An FXPRLamide Neuropeptide Induces Seasonal Reproductive Polyphenism Underlying a Life-History Tradeoff in the Tussock Moth

The white spotted tussock moth, Orgyia thyellina, is a typical insect that exhibits seasonal polyphenisms in morphological, physiological, and behavioral traits, including a life-history tradeoff known as oogenesis-flight syndrome. However, the developmental processes and molecular mechanisms that mediate developmental plasticity, including life-history tradeoff, remain largely unknown. To analyze the molecular mechanisms involved in reproductive polyphenism, including the diapause induction, we first cloned and characterized the diapause hormone-pheromone biosynthesis activating neuropeptide (DH-PBAN) cDNA encoding the five Phe-X-Pro-Arg-Leu-NH(2) (FXPRLa) neuropeptides: DH, PBAN, and alpha-, beta-, and gamma-SGNPs (subesophageal ganglion neuropeptides). This gene is expressed in neurosecretory cells within the subesophageal ganglion whose axonal projections reach the neurohemal organ, the corpus cardiacum, suggesting that the DH neuroendocrine system is conserved in Lepidoptera. By injection of chemically synthetic DH and anti-FXPRLa antibody into female pupae, we revealed that not only does the Orgyia DH induce embryonic diapause, but also that this neuropeptide induces seasonal polyphenism, participating in the hypertrophy of follicles and ovaries. In addition, the other four FXPRLa also induced embryonic diapause in O. thyellina, but not in Bombyx mori. This is the first study showing that a neuropeptide has a pleiotropic effect in seasonal reproductive polyphenism to accomplish seasonal adaptation. We also show that a novel factor (i.e., the DH neuropeptide) acts as an important inducer of seasonal polyphenism underlying a life-history tradeoff. Furthermore, we speculate that there must be evolutionary conservation and diversification in the neuroendocrine systems of two lepidopteran genera, Orgyia and Bombyx, in order to facilitate the evolution of coregulated life-history traits and tradeoffs.ArticlePLOS ONE. 6(8):e24213 (2011)journal articl

Seasonal polyphenism in the white spotted tussock moth, <i>Orgyia thyellina</i>.

<p><i>Orgyia</i> exhibits seasonal changes in various morphological, physiological, and behavioral traits, which are determined by the photoperiod during the late larval stages from 4^th to 5^th instar larvae. In the long-day condition (LD), the larval integument becomes light colored in the final instar larva (6^th instar) as does the pupae of the females and their cocoons. The larval integuments and cocoons are darkly colored under short-day conditions (SD). In the adult stage, LD females are flight-capable long-winged morphs, but SD females are flightless short-winged morphs. Furthermore, LD female lay non-diapause eggs, whereas the SD female lay diapause eggs, which are arrested in early embryonic development. These diapause eggs are heavier in weight, larger in size, and much thicker in the chorion than non-diapause eggs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024213#pone.0024213-Kimura1" target="_blank">[3]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024213#pone.0024213-Sato1" target="_blank">[4]</a>.</p

Developmental expression profiles of <i>OtDH-PBAN</i>.

<p>A; RT-PCR analysis was performed on day 3 in sixth instar larvae (LVI3) as well as on day 1 in pupae (P1). Levels of DH-PBAN (DHP, lanes 1–13) and actin (lanes 14–26) mRNAs were examined. BS, brain-subesophageal ganglion complex; MG, midgut; FB, fat body; SL, silk gland; OD, ovarian disc; WD, wing disc; IM, integument and muscle; WG, wing; OV, ovary. Whole-mount <i>in situ</i> hybridization was performed in larval (B) and pupal (F) brain-SG complexes by using antisense RNA of <i>OtDH-PBAN</i> as a probe. Using anti-FXPRLa antibody, immunohistochemistry was performed in larvae (C) and pupae (G) stages. Magnified double stained images are shown in magenta (DH-PBAN RNA) and green (anti-FXPRLa) in larval (D) and pupal (H) SG, and larval corpus cardiacum, CC (E). Immunostaining was performed in pupal CC (I). FXPRLa immunoreactive somata were detected in three neuromeres, mandibular cells (SMd), maxillary cells (SMx), and labial cells (SLb), located along the ventral midline. The projective axons (arrowhead) from these somata run into the CC via the circumesophageal connective (CoC). Scale bar = 10 µm.</p

Effects of other <i>Orgyia</i> FXPRLa on diapause induction in both <i>Orgyia</i> and <i>Bombyx</i>.

<p>The <i>Orgyia</i> DH, α-, β-, and γ-SGNPs, and PBAN were injected into LD-pupae of <i>Orgyia</i> (A) and the non-diapause type of <i>Bombyx</i> (B) at various doses [3 (0), 33 (1), 333 (2), and 3333 (3) pmol/pupa], and subjected to analysis of diapause egg inducing activity. Each bar represents the mean value of 20 samples ± SD. Asterisks indicate statistically significant differences at the 5% level.</p

Schematic drawing of the DH-PBAN precursor polyprotein in <i>Orgyia</i>.

<p>A; <i>DH-PBAN</i> cDNA encoding pre-prohormone consisting of 199 amino acids. It seems to undergo post-translational processing via a series of enzymatic steps that cleave and further modify by amidation the GKR, KK, GRR, and 3 GR sequences at the C-terminal amino acid of the intermediate peptide substrates to yield the signal sequence (SS), DH, α-, β-, and γ-SGNP, and PBAN, similar to other Lepidopteran DH-PBAN precursor polyproteins. B; Alignment of <i>Orygia</i> DH-PBAN with <i>Bombyx</i> DH-PBAN. Conserved amino acids are indicated with shadow boxes; highly conserved amino acids in FXPRLa sequences are indicated with dark shadow boxes. The percentages of identical amino acids is represented on the right side of the peptide sequences. A glutamine residue at position 19 in <i>Orygia</i> DH is shown by an asterisk (*).</p