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-NH2 (FXPRLa) neuropeptides: DH, PBAN, and α-, β-, and γ-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.


Introduction
A seasonal polyphenism is a developmental phenotypic plasticity that has evolved for seasonal adaptation, and consists of the differential expression of alternative phenotypes from a single genotype depending on environmental conditions, including the photoperiod, temperature, and nutrition [1,2]. The white-spotted tussock moth, Orgyia thyellina, which belongs to Lymantriidae, has two or three generation per year, and pass winter season as diapause egg. Further, only females of this moth, but not males, exhibits seasonal polyphenism with various phenotypes of morphological, physiological, and behavioral traits in response to photoperiod ( Fig. 1) [3,4]. This sexual difference in photoperiodic response is very unique among many moth species because the sexual dimorphism of other moths is genetically determined [5,6,7,8]. In the adult stage, flight-capable, long-winged females of O. thyellina are produced from larvae reared at a long photoperiod, while flightless, short-winged females are formed at a short photoperiod. Females emerging in the summer are of the normal, long-winged morph, but those in the autumn are of the shortwinged morph. The summer long-winged female lays non-diapause eggs, whereas the autumnal short-winged female lays diapause eggs, which arrest at early embryonic stage [9]. In addition, various reproductive polyphenisms are exhibited in egg size, eggshell thickness, ovarian weight, and egg number. The diapause eggs are heavier in weight, larger in size, and much thicker in the chorion than the non-diapause eggs, presumably providing richer reserves and a more protective chorion to enhance the adaptation to low temperatures in the overwintering state [3].
In general, long-winged and short-winged morphs have different energetic profiles reflecting their different propensities for migration versus reproduction [10]. The polyphenism consists of a flight-capable morph that delays reproduction, and a flightless morph that exhibits substantially elevated fecundity; this is a lifehistory tradeoff called the ''oogenesis-flight syndrome'' in various insects [10,11]. These life-history traits alter the energy allocation of limited internal resources to maximize reproductive success [12,13,14]. However, the developmental processes and molecular mechanisms that are integrated with environmental stimuli for seasonal polyphenisms and tradeoff strategies are largely unknown, although presumably the developmental plasticity of a lifehistory trait has evolved to integrate signals from the environment into normal developmental processes through transcriptional and/ or neuroendocrine regulators [2].
The silkworm, Bombyx mori (Bombycidae) is a typical insect entering diapause at an early embryonic stage, similar to Orgyia as described above [15]. Research on the diapause mechanisms of the silkworm has contributed to the understanding of insect neuroendocrinology and to the technical development of the sericultural industry [16]. Diapause hormone (DH) is a 24 aa peptide amide belonging the Phe-X-Pro-Arg-Leu-NH 2 (FXPRL amide; FXPRLa) neuropeptide family, which is responsible for embryonic diapause, and functions by acting on a G proteincoupled receptor in the developing ovaries during pupal-adult development in females [16,17]. The DH-pheromone biosynthesis activating neuropeptide (DH-PBAN) gene encodes a polypeptide precursor consisting of five FXPRLa neuropeptides: DH, PBAN, and a-, b-, and c-SGNPs in various insects, including B. mori [18,19], which is exclusively expressed in eight pairs of neurosecretory cells (DH-PBAN-producing neurosecretory cells; DHPCs) located within the subesophageal ganglion (SG) [18,20,21].
In this study, we cloned and characterized the Orgyia DH-PBAN cDNA, and showed that Orgyia DH has a pleiotropic effect in the seasonal reproductive polyphenism, including diapause induction, which may be orchestrated via several signaling pathways to integrate various traits to accomplish the seasonal adaptation. These are the first results to 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 lifehistory traits and tradeoffs.

