Disruption of diapause induction by TALEN-based gene mutagenesis in relation to a unique neuropeptide signaling pathway in Bombyx

The insect neuropeptide family FXPRLa, which carries the Phe-Xaa-Pro-Arg-Leu-NH2 sequence at the C-terminus, is involved in many physiological processes. Although ligand–receptor interactions in FXPRLa signaling have been examined using in vitro assays, the correlation between these interactions and in vivo physiological function is unclear. Diapause in the silkworm, Bombyx mori, is thought to be elicited by diapause hormone (DH, an FXPRLa) signaling, which consists of interactions between DH and DH receptor (DHR). Here, we performed transcription activator-like effector nuclease (TALEN)-based mutagenesis of the Bombyx DH-PBAN and DHR genes and isolated the null mutants of these genes in a bivoltine strain. All mutant silkworms were fully viable and showed no abnormalities in the developmental timing of ecdysis or metamorphosis. However, female adults oviposited non-diapause eggs despite diapause-inducing temperature and photoperiod conditions. Therefore, we conclude that DH signaling is essential for diapause induction and consists of highly sensitive and specific interactions between DH and DHR selected during ligand–receptor coevolution in Bombyx mori.

activate related groups of receptors 2 . However, the relationship between these ligand-receptor interactions and in vivo physiological functions remains unclear.
In the silkworm (Bombyx mori) genome, FXPRLa neuropeptides are encoded by two genes: diapause hormone (DH)-pheromone biosynthesis activating neuropeptide (PBAN) DH-PBAN and capa (Supplementary Table S1) 9 . DH-PBAN encodes a polypeptide precursor consisting of five FXPRLa neuropeptides: DH, PBAN, and α -, β -, and γ -SGNPs (Subesophageal ganglion neuropeptides) (Fig. 1A) 10 . The capa gene encodes a polypeptide precursor consisting of three neuropeptides that contain an FXPRLa, CAPA-PK. The GPCRs related to the NMU receptor activated by these peptides are clustered in the phylogeny 11 . DH is well known as a neuropeptide hormone responsible for induction of embryonic diapause in Bombyx 12 . DH functions by acting on a GPCR, DH receptor (DHR; Fig. 1B) in the developing ovaries during pupal-adult development in females 13 . Previous studies showed that DH is a selective and sensitive ligand for DHR and is distinguished from other neuropeptides encoded by DH-PBAN 8,13 . Therefore, it was thought that diapause induction elicited by DH signaling consisted of interactions between DH and DHR. However, the relationship between DH-DHR interactions and diapause induction has not been investigated in vivo.
Bombyx embryonic diapause is a unique process of seasonal polyphenism that is induced transgenerationally as a maternal effect in the bivoltine strain. Progeny diapause is determined by the mother's experience during embryonic development. When eggs are incubated at 25 °C under continuous darkness (25DD), the resultant female moths are able to lay diapause eggs. In contrast, incubation at 15 °C under continuous darkness (15DD) causes the resultant moths to lay non-diapause eggs. If eggs are incubated at 20 °C under continuous illumination (20LL) or darkness (20DD), the resultant moths produce diapause or non-diapause eggs, respectively 14 . Thus, progeny diapause is determined by environmental factors such as photoperiod and temperature during maternal embryogenesis. Recently, we showed that the embryonic Bombyx TRPA1 ortholog (BmTrpA1) acts as a thermosensitive channel that is activated at temperatures above ~21 °C and affects diapause induction through DH release during pupal-adult development 15 . Thus, some of the molecular mechanisms in the pathway leading from reception of environmental signals to expression of the diapause phenotype have been revealed.
Here, we performed TALEN-based mutagenesis of Bombyx DH-PBAN and DHR to isolate the null mutants of those genes in a bivoltine strain. All mutant silkworms were fully viable and showed no abnormalities in the developmental timing of ecdysis and metamorphosis. However, female adults oviposited non-diapause eggs despite diapause-inducing temperature and photoperiod conditions. Therefore, we concluded that DH signaling is essential for diapause induction, which is independently accomplished by highly sensitive and specific interactions between DH and DHR selected through ligand-receptor coevolution in Bombyx mori.

