Lunar Phase-Dependent Expression of Cryptochrome and a Photoperiodic Mechanism for Lunar Phase-Recognition in a Reef Fish, Goldlined Spinefoot

Lunar cycle-associated physiology has been found in a wide variety of organisms. Recent study has revealed that mRNA levels of Cryptochrome (Cry), one of the circadian clock genes, were significantly higher on a full moon night than on a new moon night in coral, implying the involvement of a photoreception system in the lunar-synchronized spawning. To better establish the generalities surrounding such a mechanism and explore the underlying molecular mechanism, we focused on the relationship between lunar phase, Cry gene expression, and the spawning behavior in a lunar-synchronized spawner, the goldlined spinefoot (Siganus guttatus), and we identified two kinds of Cry genes in this animal. Their mRNA levels showed lunar cycle-dependent expression in the medial part of the brain (mesencephalon and diencephalon) peaking at the first quarter moon. Since this lunar phase coincided with the reproductive phase of the goldlined spinefoot, Cry gene expression was considered a state variable in the lunar phase recognition system. Based on the expression profiles of SgCrys together with the moonlight's pattern of timing and duration during its nightly lunar cycle, we have further speculated on a model of lunar phase recognition for reproductive control in the goldlined spinefoot, which integrates both moonlight and circadian signals in a manner similar to photoperiodic response

Hypothalamic expression and moonlight-independent changes of Cry3 and Per4 implicate their roles in lunar clock oscillators of the lunar-responsive Goldlined spinefoot.

Lunar cycle-associated physiology has been found in a wide variety of organisms. Studies suggest the presence of a circalunar clock in some animals, but the location of the lunar clock is unclear. We previously found lunar-associated expression of transcripts for Cryptochrome3 gene (SgCry3) in the brain of a lunar phase-responsive fish, the Goldlined spinefoot (Siganus guttatus). Then we proposed a photoperiodic model for the lunar phase response, in which SgCry3 might function as a phase-specific light response gene and/or an oscillatory factor in unidentified circalunar clock. In this study, we have developed an anti-SgCRY3 antibody to identify SgCRY3-immunoreactive cells in the brain. We found immunoreactions in the subependymal cells located in the mediobasal region of the diencephalon, a crucial site for photoperiodic seasonal responses in birds. For further assessment of the lunar-responding mechanism and the circalunar clock, we investigated mRNA levels of Cry3 as well as those of the other clock(-related) genes, Period (Per2 and Per4), in S. guttatus reared under nocturnal moonlight interruption or natural conditions. Not only SgCry3 but SgPer4 mRNA levels showed lunar phase-dependent variations in the diencephalon without depending on light condition during the night. These results suggest that the expressions of SgCry3 and SgPer4 are not directly regulated by moonlight stimulation but endogenously mediated in the brain, and implicate that circadian clock(-related) genes may be involved in the circalunar clock locating within the mediobasal region of the diencephalon

Immunohistochemical localization of <i>Siganus guttatus</i> Cryptochrome3 (SgCRY3) in the diencephalon.

<p>(A) Drawing of the lateral view of the brain of <i>Siganus guttatus</i>. Lettered dotted lines indicate the levels of the transverse sections shown in panels C–J. Ce, cerebellum; Di, diencephalon; OpN, optic nerve; OT, optic tectum; Te, telencephalon. (B) Drawing of the transverse sections at the level of panel A. (C, D) αSgCRY3CT staining without a competitive peptide. (E, F) control sections (mouse IgG was used instead of αSgCRY3CT). (G, H) αSgCRY3CT staining in the presence of 100 µM CT17 epitope peptide. (I, J) αSgCRY3CT staining in the presence of 100 µM CT17S epitope-shuffled peptide. In the preabsorption experiment (panels G–J), CT17 or CT17S peptide (100 µM) had been incubated for 16 h at 4°C with αSgCRY3CT before the primary antibody reaction. Panels D, F, H, and J are magnified view of panels C, E, G, and I, respectively. Wash solution; PBS containing 0.25% (panels C–F) or 0.05% (panels G–J) of Triton X-100. Blocking solution; Wash solution containing 1.5% horse normal serum. Each tissue was sampled either March 23, 2012 (new moon) or June 27, 2014 (new moon).</p

Lunar phase-dependency of <i>Siganus guttatus Period4</i> (<i>SgPer4</i>) and <i>Period2</i> (<i>SgPer2</i>) mRNA expression in the brain.