Characterization of the Orgyia thyellina DH-PBAN cDNA
The DH-PBAN cDNA had already been cloned in various insect species, including the Lepidoptera [19,22]. We cloned the Orgyia thyellina DH-PBAN (OtDH-PBAN) cDNA from brain-SG complex mRNA using a PCR-based strategy. By aligning the deduced amino acid sequence with the DH-PBAN precursor polyprotein from various insects, we found highly similar characteristics of those precursor polyproteins, especially in the Lepidoptera (data not shown). The DH-PBAN cDNAs were highly conserved in all five encoded FXPRLa neuropeptides, including those of Bombyx (Fig. 2) [19]. These seemed to include processing sites for the molecular maturation of DH and other FXPRLa neuropeptides through tryptic cleavage and amidation of the GKR, KK, GRR, and 3 GR sequences, as well as a signal peptide cleavage site ( Fig. 2A) [18]. Compared with that in Bombyx, the C-terminal amino acid sequence, FXPRLa, was completely conserved in each peptide (Fig. 2B). In addition, a glutamine residue at position 19 in DH was inserted in Orgyia, although this insertion was not found in other insect species (Fig. 2B). Thus, we first identified the DH-PBAN cDNA in Lymantriidae insects, and found that it was highly conserved in other species.

Developmental expression profiles of DH-PBAN
We looked at the expression of OtDH-PBAN in various tissues during larval-pupal and pupal-adult development using RT-PCR analysis. OtDH-PBAN mRNA was exclusively expressed in the brain-SG complex during post-embryonic development (Fig. 3A, lanes 1 and 8), although actin mRNA was expressed ubiquitously throughout post-embryonic development (Fig. 3A, lanes 14-26). Next, we examined the localization of OtDH-PBAN in the central nervous system by whole-mount in situ hybridization and immunohistochemistry (Figs. 3B-I). The intensity signal was detected in large somata along the ventral midline within the larval and pupal SG, respectively, in in situ hybridization ( Fig. 3B and F). Furthermore, immunohistochemical staining with an anti-FXPRLa antibody identified somata in the SGs of both larval and pupal stages ( Fig. 3C and G) whose immunofluorescence overlapped with the HNPP/FastRed TR fluorescences of the DH-PBAN mRNA probe ( Fig. 3D and H). We found that the arrangements of these neuromeres were similar to DHPCs conserved among insect species, which contain three neuromeres, four mandibular cells (SMd), six Orgyia 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 [3,4]. doi:10.1371/journal.pone.0024213.g001 maxillary cells (SMx), and two labial cells (SLb) located along the ventral midline [23,24,25]. Using an anti-FXPRLa antibody, the axons projecting from the DHPCs extended to the circumesophageal connective (Fig. 3H), and their axonal projections reached the neurohemal organ corpus cardiacum (CC) in the larval and pupal SG, respectively ( Fig. 3E and I). Therefore, we concluded that OtDH-PBAN is expressed in the DHPCs of SG, and the FXPRLa peptides are transported into the CC where they are released into the hemolymph, and that these neuroendocrine processes conserved in other lepidopteran insects.

Effects of Orgyia DH on diapause induction and its metabolism
To determine whether Orgyia DH induces embryonic diapause, we injected chemically synthetic DH into pupae kept under longday condition (LD; non-diapause type) (Fig. 4). When DH was injected into female pupae at 333 pmol/pupa, oviposited eggs took on a dark brown color (Fig. 4C), similar to diapause eggs induced by short-day condition (SD) (Fig. 4B), and were bigger than nondiapause eggs induced by LD (Fig. 4A). At 8 days after oviposition, non-diapause eggs took on pigmentation in the head and body of developing embryos ( Fig. 4E and I). In other words, some of the eggs oviposited from DH-injected pupae had the appearance of diapause eggs (Fig. 4G, arrow) obtained by SD (Fig. 4F), although some of the eggs took on pigmentation in the head and body of developing embryos similar to non-diapause eggs (Fig. 4G, arrowhead). In SD animals, we observed that the embryos were arrested and entered diapause in the early embryonic stage, with cephalic lobe and talson formation (Fig. 4J). We also observed variation in diapause stages in DH-injected animals, not only in cephalic lobe and talson formation similar to SD animals, but also in segmentation of the mesoderm in early embryonic development in Orgyia (Fig. 4K). These diapause eggs never hatched, even after a year kept at 25uC. However, when the eggs were kept at 5uC for 3 months starting from 7 days after oviposition, and then returned to 25uC, larvae hatched within 10 days (data not shown). Therefore, we categorized these eggs injected with DH as diapause the same as those induced by SD. Next, we injected various amounts of Orgyia DH into LD-types. DH caused a dose-dependent increase in the percentage of diapause eggs in a range of 12-333 pmol/pupa (Fig. 5A). The activity was saturated at more than 333 pmol/pupa, and the half-maximal dose was estimated to be 111 pmol/pupa. In addition to DH injection, we examined whether the injection of anti-FXPRLa antibody affects diapause induction. We injected anti-FXPRLa serum 1 day after pupation in SD-pupae, and observed the diapause nature of the eggs (Fig. 4D, H, and L). Although pre-immune serum had no effect on the diapause status, so that all eggs entered diapause as did the non-treated controls (Fig. 5A, 2), injection of anti-FXPRLa caused non-diapause eggs to become light yellow and smaller than diapause eggs in most of the egg batch just after oviposition ( Fig. 4D and 5A, +). With advancing embryogenesis, non-diapause eggs took on pigmentation in the head and body of developing embryos (Fig. 4H). The larvae were hatched within 10 days (Fig. 4L). Consequently, these results suggest that the anti-FXPRLa serum acts in the hemolymph to inactivate DH through immunoneutralization.
In general, insects accumulate reserves prior to diapause. Sufficient reserves must be sequestered to both survive the diapause period and enable postdiapause development [26]. In Bombyx, diapause eggs are associated with qualitative and quantitative shifts in glycogen metabolism, which accumulate larger glycogen reserves than their nondiapausing counterparts [15]. Therefore, we investigated whether the Orgyia DH affects glycogen content in the ovary. The Orgyia DH caused an increase in glycogen content in ovaries in a dose-dependent manner, and the injection of anti-FXPRLa suppressed that accumulation (Fig. 5B). Collectively, we demonstrated that the neuropeptide DH induced not only embryonic diapause, but also shifts to diapause metabolism in Orgyia.