Construction of TALEN-based mutants of DH-PBAN and DHR. For mutagenesis of DH-PBAN,
we selected a TALEN target in the sequences of the first exon that encoded DH (Fig. 1A,C). We isolated the two homozygous mutants containing 4-or 5-base deletions and designated the mutants as Δ DHP33 and Δ DHP531, respectively (Fig. 1C). These were considered null mutants, which could not translate the DH-PBAN preprohormone by frame-shift of DH-PBAN cDNA. We obtained two mutants of DHR, designated Δ DHR96 and Δ DHR111 (Fig. 1D), which carried a 7-or 24-base deletion, respectively, of the sequences in the anterior region encoding the transmembrane domain 1 (TM1) of the second exon (Fig. 1B). The Δ DHR96 mutant translated the truncated protein containing a partial DHR signal sequence and was considered the null mutant. The Δ DHR111 mutant was thought to cause an in-frame mutation; DHR111 was missing eight amino acids, which led to the production of truncated protein defective in the extracellular domain at the N-terminus of DHR. Thus, we isolated four mutants related to DH signaling.

DH and glycogen contents in ΔDHP and ΔDHR mutants.
To confirm the null mutagenesis of DH-PBAN, we first investigated the immunoreactivity of DH and PBAN in pupal subesophageal ganglion (SG). Previous reports showed that DH-PBAN is exclusively expressed in seven pairs of neurosecretory cells (DH-PBAN-producing neurosecretory cells; DHPCs) located within the SG 18,19 . In wt (25DD) pupal SG, namely wt pupal SG that incubated at 25 °C under continuous darkness during embryogenesis, immunoreactive signals to anti-DH[N] antibody were detected in DHPC somata, including the SMd, SMx, and SLb neuromeres along the ventral midline and in SL cells ( Fig. 2A), similar to previously reported results 20  To further accurately determine the levels of circulating DH in the hemolymph, we developed a new, sensitive time-resolved fluoroimmunoassay (TR-FIA). By using synthetic DH as a standard, we found the detection limit for a 150-μ L hemolymph sample from one or two animals to be ≈ 0.57 pM (≈ 85.8 amol). We measured DH levels in the hemolymph of pupa at 4 days after pupation (Fig. 2K). In wt (25DD) pupa, DH titer was 8.85 ± 2.71 pM, which was two-fold higher than in wt (15DD), suggesting the active release of DH in wt (25DD). DH was undetectable in the Δ DHP33 and Δ DHP531 lines despite rearing under 25DD conditions. Furthermore, DH levels in DHR mutants were two-fold higher than those in wt (25DD). These results suggest changes in DH titer in diapause of wt; in addition, DH levels were affected by the disruption of DH and DHR, indicating that DH signaling itself regulates the hemolymph DH levels.
Bombyx DH stimulates transcription of the trehalase gene in ovaries, thereby increasing trehalase activity, which facilitates greater accumulation of glycogen in eggs-a prerequisite for diapause initiation 21,22 . The glycogen content in ovaries of wt (25DD) pupa was high compared with that in wt (15DD) (Fig. 2L). The four 25DD Δ DHP and Δ DHR mutants had similar glycogen content to wt (15DD), but glycogen differed significantly from that in the wt (25DD). These results suggested that DH signaling affects glycogen accumulation in ovaries during the preparative phase of diapause.
Phenotypic analyses of ΔDHP and ΔDHR mutants. In general, diapause eggs have dark brown pigmentation because of 3-hydroxykynurenine (3-OHK) in their serosa, whereas non-diapause eggs lack this pigment, and thus, appear light yellow. Notably, DH was suggested to facilitate the accumulation of 3-OHK in pupal ovaries 14 . Progeny eggs of wt (25DD) showed light brown pigmentation on day 2 after oviposition, became dark brown on day 10 after oviposition, and, as they were diapause eggs, eventually failed to hatch (Fig. 3A, wt). Progeny eggs of Δ DHP33 and Δ DHR96 showed no pigmentation, and the larvae hatched on day 10 after oviposition despite 25DD rearing conditions (Fig. 3A, Δ DHP33, Δ DHR96). The percentage of diapause eggs was counted in 50 batches of progeny eggs among the wt and four mutants ( Fig. 3C and Supplementary Fig. S1A). In the wt, 25DD and 20LL adults oviposited diapause eggs, and 15DD and 20DD eggs mostly became non-diapause eggs. However, all four mutant adults oviposited non-diapause eggs, mostly under 25DD, 15DD, 20LL, and 20DD conditions (Fig. 3C). Thus, disruption of the DH signaling pathway appeared to block diapause induction in the mutants despite diapause-inducing temperature and photoperiod conditions.
