCry genes in the pituitary gland, diencephalon, and telencephalon were measured using qPCR.Open and solid circles indicate the moonlight-exposed and moonlight-disrupted groups, respectively.The transcript levels of mgCrys were measured using qPCR.The data were normalized by determining the amount of mgEf1α.Each value is expressed as mean ± standard error of the mean (SEM).Asterisks between moon phases indicate significant difference in the transcript levels of each gene at P < .01.
appears to contribute little to the circadian system in the Malabar grouper.This is in line with a previous study in which a luciferase reporter gene assay showed that zebrafish Cry3 failed to inhibit CLOCK:BMAL1-induced transcription, suggesting that it may be a Drosophila-type CRY and have different functions from Cry1 and Cry2, although its function remains unidentified (Kobayashi et al. 2000).By comparing the diurnal variation of mgCry genes, we noticed the differences in their transcriptions at the peaks and troughs among moon phases.We tested if mgCry levels at daily peak and trough significantly differ across moon phases (Figure 3).The transcript levels of mgCry2, but not the other mgCry genes, showed weekly fluctuations, with the difference between the highest (19:00) and lowest expression (03:00) being more pronounced during the new moon period than at other times (Figure 3).These observations indicate moon dependency of the amplitude of mgCry2 expression.We confirmed these differences by comparing the abundances of mgCry2 mRNA in the pituitary gland between the new moon and full moon periods (Figure 4).We focused on the transcript levels of mgCry genes only in the pituitary because little difference in their transcription was seen in the diencephalon in the preliminary experiment, which is confirmed in the results from the moonlight blocked condition (Figure 5).This comparison further highlighted the distinct variation in expression supporting the suggestion of moondependent differences in mgCry2 expression.Moon phase-dependent expression patterns of clock genes have been reported in certain fishes that inhabit coral reefs (Fukushiro et al. 2011;Toda et al. 2014;Fukunaga et al. 2019).For example, increases in Cry3 transcript levels in the brain (mesencephalon and diencephalon) of the mature goldlined spinefoot are observed around the first quarter moon (Fukushiro et al. 2011), and a steady increase in Cry1 transcript level in the brain (diencephalon and pituitary gland) of honeycomb grouper occurs from the first quarter moon to the full moon (Fukunaga et al. 2019).The period of peak Cry transcript level coincides with spontaneous spawning events observed in the goldlined spinefoot (occurring around the first quarter moon period) and the honeycomb grouper (occurring around the full moon period) (Rahman et al. 2000;Lee et al. 2002).It is possible that the Malabar grouper possesses a similar system, because the peaks of mgCry gene expression occurred around the new moon period when the spontaneous spawning of this species occurs.Therefore, it seems that weekly changes in the transcript levels of some clock genes are related to the perception and entrainment of moon phase-related activity.This may also be applicable in invertebrates, because the stony coral Acropora millepora, a full moon spawner, shows different levels of Cry gene transcripts between the full moon and new moon periods (Levy et al. 2007).
Our findings suggest that differences in brightness at night have an impact on the transcript levels of some clock genes.To examine this further, fish were reared under moonlight-exposed or moonlight-blocked conditions during the full moon period.The results showed that the transcript levels of Cry genes were higher in the pituitary gland, but not in the diencephalon, of the moonlight-blocked fish, although the expression profiles seemed to be different among the Cry genes (Figure 5).These observations suggest that the pituitary gland is a key organ influenced by changes in moonlight.Similar findings were obtained in the honeycomb grouper, in which the transcript levels of Cry1, Cry2, and Cry3 in the pituitary gland were higher around the new moon compared to the other moon phases (Fukunaga et al. 2020), although different profiles of Cry1, Cry2, and Cry3 expression were seen in the diencephalon.Similarly, in situ hybridization analyses have revealed that signals of Cry3 at noon decrease from the first quarter moon to full moon periods in the diencephalon of goldlined spinefoot with blockade of moonlight at night (Toda et al. 2014).
Our findings suggest two possible mechanisms by which moonlight may upregulate the expression levels of Cry genes in the pituitary gland.First, the transcription of Cry genes in the pituitary gland may be influenced by an internal transducer in response to changes in moonlight, such as melatonin, which is the time-keeping hormone that increases at night and is synthesized mainly in the pineal organ and retina (Falcón et al. 2011).In mammals, for example, the melatonin receptor Mel 1 is highly expressed in the pituitary gland, where melatonin plays an important role in synchronization of seasonal activities regulated by pituitary hormones (Johnston et al. 2003;Jilg et al. 2005;Dardente et al. 2010;Dupré 2011).Melatonin also has an impact on the expression of clock genes in the pituitary gland (Messager et al. 1999;Jilg et al. 2005;Dardente 2007).Moreover, subcutaneous administration of melatonin (1 mg/kg) in mice increases the expression of Cry1 but decreases the expression of Period1 (Per1) in the pituitary gland (Dardente et al. 2003).Melatonin is also likely to be involved in regulating the expression of clock genes in the pituitary gland in fish because melatonin treatment upregulates Cry1 and Cry2 in the cultured pituitary gland of European sea bass (Herrero and Lepesant 2014).
It is possible that melatonin is a transducer of moonlight signals in fish (Takemura et al. 2004).This is partially supported by experimental evidence that moonlight affects the synthesis of melatonin and the expression of its receptors in the pineal organ of spinefoot: exposing fish to moonlight around the full moon period resulted in a decrease in the plasma level of melatonin (Rahman et al. 2004;Takemura et al. 2004); culture of the pineal organ of this species under moonlight lowered in vitro production of melatonin (Takemura et al. 2006); and the transcript levels of melatonin receptors (MT1 and Mel 1c ) in the pineal gland were significantly lower during the full moon period than the new moon period (Park et al. 2014).It is likely that gonadal development is influenced by melatonin fluctuations at night because implantation of an osmotic pump containing physiological levels of melatonin into the body cavity suppresses ovarian development in the honeycomb grouper, which spawns around the full moon (Fukunaga et al. 2019).Hence, it is likely that the increased synthesis of melatonin in the pineal gland driven by darkness during the new moon period serves as a key regulator of the levels of clock gene transcripts in the pituitary gland of Malabar grouper.
The second possibility is that the moon-related transcription of Cry genes in the pituitary gland may be influenced by the master clock system.However, there is some debate about the master clock in fish.The master clock is located in the pineal gland in zebrafish and grass puffer Takifugu niphobles (Delaunay et al. 2003;Ziv et al. 2005;Ikegami et al. 2015), while those of the bastard flounder Paralichthys olivaceus and greater amberjack Seriola dumerili are located in the suprachiasmatic nucleus (Watanabe et al. 2012).Due to the lack of available research on the coexistence of the circadian and circalunar clocks in the brain, we are unable to establish the involvement of the master clock in the moon cycle for fish.Further investigations are therefore needed to explore the potential interactions between these two systems.Fukunaga et al. (2020) noted that in the honeycomb grouper, a species known to spawn around the full moon, exposure to artificial full moon light led to significant increases in the transcript levels of Cry1 and Cry2 in the pituitary at one and 4 weeks after moonlight exposure.On the other hand, acute interruptions of moonlight around the waxing gibbous moon resulted in increases in the transcript levels of Cry1b and Cry2 in the diencephalon and pituitary gland of the goldlined spinefoot, a species that spawns around the first quarter moon (Takeuchi et al. 2018).In the case of the Malabar grouper, which is a new moon spawner, the transcript levels of mgCry genes showed a response to darkness at night (this study).Overall, these results highlighted the difference in the utilization of moonlight between full moon users and new moon users, although the darkness at night is thought to be the basal point for the expression of clock genes (Figure 6).
In summary, the transcript levels of clock genes, such as Cry1 and Cry2, in the pituitary gland exhibit weekly fluctuations according to the moon cycle and are influenced by darkness at night.These observations suggest that the darkness around the new moon period may play a role in establishing moon periodicity in the reproduction and behavior of the Malabar grouper.In addition to circadian oscillation of the master clock in the pineal organ and/or suprachiasmatic nucleus, certain clock genes are peripherally involved in driving moon-related activities in fish through the action of melatonin.Further studies are required to elucidate how clock genes regulate the synthesis and secretion of pituitary hormones to modulate moon-related reproduction and behavior in fish.Finally, our findings not only advance the understanding of circalunar rhythms in vertebrates but also offer practical benefits for aquaculture of fish with lunar-related spawning rhythmicity.(1) during the new moon period, the "darkness at night" is perceived by fish, leading to an increase in the transcript levels of mgCry2 in the pituitary, (2) changes in the transcript levels of mgCry2 among different moon phases are utilized for moon-related reproduction and behavior in fish.