Effects of Orgyia DH on oogenesis involved in seasonal polyphenism
We further examined whether DH induces various traits in seasonal polyphenism in oogenesis, although none of those changes are observed in Bombyx. First, we observed various traits of oogenesis in LD-and SD-pupae. The eggs of SD-animals were larger, heavier, had a thicker chorion, had a greater number of follicles, and were heavier in the whole body compared to those of LD-animals ( Fig. 5C-G). Next, we investigated whether the Orgyia DH affected those phenotypes. Orgyia DH caused an increase in the maximum diameter, eggshell thickness, and ovary weight in a dose-dependent manner (Fig. 5C-E), although there were differences in response for each trait (Fig. 5D vs. A-C and E). In addition, injection of the anti-FXPRLa into SD animals induced dwarfness in eggs including smaller size, thinner maximum eggshell thickness, and lighter ovaries similar to non-diapause eggs (Fig. 5C-E). However, no changes were observed in the number of follicles and body weight by Orgyia DH and antiserum injections ( Fig. 5F and G).
Consequently, Orgyia DH acts on the ovarian mass ( Fig. 5E and  H). Interestingly, although the ovarian masses of LD-and SDpupae were 20% and 35%, respectively (Fig. 5H), DH-injected animals in LD-pupae were similar to SD animals in terms of ovary mass and whole body weight irrespective of the injection of excess amounts of DH. Thus, Orgyia DH induced various traits involved in the seasonal reproductive polyphenism underlying the lifehistory tradeoff known as oogenesis-flight syndrome.

Effects of other Orgyia FXPRLa on diapause induction in both Orgyia and Bombyx
Next, we examined whether other FXPRLa (a-, b-, c-SGNPs, and PBAN) induced embryonic diapause with a similar doseresponse as DH (Fig. 6). We found that all FXPRLa had diapause egg inducing activity, which increased dose-dependently in a range of 33-333 pmol/pupa similar to Orgyia DH in Orgyia (Fig. 6A). These diapause eggs inducing activities were saturated at more than 333 pmol/pupa. Furthermore, we tested whether each Orgyia FXPRLa also induced embryonic diapause in Bombyx (Fig. 6B). Interestingly, only Orgyia DH had a similar effect with the dose used in Orgyia, but PBAN showed diapause egg inducing activity only at 10 times amount of DH. No activity was found in a-, b-, or c-SGNPs in this experiment (Fig. 6B). These results showed that the diapause inducing activity of Orgyia FXPRLa is different between Orgyia and Bombyx, suggesting that the mechanism of hormonal reception is different in the two species, given that DH and the other FXPRLa have similar structures in both species.

