Deciphering the Function of Octopaminergic Signaling on Wing Polyphenism of the Pea Aphid Acyrthosiphon pisum

Abstract

Aphids exhibit wing polyphenism (winged or wingless) for adaption to predictable or temporally heterogeneous environmental changes; however, the underlying mechanism is still unclear. This morphological change could be stimulated by high aphid density, which in turn could affect octopaminergic signaling in aphids. Octopamine is a neurotransmitter synthesized in insects that can modify their physiological metabolism, locomotion, and other behaviors. We designed experiments to determine whether octopamine functions in wing formation of the pea aphid, Acyrthosiphon pisum (Harris). We determined gene expression of tyramine β-hydroxylase (TβH), a key enzyme in octopamine synthesis at different developmental stages, in different body parts, and in different densities of aphids. We also used TβH RNAi, octopamine receptor agonists (octopamine and synephrine), and an antagonist (mianserin) to modify octopaminergic signaling. We found that transcription of TβH was related to aphid density, which affected the proportion of winged offspring. By manually modifying the mother's octopaminergic signaling, TβH expression was suppressed, and TβH (enzyme) activity decreased. The proportion of winged offspring was also affected. Our results showed that octopamine could be a link in the wing determination system, as well as environmental stimulation. The RNAi results showed that the decrease of TβH expression increased aphid's reproduction; however, the decrease of TβH expression declined the numbers of winged-offspring producers, but did not affect the proportion of winged nymphs produced by the winged-offspring producer. In conclusion, the decline in the proportion of winged daughters in the next generation was caused by the decline of winged nymph producers.

Keywords: RNA interference; alate; apterae; neurotransmitter; tyramine β-hydroxylase; winged and wingless.

Figure 1

TβH expression patterns in Acyrthosiphon pisum under different conditions. TβH expression analysis in different stages of winged and wingless A. pisum, were detected separately in the head (A) and in the abdomen (B). The analysis of TβH expression in head, abdomen and whole body of wingless A. pisum raised at different densities (C) and TβH expression difference in heads between winged and wingless morphs (D). ^** in (A–D) indicate significantly different at P < 0.01, respectively (Student's t-test), and different letters in (A,B) on top of the bars indicate significantly differences (P < 0.05, Duncan's test). The schematic of winged-stimulation protocol based on densities (E) and dissection positions (dotted line) in A. pisum are shown in (F).

Figure 2

Sequence of TβH in Acyrthosiphon pisum, and two designed dsRNA were marked (positions of primers were marked in bold); fragment for qRT-PCR test was also marked in the sequence (A). Dissection (dotted line) and injection (dark spots) position are shown in (B), and the TβH assay protocol is shown in (C).

Figure 3

Full daughter scan MS Spectra and selected ion retention time (min) of octopamine (A–C) and tyramine (D–F). The mass spectrometer was set in the positive electrospray ionization mode. Nitrogen was used as the sheath gas (40 arbitrary units) and auxiliary gas (10 arbitrary units). The spray voltage was set at 4.5 kV and the ion transfer capillary temperature was 275°C. MS Spectra information was referred to the database of METLIN (Scripps Center for Metabolomics METLIN: Metabolite and Tandem MS Database http://metlin.scripps.edu/terms.php).

Figure 4

TβH expression under RNAi experiments in wingless Acyrthosiphon pisum. TβH expression in the head, abdomen and whole body of wingless A. pisum under TβH RNAi (A); TβH expression (two dsRNA) in the head after dsRNA repression for 6 days (B), and TβH expression in the newborn nymphs after TβH RNAi of their mothers for 3 days (C). Each value represents the mean ± SEM, and different letters in a and b on top of the bars indicate significantly different (P < 0.05, Duncan's test).

Figure 5

dsRNA, octopamine, octopamine receptor agonist and antagonist injection in Acyrthosiphon pisum. Reproduction abilities changes of A. pisum 7 days after injection (A); proportions of winged offspring of each aphid after injection (B); proportions of winged producers of all experimental aphids after injection (C); proportions of winged offspring produced by winged producers after injection (D); and tyramine concentration difference in TβH bioassays under RNAi (E). Each value represents the mean ± SEM, different letters in (A,B,D,E) at the bars or points indicate significantly different (P < 0.05, Duncan's test, and the letters in (E) show comparison of three treatments at each reaction time) and ^* of (C) indicates significant different based (P < 0.05, χ²-test).

Figure 6

The hypothesis of octopamine functions in wing formation of Acyrthosiphon pisum.