Clock genes and photoperiodism in the pea aphid acyrthosiphon pisum

Resumen

Aphids have a strong seasonal response, namely the development of a sexual morphs triggered by the shortening of photoperiod in autumn that produce an overwintering egg in which an embryonic diapause takes place. From this egg, an asexual parthenogenetic female emerges giving way to the asexual phase of the aphid life cycle in which several asexual generations occur. Based on the response to short photoperiods, two strains of aphids can be found: the holocyclic develop the seasonal response under short photoperiods, while the anholocyclic do not. The sequencing of the genome of the pea aphid Acyrthosiphon pisum places this species as an excellent model to investigate the the involvement of the circadian clock in the seasonal response. In the present thesis, the characterisation of the circadian clock genes revealed an extensive alternative splicing, although it could not be associated to any of the strains or photoperiods. Moreover, the expression of circadian clock genes analysed at different moments of the day showed a robust cycling of period and timeless. Furthermore, the rhythm in expression of period and timeless was rapidly dampened under DD (continuous darkness conditions), thus supporting the model of a seasonal response based on a heavily dampened circadian oscillator. Additionally, changes in expression of some of the circadian clock genes were associated to the induction of the seasonal response. The localisation of transcripts of period and timeless in the aphid brain revealed two groups cells: the Dorsal Neurons (DN) and the Lateral Neurons (LN) in the protocerebrum. The location of DN coincided with a region previously described as essential for the sesasonal response. The involvement of several elements that may participate as output of the circadian clock were also studied. We failed to detect the aphid Pdf gene in the genome and PDH with antibodies, suggesting that PDF may be genuinely absent in aphids. We show the localisation of melatonin in the aphid nervous ganglia, which is the second report of in situ localisation of melatonin in insects and the first in Hemimetabola. Moreover, we observed an increase of melatonin content associated to the induction of the seasonal response. The analysis of the aphid AANAT genes, candidates to regulate the synthesis of melatonin, revealed concomitant levels of expression with those of melatonin in some cases. Furthermore, despite the localisation of some of the AANAT transcripts in the nervous ganglia, none of them colocalised with melatonin. Finally, the aphid Ptth gene was identified and characterised showing typical insect PTTH features. Additionally, Ptth transcripts were localised in two pairs of neurons in the aphid brain allowing to identify them as the NSC group II and consequently discarding them as essential for the seasonal response. The results presented in this thesis provide support to the involvement of at least some of the circadian clock elements in the seasonal response.