Discussion
Previously, Bombyx DH was known as the only hormone that induces diapause among known FXPRLa neuropeptides [16]. Furthermore, Bombyx DH stimulates the transcription of the trehalase gene in ovaries, and thereby increases trehalase activity which facilitates higher accumulation of glycogen in eggs, a prerequisite for diapause initiation [27,28] that sequesters sufficient reserves for survival of the diapause period and postdiapause development [26]. We found that Orgyia DH also acts as a diapause inducer during embryogenesis causing development to be arrested, and enhances the glycogen content, which seems to affect the conserved neuroendocrine system in both Bombyx and Orgyia. We think it is most likely that in SD-type morphs, transportation of DH from the DHPCs somata is actively modified in response to the photoperiod during late larval stages and results in active release into the hemolymph during the pupal stages, likely as in Bombyx [29]. Thus, we showed that the DH acts as a diapause inducer beyond the families Bombycidae and Lymantriidae in lepidopteran insects.
DH and other FXPRLa are multifunctional peptides that play a conserved role in the coordination of life-cycles among and within species. DH acts not only during diapause induction in Bombyx [16], but is also thought to function during ecdysteroidgenesis [30]. Furthermore, the neuropeptides of this family are involved in pheromone biosynthesis, cuticular tanning, myostimulation, pupariation behavior, and the termination of pupal diapause in several lepidopteran insects [31,32,33,34,35]. In addition to diapause induction, Orgyia DH affects various traits in ovarian growth and maturation such as follicle size, eggshell thickness, and ovarian mass in different dose-dependent manner, although none of these changes are observed in Bombyx. These results suggest that there are several signaling pathways involved in the induction of   (2) were injected into SD-pupae to examine the DH functions as described in the text. Each bar represents the mean value of 10 samples 6 SD. Lower-case letters indicate statistically significant differences at the 5% level in DH-injected animals at 0 pmol in LD vs. pre-immuno serum-injected these various traits. In general, the developing follicle processes the transition among broad developmental periods, which is mediated by hormones such as ecdysteroid and a sesquiterpenoid lipid-like hormone, JH, in insects [36]. In Bombyx, ecdysteroid was shown to be essential and sufficient for ovarian development through the activation of the ecdysone regulatory cascade [37,38]. Furthermore, the injection of large amounts of 20-hydroxyecdysone (20E) into normal female pupae of Bombyx alters egg production, so that these eggs are larger and heavier than normal eggs [39], suggesting that the ecdysteroid may induce an increase in the number of oocytes by functional activation of nurse cells, to accelerate the growth of oocytes accompanied by enhanced accumulation of yolk proteins and accelerated choriogenesis. Taken together, this suggests that Orgyia DH may induce egg polyphenism directly and/or indirectly via several signaling pathways, including 20E signaling, which may function via ecdysteroidgenesis as in Bombyx [30]. Thus, this is the first evidence suggesting that a neuropeptide (i.e., Orgyia DH) alone has a pleiotropic effect in seasonal reproductive polyphenims, which may be orchestrated via several signaling pathways to integrate various traits, since these polyphenisms are considered to provide richer reserves and more protective chorion to enhance adaptation to low temperatures.
Neuroendocrine systems are known to coordinate the integrated expression of multiple life-history traits across environmental conditions as developmental phenotypic plasticity, which mediates pleiotropy, life-history correlations, and tradeoffs in organisms as diverse as lizards, birds, insects, and echinoderms [13,40]. In insects, JH and ecdysteroid are well known to be important mediators of life-history tradeoffs [13,14,41]. Indeed, it has been suggested that JH and ecdysteroid are involved not only in the tradeoff between flight capability and reproduction in wingdimorphic crickets, Gryllus firmus [42], but also in tradeoffs in various insects including Drosophila, butterflies, and beetles [14,43]. Furthermore, it is known that JH alters lipid metabolism that contributes significantly to the tradeoff in G. firmus [44]. We have also confirmed that various traits with respect to ovarian and embryonic development change between long-winged and shortwinged morphs in Orgyia (Fig. 1) [3,4]. Furthermore, it has been observed that the flight muscles become extended and increase in thickness in all long-winged morphs, while the flight muscles remain thin in short-winged morphs (Y. K, unpublished results). Thus, Orgyia exhibits an extreme seasonal polyphenism resulting in a tradeoff between flight capability and reproduction, resulting in differential allocation of internal reserves to ovarian growth versus somatic growth, maintenance, or storage. These are the first results to show that a factor (i.e., DH neuropeptide) other than a sesquiterpenoid lipid-like hormone or steroid hormone (i.e., JH and ecdysteroid) can act as an important inducer of developmental phenotypic plasticity underlying a life-history tradeoff, although it is still unknown what the mechanism of energy allocation is and its involvement in wing development.
Tight control is necessary to produce an animal in which each organ is of an appropriate size relative to the whole body. Different organs typically grow at different rates in developing animals, a phenomenon called ''allometry'' [45,46]. The flexibility of allometry is necessary to produce an animal in which the whole body is of an appropriate size relative to environmental conditions such as nutrition, temperature, and population density [47,48]. Recent studies have indicated that the insulin-signaling pathway controls body and organ size in Drosophila and most metazoans by signaling nutritional conditions to the growing organs [49,50]. In this study, the rates of ovarian mass to body increased to accompany the increase in ovarian weight by DH injection, so that LD animals injected with DH, even in excess doses, shifted to become similar to SD animals. Therefore, we speculate that there is inherent allometry in ovarian mass in both LD-and SD-types, which determine the photoperiodism. Additionally, the DHsignaling pathway might be involved in the regulation of ovarybody allometry, although it is unknown whether other signals are involved, such as the insulin-signaling pathway.
It has been suggested that variations in endocrine mechanisms may be an important substrate for the evolution of coregulated life-history traits and tradeoffs [14]. Evolutionary modifications of the hormone response may be facilitated by its modular structure [40]. In a previous study, Bombyx DH was found to be solely responsible for embryonic diapause and functions by acting on a specific G protein-coupled receptor in developing ovaries, as other FXPRLa never act at similar doses to DH [16,17]. Likewise, Orgyia FXPRLa, except for DH, never induce embryonic diapause at similar doses of DH in Bombyx. Interestingly, in the present study we found that Orgyia FXPRLa functions in diapause induction in a animals in SD (a), DH-injected animals at 0 pmol in LD vs. DH-injected animals at various doses (b), and pre-immuno serum-injected animals in SD vs. anti-FXPRLa-injected animals (c). doi:10.1371/journal.pone.0024213.g005 similar dose-dependent manner to DH in Orgyia. This result suggests that the structure and/or densities of receptor protein(s) may be different with evolutionary diversification in the two species. Variation in the expression of the receptor protein(s) of the Orgyia FXPRLa family may occur in the two species, so that those differences may promote the hormonal pleiotropy in the polyphenism. Therefore, the present study may provide not only clues for solving the molecular mechanisms of life-history evolution, but also provide new insights into comparative neuroendocrinology in insects.

