Photoperiodic and clock regulation of the vitamin A pathway in the brain mediates seasonal responsiveness in the monarch butterfly

Abstract

Seasonal adaptation to changes in light:dark regimes (i.e., photoperiod) allows organisms living at temperate latitudes to anticipate environmental changes. In nearly all animals studied so far, the circadian system has been implicated in measurement and response to the photoperiod. In insects, genetic evidence further supports the involvement of several clock genes in photoperiodic responses. Yet, the key molecular pathways linking clock genes or the circadian clock to insect photoperiodic responses remain largely unknown. Here, we show that inactivating the clock in the North American monarch butterfly using loss-of-function mutants for the circadian activators CLOCK and BMAL1 and the circadian repressor CRYPTOCHROME 2 abolishes photoperiodic responses in reproductive output. Transcriptomic approaches in the brain of monarchs raised in long and short photoperiods, summer monarchs, and fall migrants revealed a molecular signature of seasonal-specific rhythmic gene expression that included several genes belonging to the vitamin A pathway. We found that the rhythmic expression of these genes was abolished in clock-deficient mutants, suggesting that the vitamin A pathway operates downstream of the circadian clock. Importantly, we showed that a CRISPR/Cas9-mediated loss-of-function mutation in the gene encoding the pathway's rate-limiting enzyme, ninaB1, abolished photoperiod responsiveness independently of visual function in the compound eye and without affecting circadian rhythms. Together, these results provide genetic evidence that the clock-controlled vitamin A pathway mediates photoperiod responsiveness in an insect. Given previously reported seasonal changes associated with this pathway in the mammalian brain, our findings suggest an evolutionarily conserved function of vitamin A in animal photoperiodism.

Keywords: CRISPR/Cas9; circadian clocks; insect photoperiodism; monarch butterfly; vitamin A.

Fig. 1.

Circadian clock genes are required for photoperiodic responses. Number of mature oocytes produced 14 d post adult emergence in loss-of-function hemizygous Clock (Clk; A), hemizygous Bmal1 (Cyc-like; B), and homozygous Cryptochrome 2 (Cry2; C) mutant females, and in their respective wild-type siblings, all raised in LP and SP at 21 °C. Monarchs have a ZW sex-determination system; males are the homogametic sex (ZZ) while females are the heterogametic sex (ZW), and Clk and Bmal1 are located on the Z chromosome. For each condition, box plots and raw data points are shown. Error bars on box plots represent 1.5 times the interquartile range. LP: 15 h light, 9 h dark; SP: 10 h light, 14 h dark. Different letters over bars indicate groups that are statistically significant (interaction genotype × photoperiod, 2-way ANOVA, Tukey’s pairwise comparisons, P < 0.05), and numbers of dissected females are indicated.

Fig. 2.

Rhythmic gene expression analysis reveals photoperiodic regulation of the vitamin A pathway in the monarch brain. (A) Heatmaps of relative RNA levels for genes rhythmically expressed in brains of monarchs raised in either LP or SP at 21 °C (Left) and in brains of either summer-like or wild-caught migrant monarch (Right). R-R, genes rhythmic in both conditions; R-AR: genes rhythmic only in summer-like or LP; AR-R, genes rhythmic only in migrants or SP. Columns represent samples collected over a 24-h LD cycle. Transcripts are arranged by phase, and their order along the vertical axis is conserved in both conditions. White bars: light conditions; black bars: dark conditions. (B) Number of mature oocytes produced by females 9 to 15 d post adult emergence for summer-like monarchs and 7 to 11 d for LP/SP monarchs. Wild-caught migrants of unknown age were dissected 8 d after capture. Boxplots as in Fig. 1. One-way ANOVA, post hoc Tukey test, P < 0.05. (C) Table showing the number of genes found in any given category and the number of overlapping genes between comparisons. Genes involved in the vitamin A pathway are shown. The complete lists are shown in SI Appendix, Tables S1, S3, and S5. (D) Proposed model of the vitamin A pathway in insect brain cells. Beta-carotene is transported into extraretinal neural cells of the adult brain via santa maria and converted to retinal by ninaB. Retinal can either be interconverted into retinol by a retinol dehydrogenase (RDH) or converted into RA by a retinaldehyde dehydrogenase (RALDH). RA binds to retinoid receptors to regulate transcription of target genes.

Fig. 3.

The vitamin A pathway is under both photoperiodic and clock regulation. (A) Temporal mRNA expression profiles of santa maria 1, santa maria 2, ninaB1, and retinol dehydrogenase 13 in brains of monarch raised in LP and SP (Top) and summer-like and migrant monarchs (Bottom). R-AR: rhythmic in LP and summer-like monarchs, arrhythmic in SP monarchs and migrants; R-R: rhythmic in LP, SP, summer-like monarchs, and migrants. The R-R and R-AR categories were defined based on the analysis of rhythmic gene expression reported in Fig. 2. (B) Temporal mRNA expression profiles of santa maria 1, santa maria 2, ninaB1, and retinol dehydrogenase 13 in brains of wild-type summer-like monarchs (plain lines), and Clk (dotted lines) and Cry2 (dashed lines) loss-of-function mutants raised in summer-like conditions. For each condition, 2 biological replicates are plotted.

Fig. 4.

Disruption of the vitamin A pathway abolishes photoperiodic responses. (A) Generation of a ninaB1 loss-of-function mutant using CRISPR/Cas9-mediated targeted mutagenesis. (Top) Genomic structure of ninaB1 in the monarch. Red star, position of the site targeted by sgRNA. (Bottom) Nucleotide sequence of the targeted site (underlined) and introduced frame-shifting mutation (7-bp deletion). (B) Number of mature oocytes produced 10 d post adult emergence in ninaB1 homozygous mutant and wild-type sibling females. Boxplots as in Fig. 1. Interaction genotype × photoperiod, 2-way ANOVA, Tukey’s pairwise comparisons, P < 0.05. (C) Number of mature oocytes produced 10 d post adult emergence in wild-type female monarchs with unpainted eyes or eyes covered with either clear or black paint at the day of emergence. Interaction genotype × painting condition, 2-way ANOVA, Tukey’s pairwise comparisons, P < 0.05. (D) Associative visual conditioning of the proboscis extension reflex (PER) of ninaB1 homozygous mutants (red line, n = 6) and wild-type monarchs with eyes painted clear (gray line, n = 6) or black (black line, n = 6) for 14 training trials on day 3 of training. (E) Profiles of adult eclosion in DD of wild-type (blue) and ninaB1 loss-of-function siblings (red). Gray bars: subjective day; black bars: subjective night. Kolmogorov–Smirnov test, P > 0.05. (F) Circadian expression of period in brains of wild-type (solid black lines) and ninaB1 loss-of-function (dashed gray lines) siblings. Values are mean ± SEM, and the numbers of animals for each genotype and time point are indicated. Interaction genotype × time, 2-way ANOVA, P > 0.05.