Many insects exhibit daily rhythms in their physiological functions and behaviors. Many of these daily oscillations are regulated by an endogenous circadian clock, a biological clock with a free-running period of approximately 24 h. A circadian clock is entrained by environmental time cues (zeitgebers) such as light and temperature (Saunders, 2002). Once-in-a-lifetime events, such as hatching, pupation, and eclosion, are also regulated by the circadian clock, and their daily rhythms can be expressed at the population level.

The molecular mechanisms of the circadian clock in insects consist of negative transcriptional/translational feedback loops. In the major loop, the positive elements, cycle (cyc) and Clock (Clk), activate the transcription of the negative elements, period (per), timeless (tim), and/or cryptochrome 2 (cry2), which repress the action of the positive elements. In the minor loop, the rhythmic expression of Clk is regulated by the positive element PAR-domain protein 1 (Pdp1) and the negative element vrille (vri) (Sandrelli et al., 2008, Tomioka and Matsumoto, 2019).

Photoperiods are major environmental cues used by temperate insects for coordinating their development and reproduction with seasonal environmental changes (Nelson et al., 2010, Saunders, 2002). It is widely accepted that the circadian clock plays a role in photoperiodic time measurement in many insect species (Goto, 2022, Koštál, 2011, Meuti and Denlinger, 2013). The photoperiodic response of aphids (Hemiptera: Aphididae) has long been a subject of debate. Most aphid species reproduce parthenogenetically and viviparously during long days in spring and summer, whereas oviparous sexual females and males are induced by short days in autumn and lay overwintering eggs, which hatch the next spring as fundatrices (Moran, 1992, Simon et al., 2002). Extensive studies by Lees, 1966, Lees, 1973 have suggested that the photoperiodic response is based on a non-circadian or hourglass mechanism in the vetch aphid, Megoura viciae. Such an hourglass-like clock is currently regarded as a heavily damped circadian oscillator (Saunders, 2021, Vaz Nunes and Saunders, 1999). Additional experiments have revealed the involvement of the circadian clock in the photoperiodic response of M. viciae (Vaz Nunes and Hardie, 1993). However, empirical data demonstrating the damping of circadian oscillations are scarce (Beer et al., 2017).

Several studies have reported the existence of diel rhythms in various aphid species at the behavioral level (Beer et al., 2017, Eisenbach and Mittler, 1980, Hodgson and Lane, 1981, Jeon et al., 2003, Joschinski et al., 2016), gene expression levels (Barberà et al., 2017, Barberà et al., 2022, Cortés et al., 2010), and protein expression levels (Colizzi et al., 2021); however, the possibility that host plants affect these rhythms has not been completely excluded in most of these studies. By feeding aphids an artificial diet, Beer et al. (2017) showed that circadian rhythms of locomotor and metabolic activity in the pea aphid, Acyrthosiphon pisum are independent of the host plant. These oscillations persisted for a few days and then quickly damped under constant conditions. Notably, similar damping oscillations were observed in the mRNA abundance of per, tim, and cry2 after aphids were transferred to constant conditions (Barberà et al., 2017, Barberà et al., 2022). These features support the hypothesis that aphids have a heavily damped circadian clock. In contrast, the circadian rhythmicity of immature stages, such as hatchlings, has not been studied in aphids, and thus the ontogeny of a damped circadian clock is mostly unknown. Particularly, hatching time is thought to have evolved to maximize the survival of hatchlings against predators and weather conditions and has been investigated in various insects (Lockwood and Story, 1985, Minis and Pittendrigh, 1968, Tomioka et al., 1991). However, the time at which aphids hatch has not yet been examined.

The present study elucidated the daily hatching patterns in A. pisum under light-dark cycles. During these observations, hatching occurred intensively during the early photophase. Therefore, the endogenous rhythmicity in hatching was evaluated under constant darkness (DD). If such a hatching rhythm is driven by a circadian clock, robust oscillations might be observed in the mRNA abundance of circadian clock genes. To test this hypothesis, the temporal expression patterns of six clock genes, cyc, Clk, per, tim, Pdp1, and vri, in the late embryonic stage were examined under light-dark (LD) cycles and DD. Based on the results obtained, quick damping of the circadian clock in A. pisum eggs is discussed.