Interestingly, a few eggs in the mutants had light-brown pigmentation on day 2 after oviposition ( Fig. 3B, black asterisk); these pigmented eggs hatched, but embryonic development was slightly delayed (Fig. 3B, white asterisk). Further, some of the pigmented eggs failed to hatch, and embryonic development was arrested at a specific stage during embryogenesis, immediately after formation of the cephalic lobe and telson and after segmentation of mesoderm, known as the diapausing stage in wt (Fig. 3D) 23 . The resulting mutants oviposited diapause eggs at ratios of 0.03-0.50% ( Fig. 3C and Supplementary Fig.  S2A). Next, we attempted rescue experiments of mutant lines by injecting synthetic DH or other FXPRLa to confirm whether only DH was responsible for diapause induction (Fig. 3E). In wt (15DD), DH had a significant effect on diapause egg inducing activity, which increased in a dose-dependent manner at the range of 10-1000 pmol/pupa; however, PBAN showed diapause egg inducting activity only at 100 times the amount of DH. Furthermore, almost no activity was observed after injections of α -, β -, and γ -SGNPs. In Δ DHP33 and Δ DHP531 lines, as well as in wt (15DD), high diapause eggs inducing activities were noted only after DH injection. In addition, almost no activity was observed in Δ DHR96 and Δ DHR111 even after DH injection.
Previous studies using in vitro assays showed that Bombyx DHR is also expressed in the prothoracic gland, the organ that synthesizes and releases the insect molting hormones (ecdysteroids), which may be activated by DH to function ecdysteroidogenesis in larval instars 8 . Therefore, we tested for effects on the developmental timing of ecdysis and metamorphosis in the mutant lines ( Fig. 3F-H). Generally, wt (15DD) larvae spent less time in the larval period than did wt (25DD) larvae; wt (15DD) larvae initiated spinning earlier than did wt (25DD) larvae 23 , as indicated by the 3-day shift in wt (15DD) compared to that in wt (25DD) (Fig. 3F). We reared 100 larvae from each of the wt and four mutants, all under the same conditions, and observed the time of molting and duration of the larval period. Most wt (25DD) larvae spent 5, 4, 5, 2.5, and 7.5 d in the 1st, 2nd, 3rd, 4th, and 5th instar, respectively (Fig. 3F), and larvae initiated spinning 21 d after hatching, with a peak at 22 d ( Supplementary Fig. S1B)-similar to that of the four mutants under 25DD conditions (Fig. 3F). Under 15DD conditions, the four mutants and wt showed similar developmental timing ( Fig. 3F and Supplementary Fig. S1B). In addition, because it is known that wt (25DD) pupae have heavier bodies and cocoon shells 23 , we tested whether pupal and cocoon-shell weights are affected by DH signaling. Both female and male pupae incubated under 25DD conditions had bodies ( Fig. 3G and Supplementary Fig. S1C) and cocoon shells ( Fig. 3H and Supplementary Fig. S1D) that were heavier than those of wt (15DD) and that were similar in weight to those of all mutant pupae. Thus, we did not observe differences in the duration of the larval period or the weight of pupae and cocoons in DH-PBAN and DHR mutants, suggesting that DH signaling does not participate in ecdysteroidogenesis in vivo. Further, because the previous in vitro experiments used high concentrations of DH, we speculated that artificial effects were observed. PBAN is known to stimulate the secretion of a sex pheromone, bombykol, from the pheromone gland in Bombyx 3 . Further, we observed a slight reduction in sexual behaviors such as flapping, orientation, and attempted copulation in male Δ DHP33 and Δ DHP531 mutants but not in Δ DHR mutants, which suggests that pheromone production is suppressed by PBAN knockout in female. Each mutant eventually mated and oviposited eggs in similar numbers to the wt (Supplementary Fig. S1A).