<p>(A) Experimental design using a tank cover for nocturnal moonlight interruption. See legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109119#pone-0109119-g003" target="_blank">Figure 3A</a>. (B, C) The diencephalon (n = 4) was collected at noon from the new moon to full moon phase. <i>SgPer4</i> (panel B) and <i>SgPer2</i> (panel C) mRNA levels were calculated as values relative to those of the virtual reference control gene and were defined as the average of the threshold cycles (Ct) for <i>SgEF1α</i>, <i>SgPGK and Sgβ-actin.</i> Error bars represent ± SD. Lunar phases are indicated by schematic moon images. The p values are indicated on each graph, two-way ANOVA.</p

Multiple sequence alignment of the deduced amino acids of <i>Siganus guttatus</i> Cryptochrome1 (SgCRY1) and Cryptochrome3 (SgCRY3), competitive ELISA and immunoblot analysis.

<p>(A) Multiple sequence alignment of the deduced amino acids of SgCRY1/3. The line above the alignment indicates the antigenic region that was conjugated to KLH or BSA and the conjugate was used as an antigen. CT17 and CT17S are epitope and epitope-shuffled peptides, respectively. (B) Competitive ELISA showing the antigen-specificity. ELISA microplate wells were coated with GST-SgCRY3CT antigen, blocked with 1% skim milk, reacted with αSgCRY3CT that had been mixed with CT17 epitope or CT17S epitope-shuffled peptide at the indicated concentration in advance. Then, the unreacted antibody was washed out and the remaining antibody was detected through use of an HRP-labeled secondary antibody. (C, D) Immunoblot analyses for validating the specificity of αSgCRY3CT to antigenic agent. GST-SgCRY3CT (0.1 µg), GST (0.1 µg), BSA-SgCRY3CT (0.15 µg), and BSA (0.15 µg) proteins were subjected to 10% polyacrylamide SDS-PAGE. In the preabsorption experiment (panel D, lanes 3–6), CT17 or CT17S peptide (100 µM) had been incubated for 1 h at 37°C with αSgCRY3CT before the primary antibody reaction.</p

Lunar phase-dependency of <i>Siganus guttatus Cryptochrome3</i> (<i>SgCry3</i>) mRNA expression in the brain.

<p>(A) Experimental design using a tank cover for nocturnal moonlight interruption. Four groups of fish were contained in tanks maintained under natural (MM, natural and natural condition) conditions or constant darkness from 30 minutes after sunset to midnight (DM, dark and natural condition) or constant darkness from midnight to 30 minutes before sunrise (MD, natural and dark condition) or constant darkness from 30 minutes after sunset to 30 minutes before sunrise (DD, dark and dark condition) from May 21 (new moon) to June 4 (full moon). Illustration shows nocturnal light conditions in tanks and the time of moonlight irradiation from May 21 to June 4, 2012. Lunar phases are indicated by schematic moon images. (B–E) Lunar changes in <i>SgCry3</i> mRNA levels in the brain. The diencephalon and optic tectum (n = 4) were collected at noon from the new moon to full moon phase. <i>SgCry3</i> mRNA levels were calculated as values relative to those of the virtual reference control gene and were defined as the average of the threshold cycles (Ct) for <i>SgEF1α</i>, <i>SgPGK</i> and <i>Sgβ-actin.</i> Error bars represent ± SD. The p values are indicated on each graph, two-way ANOVA.</p

Primers for quantitative RT-PCR.

<p>Primers for quantitative RT-PCR.</p