Initial studies used methods such as the differential display reverse transcriptase-polymerase chain reaction (DD-RT-PCR) or suppression subtractive hybridization to identify transcripts differentially expressed between aphids reared under long days (producers of parthenogenetic progeny) and short days (producers of sexually-reproducing offspring). A transcript homologous to an amino acid transporter within GABAergic neurones was first identified by DD-RT-PCR as being over-expressed in short-day, sexual-offspring-producing individuals (Ramos et al., 2003). A putative role for this transcript in the transduction of the photoperiodic signal was proposed. Suppression subtractive hybridization approaches coupled with quantitative RT-PCR then allowed the identification of transcripts coding cuticular proteins and a β-tubulin that could play a role in hormone responses (Cortés et al., 2008). The precise function of these candidate genes in the regulation of photoperiodism is nevertheless unknown.
Genomic resources such as expressed sequence tag libraries from various aphid tissues were generated (Sabater-Muñoz et al., 2006). These libraries were used to build two generations of cDNA microarrays containing, respectively, 1700 (Le Trionnaire et al., 2007) and 7000 transcripts (Le Trionnaire et al., 2009, 2012). Heads of aphids reared under long-day or short-day photoperiods were collected at five stages of development during the process of sexual morph induction. By focusing on heads and cerebral tissues, the aim was to capture the genetic programmes set up during the initial steps of photoperiodic signal detection and transduction (Le Trionnaire et al., 2007, 2009). Microarray hybridizations combined with proteomics approaches (two dimensional differential in gel electrophoresis) revealed the differential expression of a significant number of transcripts (10% of spotted cDNAs) and peptides within the heads of aphids in response to short photoperiods, allowing the identification of several genetic programmes that could be associated with the photoperiodic response (Fig. 2). Among these, a subset of transcripts showed homologies with Drosophila melanogaster genes involved in the visual system such as Arrestin and Calnexin, known to play a role in rhodopsin phototransduction and maturation. This confirmed an earlier study showing that antibodies against a vertebrate arrestin strongly labelled the putative brain photoperiodic photoreceptors (Gao et al., 1999). Another set of transcripts were related to the nervous system, with several transcripts differentially expressed displaying homologies with Drosophila genes involved in axon guidance (Rho I, NLaz, Capulet and Wunen) and neurotransmission (Kinesin, Dunc 10-4A, Dunc 13-4A and a DEP-containing domain protein), strongly suggesting an involvement of the nervous system in the transduction of the photoperiodic signal. Insulin signalling might also play a role because one transcript encoding an insulin-degrading enzyme and another one coding for an insulin receptor were found to be differentially expressed in response to short photoperiods. Unexpectedly, a large number (n = 38) of cuticular protein transcripts appeared to be regulated. Most of them (n = 25) contained a RR domain (RR1 or RR2) that allows chitin-cuticular protein linkage (Gallot et al., 2010). Most of these transcripts were down-regulated under short-day photoperiods, suggesting a putative relaxing of the chitin-cuticular protein network in response to short days. Cuticle also contains N-β alanyl dopamine (NBAD) that allows linkage between cuticular proteins to produce hard-cuticle or sclerotization. NBAD is made of dopamine and β-alanine and the enzyme responsible for this conjugation is coded by the ebony gene. β-Alanine is synthesized from aspartate by the action of an enzyme coded by black gene. Transcriptomic analyses revealed that ebony and black transcripts were down-regulated in short-day reared aphids. Consequently, it can be hypothesized that less NBAD is synthesized under short-day conditions. This suggests that short photoperiods could result in the reduction of sclerotization level in the aphid heads, thereby modifying cuticle structure. These observations also raise the question of the level of dopamine in aphid heads under short-day conditions. Indeed, if less NBAD is synthesized, is the general level of dopamine affected? Dopamine synthesis involves two main enzymes: tyrosine hydroxylase (th), which metabolizes tyrosine into l-3,4-dihydroxyphenylalanine (l-DOPA), and dopa-decarboxylase (ddc), which metabolizes l-DOPA into dopamine. RT-PCR experiments showed that th and ddc transcripts were down-regulated in short-day reared aphid heads, suggesting that short photoperiods could result in a diminution of dopamine synthesis within aphid brains (Gallot et al., 2010). Because dopamine is a neurotransmitter (and a neurohormone), it is tempting to speculate that this molecule might be involved in the transduction of the photoperiodic signal. A recent study in Locusta migratoria demonstrated that the dopamine synthesis pathway was involved in the transition from the solitary to the gregarious phase (Ma et al., 2011). More precisely, the data showed that th (tyrosine hydroxylase), henna and vat1 (vesicle amino-acid transporter), three genes coding for enzymes involved in dopamine biosynthesis and synaptic release, were significantly down-regulated during the solitary phase. Functional and pharmacological analyses confirmed that the dopamine pathway was clearly involved in the behavioural transition (Ma et al., 2011). Because such a behavioural change in the locust is a case of phase polyphenism (but not triggered by day length changes), a clear parallel with reproductive polyphenism (triggered by photoperiod shortening) can be made and the dopamine biosynthesis pathway might also be involved in the transition from asexual to sexual reproduction in response to short days in aphids. To address this, the level of expression, the localization and the functional characterization of pale, vat1 and henna transcripts in both long- and short-day reared aphids all have to be investigated. It is striking to emphasize that some of these transcriptomic modifications observed on aphids reared under controlled conditions were also detected in aphids reared outdoor under natural photoperiodic conditions. However, the differential expression of several heat-shock protein transcripts also suggested a strong response of aphids to additional environmental parameters such as temperature (Le Trionnaire et al., 2012).