Discussion
We clearly showed in this study that in vivo disruption of DH-PBAN and DHR blocked diapause induction in progeny embryos. As described previously, when expressed in a Xenopus oocyte system, DHR showed the highest affinity (EC 50 , ~70 nM) for DH compared with the other FXPRLa encoded by DH-PBAN 8,13 . Furthermore, an in vivo bioassay showed that synthetic DH was more effective than other FXPRLa at inducing diapause, with threshold levels less than 1/100 that of PBAN and other peptides encoded by DH-PBAN (Fig. 3E) 10 . In Orgyia thyellina, not only DH induced embryonic diapause in progeny, but also other FXPRLa encoded by DH-PBAN induced diapause in an in vivo bioassay 24 . Taken together, we conclude that DH signaling is essential for diapause induction and that a highly sensitive and specific interaction between DH and DHR is a result of ligand-receptor coevolution in Bombyx mori.
Extensive structural-functional studies of the Bombyx PBAN receptor (PBANR) have been performed using mutant receptors and in vitro assays 25,26 . These studies revealed a number of functional domains and sites that are crucial for receptor activation and regulation; thus, it has been suggested that the extracellular loops (regions between each transmembrane domain) of PBANR, DHR, and related GPCRs function as a ligand-selection filter 26 . Therefore, the extracellular loop domains of DHR may have evolved to interact selectively with DH as well as to fulfill the functional requirements for diapause induction in Bombyx. The Δ DHR111 mutant, which was defective in eight amino acids of the extracellular N-terminus, had a similar phenotype as did the null mutant Δ DHR96. Since domain swaps in the Helicoverpa zea PBANR suggested roles for the N-terminus in ligand binding 5 , the Δ DHR111 mutant may be defective in ligand binding ability. Furthermore, we showed that the DH titer increased in DHR mutants compared to the wt. It is probable that these mutant receptors were unable to internalize the ligand, similar to that reported for PBANR 27 , or were unable to trigger negative feedback regulation of DH release, resulting in an abnormal increase in DH titer. Thus, we propose, based on our TALEN-mediated in vivo analysis, that the extracellular N-terminus is critical for Bombyx DHR function.
The structural similarity between DH and CAPA-PK, which carries the WFGPRLa sequences in the C-terminus (Supplementary Table S1), has been assumed to explain the highly sensitive cross-reactivity of CAPA-PK to DHR. We clearly demonstrated the role of DH in diapause induction. It may be likely that there are differences in spatiotemporal dynamics between DH and CAPA-PK. Therefore, CAPA-PK might not interact with DHR in ovaries during pupal-adult development.
In facultative diapause, the decision to enter diapause is generally determined by environmental factors such as photoperiod, temperature, and nutrition received by that individual or its mother at an earlier developmental stage 28 . Although many links in the pathway leading from reception of environmental signals to expression of diapause phenotype remain poorly understood, it has been proposed that environmental information is stored, integrated, and later translated into neuroendocrine functions in the form of diapause induction 29 . The duration over which the information is stored may span numerous developmental stages or even generations 29 . We demonstrated that this hypothesis is well adapted to embryonic diapause in Bombyx. Recently, we showed that the embryonic BmTRPA1 acts as a thermosensitive channel that is activated at temperatures above ~21 °C and affects diapause induction through DH release during pupal-adult development 15 . In this study, we demonstrated that both thermal and photoperiod information was stored until the mid-pupal stage and was integrated with DH signaling to determine diapause phenotype, although the molecular mechanism(s) participating in light (photoperiod)-sensing and storage and integration of information remain unknown (Fig. 4). Furthermore, because it has been speculated that innervation from the brain controls the release of DH 30 , integrated information may affect brain plasticity in the control of DH release. Here, we have attempted to resolve the molecular mechanisms described in Fig. 4 using TALEN-based mutagenesis.
Diapause is accompanied by complex physiological and biochemical changes (referred to as diapause syndrome) in which reserves are accumulated prior to diapause to enable survival during diapause and post-diapause development 31 . In Bombyx, there are dramatic metabolic differences between 25DD and 15DD during the preparative phase of diapause in the maternal generation. Namely, 25DD eggs accumulate greater glycogen and become pigmented. As suggested in previous reports 14,21,22 , we revealed that DH signaling alone facilitates greater accumulation of glycogen as well as accumulation of 3-OHK in 25DD eggs. Because the removal of SG from diapause-type animals in mid-pupal stages can induce the production of non-diapause eggs that are occasionally light pink 32 , the mutant phenotypes of light-colored pigmented eggs obtained here are consistent with the idea that the animals were deficient in DH signaling.