Insects
Eggs of the white spotted tussock moth, Orgyia thyellina, were collected at an apple orchard in the Nagano Fruit Tree Experiment Station (Suzaka, Japan). The hatched larvae were reared on an artificial diet (Silkmate-2S, Nosan Co., Yokohama, Japan) from first instar to third instar, and fresh apple leaves from fourth instar to final instar (sixth instar in females and fifth instar in males) at 22uC under LD (16-h light/8-h dark cycle; 16L:8D) or SD (8L:16D). Pupae were collected within the day just after pupation (referred to as day 0) to synchronize their subsequent development.
Pupae were kept at 25uC to allow adult development.
The polyvoltine (N4) strain of Bombyx mori was used in this study. Eggs were incubated at 25uC under continuous darkness. Larvae were reared on an artificial diet (Kuwano-hana, JA Zennoh Gunma, Gunma, Japan). During larval stages, silkworms were reared at 25 to 27uC under a 12L:12D. Pupae were kept at 25uC to allow adult development. N4 moths laid only non-diapause eggs genetically, so that eggs never entered diapause.
The ORF sequence of the actin cDNA was amplified using primers based on sequences of the actin cDNA cloned from various insects: 59-ATGTGCGACGARGAAGTTGC-39 (forward) and 59-TTAGAAGCACTTCCTGTGNAC-39 (reverse). The fulllength sequence (Accession no. AB283042) was determined using a SMART RACE cDNA amplification kit (Clontech).

