Comparative Analysis of Diel and Circadian Eclosion Rhythms and Clock Gene Expression Between Sexes in the Migratory Moth Spodoptera frugiperda

Abstract

The circadian clock orchestrates behavioral and molecular processes such as eclosion. Understanding eclosion timing may offer insights into circadian mechanisms underlying migratory timing. Here, we characterize the diel and circadian patterns of eclosion and core clock gene expression in the fall armyworm (FAW), Spodoptera frugiperda, a globally distributed migratory moth. Using a custom-designed eclosion monitoring system under 14 h light: 10 h dark (L14: D10) and constant darkness (DD) conditions, we observed robust diel eclosion rhythms peaking shortly after lights-off under L14: D10, which became delayed and damped over three consecutive days in DD. Males showed a tendency toward more dispersed emergence patterns and exhibited statistically distinguishable eclosion distributions from females under both conditions. Expression of five canonical clock genes (cyc, clk, tim, per, cry2) displayed significant 24 h rhythmicity, with generally higher mesors in males. However, sex-specific differences in amplitude and phase were detected only for clk and cyc under L14: D10, not in DD. These findings suggest that sex-specific differences in circadian regulation are limited. Nonetheless, subtle variations in clock gene output and emergence timing in the FAW population established in China may contribute to sex-specific ecological strategies in the novel migratory arena.

Keywords: Spodoptera frugiperda; circadian rhythm; clock gene expression; eclosion timing; light regime; migratory insect pest; sex differences.

Figure 1

The assay for eclosion rhythm monitoring and adult tissue collection of S. frugiperda. (A) The real-time eclosion monitoring apparatus for insects. Key components include: 1. Glass tube; 2. Camera; 3. Acrylic stand; 4. Screen, displaying the real-time camera view (powered off during experiments); 5. Video recorder, equipped with a hard drive and control unit for data storage. Cameras are connected to a video recorder via network cables. Granted Chinese patent: ZL202120866706.1. (B) Circadian eclosion monitoring assay under the first (DD1), second (DD2), and third (DD3) dark–dark (DD) cycles after entrainment to 14 h light: 10 h dark (L14: D10) cycle. Dark grey and black bars represent subjective day and night, respectively. (C) Entraining adults under L14: D10 for seven cycles, followed by sample collection for gene expression analysis after two additional LD or DD cycles. Heads of S. frugiperda were collected every 4 h under LD condition and every 3 h under DD3 condition over 24 h.

Figure 2

Diel and circadian eclosion rhythms of S. frugiperda entrained to L14: D10 and DD. (A) Pooled diel eclosion rhythm over five consecutive days of recording under 14 h light: 10 h dark (L14: D10) condition. (B) Pooled circadian eclosion rhythm of females and males over three consecutive days of recording under constant darkness (DD). (C) Profiles of female and male adult eclosion across the first, second, and third days of constant darkness (DD1, DD2, and DD3 respectively). Data are binned in 1-h intervals. Horizontal bars show objective day (white) and night (black) in panel (A) and subjective day (gray) and night (black) in panels (B,C). N, sample number.

Figure 3

Central tendency comparison for sex differences in eclosion rhythm of S. frugiperda under 14 h light: 10 h dark (L14: D10) and constant darkness (DD) conditions. Red, blue, purple, orange, and green dots represent data from five consecutive days under 14 h light: 10 h dark (pooled LD), three consecutive days under constant darkness (pooled DD), the first (DD1), second (DD2), and third (DD3) day of constant darkness, respectively. Solid lines represent the median value, and error bars represent the interquartile range. F, female; M, male. Uppercase and lowercase letters indicated significant differences between sexes for the same photoperiod condition (LD or DD), and between the light regimes for females or males by the Mann–Whitney test at p < 0.05, respectively.

Figure 4

The expression of clock genes of cyc (A), clk (B), tim (C), per (D), and cry2 (E) in the heads of adults under 14 h light: 10 h dark (LD) and the third day of constant darkness (DD3) after 7 days of adult entrainment to LD. Horizontal bars show objective day (white) and night (black) in the left panels and subjective day (gray) and night (black) in the right panels. Pink and blue lines indicate females (LD-female) and males (LD-male) sampled under LD condition, and red and black lines indicate females (DD3-female) and males (DD3-male) sampled under DD3. Solid lines represent the mean value, and error bars represent the standard error of the mean (SEM). The first and last data points are intentionally duplicated to facilitate visualization of rhythmic patterns across the 24 h cycle. Asterisks indicate significant sex differences at individual ZT or CT time points (p < 0.05, one-way ANOVA), conducted only when a significant or marginal interaction was detected in the two-way ANOVA. In the absence of interaction, only main effects were interpreted. Different lowercase letters indicate significant differences among time points within the same sex and light regime (Tukey’s HSD, adj p < 0.05). No letters are shown when differences are not statistically significant.