Although the DH signaling cascade was blocked in the DH-PBAN and DHR mutants under 25DD, 20LL, and 20DD conditions, we obtained a few pigmented diapause eggs from these mutants. Largely unknown signaling pathway(s) may be active in the preparative phase of diapause induction during Scientific RepoRts | 5:15566 | DOi: 10.1038/srep15566 embryonic development at temperatures above 20 °C. For example, we observed extended developmental periods and heavier bodies and cocoon shells in 25DD silkworms, consistent with a previous report 23 . We suggest that the activation of BmTRPA1 signaling pathway(s) might affect the duration of growth in the larval period and the weight of the pupal body and cocoon shell. This signaling might be involved in preparing the diapause phenotype to facilitate entering diapause, and it might participate in the storage and integration of information linked to DH signaling. Therefore, 25DD conditions increase the potential for silkworms to enter diapause. Occasionally, diapause may be accidentally induced in a subset of eggs, without DH signaling. Although DH signaling is essential for diapause induction, unknown signaling pathway(s) including the BmTRPA1 pathway may help in preparing for diapause induction at 25 and 20 °C during embryogenesis. These pathways might also participate in the storage and integration of environmental information linked to DH signaling in the maternal generation in order to induce diapause. To screen the knockout strain, eggs were incubated at 25 °C under high humidity (approximately 80%) until hatching; larvae were reared at 25 to 27 °C under long-day conditions (20L:4D) on an artificial diet (Product No. 404110, Kyoto Institute of Technology) to induce non-diapause eggs despite 25DD conditions, as described previously 33 .

TALEN construction and screening of knockout silkworm. Construction of TALEN mRNAs and
screening for germline mutants were performed according to Takasu et al. 17 . Briefly, TALEN targets were searched using TAL Effector Nucleotide Targeter 2.0 (https://tale-nt.cac.cornell.edu) in the coding regions of the DH-PBAN and DHR genes. DNA constructs containing the TAL segments were prepared using Golden Gate TALEN kit (Addgene). TALEN mRNAs were then synthesized using mMessage mMachine T7 Ultra kit (Ambion); mRNA of each TALEN was mixed at a concentration of 0.5 μ g/μ L for microinjection. Non-diapause eggs of the Kosetsu strain were collected within 1 h after oviposition during the syncytial blastoderm stage; the TALEN mRNA mixture was injected into the eggs using a glass needle (uMPm-02; Daiwa Union) attached to a manipulator (kaikopuchu-STDU1; Daiwa Union) and FemtoJet (Eppendorf).
For screening of germline mutagenesis, the G 0 adults were mated with wt. The oviposited G 1 eggs were collected, and approximately 10 eggs from each brood were pooled for genomic DNA extraction using Nucleospin Tissue (Macherey-Nagel). The DNA fragment containing the targeted region of interest was amplified by PCR using Takara Ex Taq (Takara) (Supplementary Fig. S2A). To test for mutagenesis, the PCR products of DH-PBAN and DHR were digested with restriction enzymes Psp1406I (Takara) and FastDigest MnlI (Thermo), respectively; the presence of an undigested PCR product would suggest that the restriction site was disrupted by TALENs ( Supplementary Fig. S2A,B). Mutated PCR products were  Figure 4. Schematic drawing of relationship between temperature and photoperiod and diapause induction via a unique peptidergic signaling system, DH signaling. Progeny diapause is determined by environmental temperature and photoperiod during maternal embryonic development. Silkworms incubated under 25DD and 20LL conditions lay pigmented diapause eggs. In contrast, incubation at 15DD and 20DD causes the resultant moths to lay non-diapause eggs. BmTRPA1 acts as a thermosensitive channel that affects diapause induction. We hypothesize that the links in the pathway from reception of environmental signals to expression of the diapause phenotype include storage, integration, and later translation of information into DH signaling in the form of diapause induction. Non-diapause eggs complete their embryogenesis approximately 9 d after oviposition at 25 °C. In contrast, diapause eggs remain in the diapause stage. subcloned using a TOPO TA cloning kit (Invitrogen) and checked by sequencing. The broods containing mutated sequences were reared, and mutated G 1 adults were crossed with the siblings that carried the same mutation. Homozygous mutants were obtained after confirmation by sequencing of the target region in the G 2 or G 3 egg genome.