RT-PCR analysis
Various tissues were dissected from day 3 sixth instar larvae and day 1 pupae of females reared in SD. Total RNAs were extracted using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and then subjected to Poly (A) + RNA purification using Dynabeads Oligo (dT) 25 (Dynal Biotech LLC.). Poly (A) + RNA from the brain-SG complex was directly purified using Dynabeads Oligo (dT) 25 . First-strand DNA was synthesized using a SMART RACE amplification kit (Clontech). PCR amplification was carried out on mRNAs for DH-PBAN and actin genes. The DH-PBAN cDNA was amplified from nucleotides +1 to +759, and the actin cDNA from +238 to +722. The PCR products were subjected to electrophoresis, and then visualized with ethidium bromide staining.

In situ hybridization and Immunohistochemistry
In situ hybridization was performed as described by [51] with some modifications. Brain-SG and retrocerebral, corpus cardiacum (CC)-corpus allatum (CA) complexes were treated for 5 min or 3.5 min with 10 mg/ml proteinase K (Roche) at 37uC on larval or pupal tissues, respectively. The DIG-labeled RNA probes were prepared with a DIG RNA labeling kit (Roche, Mannheim, Germany) using DH-PBAN cDNA as a template. DH-PBAN cDNA encoding from nucleotides +1 to +759 was amplified by PCR and inserted into the pCR-XL-TOPO vector (Invitrogen) in an antisense direction from the T7 promoter. DIG-labeled RNA was detected with an alkaline phosphatase-conjugated anti-DIG antibody using a DIG nucleic acid detection kit (Roche), and by using the HNPP fluorescent detection set (Roche) as a substrate. The immunoreaction procedures were adapted from [29] by using an anti-FXPRLa polyclonal antibody [25,29]. HNPP/FastRed TR fluorescence and anti-FXPRLamide immunofluorescent staining were detected using both a Radiance 2000 confocal microscope (Bio-Rad Lab., Hercules, CA, USA) and confocal microscope system A1 (Nikon, Tokyo, Japan). Images were adjusted and assembled in Adobe Photoshop CS3 (Adobe Systems, Inc., San Jose, CA, USA).

Injection of peptides and antibody
Purified synthetic peptides (DH, a-, b-, c-SGNP, and PBAN) of .95% purity (HPLC area percentage) were provided from Operon Biotechnologies, KK. (Tokyo, Japan). Each peptide was dissolved in distilled water, and 10 ml solutions of each at various doses were injected into pupae on the day of pupation (day 0) of Orgyia, and 4 days after pupation in the N4 strain of Bombyx. Anti-FXPRLa antiserum was injected at 10 ml into Orgyia pupae on day 0 in accordance with [27].

Analyses of oogenesis
The ovaries were dissected out with phosphate-buffered saline (PBS) 1 day before eclosion when pupae integuments became white, and the wing color patterns of the adult forms were visualized. The paired ovaries were thoroughly separated into 8 ovarioles, then blotted dry, weighed quickly, and the number of follicles was counted. In each ovariole, 20 successively developing follicles from side of the lateral oviducts were collected to measure the maximum diameter, eggshell thickness, and glycogen content. The maximum diameter and eggshell thickness were measured by scanning on a SZX-12 microscope (Olympus, Tokyo, Japan) with a micrometer. Glycogen was extracted by digesting the homogenate with 30% ( W / V ) KOH in a boiling bath for 30 min, and was precipitated with ethanol at 4uC [52], and measured by the phenol-sulfuric acid method [53].

Observation of embryogenesis and diapause
In both Orgyia and Bombyx, eclosed female moths were allowed to copulate with males overnight, and then to lay eggs overnight. The laid eggs were kept at 25uC, under which condition the nondiapause eggs hatched on day 7 to 8 after oviposition. Some eggs became light white or yellow in a few days and were recognized as non-fertilized eggs. Diapause eggs were identified as eggs that were colored dark brown on day 3 and remained unhatched at 2 weeks after oviposition, at which time all non-diapause eggs had hatched.
Diapause egg inducing activity was estimated by counting the number of diapausing and non-diapausing eggs after the nondiapause eggs hatched, and the results were expressed as a percentage of diapause eggs [54]. The embryos were stained with carbolic thionin solution according to [55]. Eggs were mounted on a slide glass sealed with adhesive tape, and then eggs were sliced with a razor and observed under a microscope.