Figure 2 .
Figure2.Daily variation of (A-D), mgCry2 (E-H), and mgCry3 (I-L) transcription in the diencephalon (including pituitary) of the Malabar grouper during four moon phases.Fish (six individuals per each sampling point) were reared under natural conditions and their brain was taken from fish at 4-hour intervals around the new moon (A, E, and I), first quarter moon (B, F, and J), full moon (C, G, and K), and last quarter moon (D, H, and L).The transcript levels of mgCry genes were measured using qPCR.The data were normalized by determining the amount of mgEf1α.The solid and open bars depicted in each figure represent the scotophase (dark phase) and photophase (light phase), respectively.Each value is expressed as mean ± standard error of the mean (SEM).Different letters indicate a significant difference in the transcript levels of each gene within a day at a significant level of P < .05.

Figure 3 .
Figure 3. Transcript levels of mgCry1 (A and B) and mgCry2 (C and D) in the diencephalon (including pituitary) of the Malabar grouper between the peak and bottom time points during four moon phases.The peak times (11:00 for mgCry1 and 19:00 for mgCry2) and the bottom times (23:00 for mgCry1 and 03:00 for mgCry2) were selected from Figure2and reorganized according to the moon phase: the new moon (NM), first quarter moon (FQM), full moon (FM), and last quarter moon (LQM).Each value is expressed as mean ± standard error of the mean (SEM).Different letters indicate a significant difference in the transcript levels of each gene within a day at a significant level of P < .05.

Figure 4 .Figure 5 .
Figure 4. Daily variation of mgCry1 (a), mgCry2 (b), and mgCry3 (c) transcription in the pituitary gland of the Malabar grouper during the new moon and the full moon periods.Fish (six individuals per each sampling point) were reared under natural conditions and their brain was taken from fish at 4-hour intervals during the new moon period (solid circle) and full moon period (open circle).The transcript levels of mgCry genes were measured using qPCR.The data were normalized by determining the amount of mgEf1α.The solid and open bars depicted in each figure represent the scotophase (dark phase) and photophase (light phase), respectively.Each value is expressed as mean ± standard error of the mean (SEM).Different letters indicate a significant difference in the transcript levels of each gene within a day at a significant level of P < .05.

Figure 6 .
Figure 6.Possible regulation of lunar clock in the Malabar grouper.(1)during the new moon period, the "darkness at night" is perceived by fish, leading to an increase in the transcript levels of mgCry2 in the pituitary, (2) changes in the transcript levels of mgCry2 among different moon phases are utilized for moon-related reproduction and behavior in fish.