Immunostaining. The immunostaining procedures were adapted from Hagino et al. 20 . Briefly, the primary antibodies, anti-DH[N] or -PBAN[N], which recognize a 12-amino acid sequence of the Nterminal region of each peptide, were used at a ratio of 1:2500, respectively, at 4 °C overnight. The signal was detected with Cy2-labeled goat anti-mouse IgG (Jackson ImmunoResearch Lab.) diluted to 1:1500 and was observed using an Olympus FV1000-D confocal microscope (Olympus). Confocal scans were performed under the same conditions for specimens in each mutant strain.
Time-resolved fluoroimmunoassay (TR-FIA). We developed a new method for measurement of the hemolymph DH titers. Hemolymph was collected on ice from one or two pupae on day 4 after pupation into a microcentrifuge tube containing small amounts of sodium diethyldithiocarbamate, a phenoloxidase inhibitor, and p-APMSF, a protease inhibitor. The final concentrations of the inhibitors were 5 mM and 20 μ M, respectively. After centrifuging at 9,200 × g for 5 min to remove hemocytes, 150 μ L of the hemolymph was added with 150 μ L of 2% acetic acid and 300 μ L of methanol, followed by boiling for 10 min. The mixture was centrifuged at 18,000 × g and 480 μ l of the resulting supernatant was concentrated to approximately 100 μ l by vacuum centrifugation for 1 h, followed by mixing with 1 ml of 1% trifluoroacetic acid (TFA). This mixture was applied to a Sep-Pak Vac 3cc C8 cartridge (Waters) equilibrated with 0.1% TFA. The cartridge was washed with 10% acetonitrile (ACN) containing 0.1% TFA and eluted with 40% ACN containing 0.1% TFA. The eluate was lyophilized and then dissolved with dilution buffer [TBS (50 mM Tris-HCl containing 0.9% NaCl) containing 0.5% BSA, 0.1% Tween-20, and 0.05% sodium azide] for use in DH determination by TR-FIA. The recovery rate of DH by this extraction method was estimated to be ≈ 80%.
TR-FIA was developed based on the method described by Mizoguchi et al. 34 . The wells of an RIA/EIA plate (Costar #3590) were filled with 80 μ L each of anti-DH[N] mouse monoclonal antibody 35 (1.5 μ g/mL in TBS) and incubated overnight at 4 °C. After discarding the antibody solution, the wells were blocked with TBS containing 4% skimmed milk and 0.1% Tween-20 for 1 h at 25 °C. After washing three times with TBS-T (TBS containing 0.05% Tween-20), 100 μ L of anti-DH[C] rabbit antibody 35 diluted 1:300 with dilution buffer and either the standard hormone (chemically synthesized DH in 50 μ L dilution buffer) or 50 μ L of the test sample (hemolymph extract) were distributed into the wells, and the plate was incubated for 2 h at 25 °C with shaking on a microplate shaker. The wells were then washed four times with TBS-T, filled with 50 μ L of DELFIA Assay buffer (PerkinElmer) containing biotinylated anti-rabbit IgG antibody (Boehringer Mannheim) and europium-labeled streptavidin (PerkinElmer) at concentrations of 83 and 200 ng/mL, respectively, and incubated for 1 h at 25 °C with shaking. After incubation, the wells were developed with DELFIA Enhancement solution and the fluorescence signals measured with an ARVO X4 plate reader (PerkinElmer).
Measurement of glycogen content. The ovaries were dissected out with phosphate-buffered saline (PBS) just after eclosion. One ovariole was thoroughly separated from each animal, blotted dry, weighed quickly, and stored at -20 °C before use. Glycogen was extracted by digesting the homogenate with 30% (w/v) KOH in a boiling bath for 30 min; the glycogen was then precipitated with ethanol at 4 °C 36 and measured by the phenol-sulfuric acid method 37 .
Thionin staining of embryo. The embryos were collected 10 d after oviposition and were stained with carbolic thionin solution according to An et al. 38 with modifications; to facilitate dechorionization, the eggs were boiled in 80% ethanol for 5 min after fixation.
Rescue experiment. Synthetic peptides (DH, α -, β -, and γ -SGNPs, and PBAN) of 95% purity (HPLC area percentage) were obtained from Operon Biotechnologies. Each peptide was dissolved in peanut oil (Sigma-Aldrich), and 10 μ l solutions of various doses were injected into pupa at 4 days after pupation. The diapause eggs inducing activity was estimated by counting the numbers of eggs in diapause and those not in diapause after the non-diapause eggs hatched. The results are expressed as the average percent diapause in each egg batch as described above.