Characterisation, analysis of expression and localisation of circadian clock genes from the perspective of photoperiodism in the aphid Acyrthosiphon pisum

Abstract

Aphids are typical photoperiodic insects that switch from viviparous parthenogenetic reproduction typical of long day seasons to oviparous sexual reproduction triggered by the shortening of photoperiod in autumn yielding an overwintering egg in which an embryonic diapause takes place. While the involvement of the circadian clock genes in photoperiodism in mammals is well established, there is still some controversy on their participation in insects. The availability of the genome of the pea aphid Acyrthosiphon pisum places this species as an excellent model to investigate the involvement of the circadian system in the aphid seasonal response. In the present report, we have advanced in the characterisation of the circadian clock genes and showed that these genes display extensive alternative splicing. Moreover, the expression of circadian clock genes, analysed at different moments of the day, showed a robust cycling of central clock genes period and timeless. Furthermore, the rhythmic expression of these genes was shown to be rapidly dampened under DD (continuous darkness conditions), thus supporting the model of a seasonal response based on a heavily dampened circadian oscillator. Additionally, increased expression of some of the circadian clock genes under short-day conditions suggest their involvement in the induction of the aphid seasonal response. Finally, in situ localisation of transcripts of genes period and timeless in the aphid brain revealed the site of clock neurons for the first time in aphids. Two groups of clock cells were identified: the Dorsal Neurons (DN) and the Lateral Neurons (LN), both in the protocerebrum.

Identification of the prothoracicotropic hormone (Ptth) coding gene and localization of its site of expression in the pea aphid Acyrthosiphon pisum

“…The distribution of Ptth‐ expressing neurones was very similar to that of a particular subset of dorsal clock neurones (DNs) known as large DNs (l‐DNs) that express the circadian clock gene timeless (Barberà et al ., ). In order to test the possibility that these l‐DNs were expressing Ptth in addition to the clock gene timeless , we carried out a simultaneous localization with two different probes against transcripts of both timeless and Ptth genes.…”

“…B–G). As already reported for other in situ hybridizations of aphid brains with different riboprobes (Barberà et al ., ), unspecific staining of tracheae with both sense and antisense Ptth probes was observed (Fig. A, D).…”

“…The DIG‐labelled riboprobe against timeless transcripts used in Fig. was obtained similarly to the Ptth probe (for details, see Barberà et al ., ).…”

Light input pathways to the circadian clock of insects with an emphasis on the fruit fly Drosophila melanogaster

“…In the aphid Megoura viciae, the site of photoreception for initiating sexual morphs under decreasing photoperiods in late summer has also been localized to the dorsal brain (Lees 1964). More recent studies in the pea aphid, Acyrthosiphon pisum, identified the clock genes and revealed that A. pisum possesses also mammalian-type and Drosophila-type clock genes that are expressed in the dorsal brain, but additionally also in the lateral brain (Cortés et al 2010;Barberà et al 2017). The location of the master clock in mosquitoes is less well studied.…”

A damping circadian clock drives weak oscillations in metabolism and locomotor activity of aphids (Acyrthosiphon pisum)

“…Several behavioural studies suggest the existence of a functional circadian clock in sexual as well as in parthenogenetic aphid forms of various aphid species 19 , 20 , although the experimental protocols did not allow to investigate damping of the clock. More recently, two studies identified putative clock genes and the location of clock gene expressing neurons in the pea aphid Acyrthosiphon pisum ( Harris ) 21 , 22 . Unfortunately the challenge to uncouple the aphid’s activity from the plant’s influence remained unattended in most studies.…”