Functional Mimicry of the Silkworm Diapause Hormone by an Insect Paralytic Peptide

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1 Journal of Insect Biotechnology and Sericology 76, (2007) Functional Mimicry of the Silkworm Diapause Hormone by an Insect Paralytic Peptide Ying An 1, Tetsuro Yamashita 1, Atsushi Seino 2, Kunio Imai 3 and Koichi Suzuki 1 1 Department of Agro-Bioscience, Faculty of Agriculture, Iwate University, Morioka , Japan, 2 National Institute of Agrobiological Science, Tsukuba , Japan, and 3 Department of Life Science, Faculty of Bioresources, Mie University, Tsu , Japan (Received September 11, 2006; Accepted October 30, 2006) Peptides of the ENF (Glu-Asn-Phe) family, which occur in the hemolymph of Lepidoptera, were reported to act on the plasmatocytes and heart, cause body paralysis, and inhibit body growth. In this paper we report on yet another, most surprising effect. An ENF peptide isolated from the wild silkmoth, Antheraea yamamai, and named Antya-ParP causes paralysis in the caterpillars but not in the pupae of Bombyx mori. However, adults that emerge from the treated pupae lay diapausing eggs. The embryonic diapause of B. mori is normally induced by the diapause hormone (Bommo-DH), which is produced in pupae from the suboesophageal ganglion. The effect of Antya-ParP is independent of this ganglion, showing that the Antya-ParP action is not mediated by Bommo- DH. The sequence of 23 amino acid residues of Antya-ParP shows no similarity to the Bommo-DH sequence of 24 residues. The mode of action of the two peptides seems to be identical but the threshold concentration of Bommo-DH is up to hundred times lower than that of Antya-ParP. Key words: Antheraea yamamai, paralytic peptide, Bombyx mori, diapause induction, diapause hormone, functional mimicry INTRODUCTION Growth, metamorphosis, reproduction and diapause are regulated by peptides secreted from the central nervous system throughout the insect development (Menn et al., 1991). However, caterpillars infested with hymenopteran parasitoids were found to contain in their hemolymph a peptide that was apparently derived from the fat body of host and that caused developmental arrest by inhibiting juvenile hormone esterase and preventing thus the onset of metamorphosis (Hayakawa, 1990, 1991). Seven similar peptides were subsequently isolated from the lepidopteran hemolymph and showed to induce body paralysis (Skinner et al., 1991). Additional effect, the spreading of plasmatocytes, was reported for the homologue identified in the hemolymph of Pseudoplusia includens (Clark et al., 1997). Finally, a cardioactive peptide of the southern armyworm is also homologous to the paralytic peptides (Furuya et al., 1999). Since all peptides under discussion are characterized by the N-terminus ENF (Glu-Asn-Phe), the name ENF peptides has recently been proposed for this peptide family (Strand et al., 2000). We have isolated an ENF peptide from the larval hemolymph of the wild silkmoth, Antheraea yamamai (Seino et al., 1998). This peptide, named Antya-ParP (we have proposed Any-ParP in the previous papers, but here use the five letter code), was shown to cause body paralysis *To whom correspondence should be addressed. Fax: Tel: anying@iwate-u.ac.jp when injected into the caterpillars of A. yamamai and to exert a paralyzing effect also in the silkworm, Bombyx mori. We also found that Antya-ParP can induce egg diapause in B. mori (Song et al., 2001). In this study, we examined pharmacological action of Antya-ParP in comparison with the structurally unrelated diapause hormone (Bommo-DH). Its effect is extremely similar to the Bommo-DH function. MATERIALS AND METHODS Animals A bivoltine race (Daizo) of the silkworm, Bombyx mori L., was used in all experiments. Oviposited eggs were incubated either in continuous light at 27 C (LW condition) or in continuous darkness at 15 C (DC condition). Hatched larvae were reared on mulberry leaves or a semiartificial diet in 12 h light: 12 h dark cycle at 25 C. The pupae and adults were also kept under this rearing regime. Adults developing from the eggs that had been incubated at DC conditons laid non-diapausing, and those from the LW-incubated eggs laid diapausing eggs. Peptide synthesis The peptides of Bommo-DH and Antya-ParP (Fig. 1) were synthesized on solid matrix on the PSSM-8 peptide synthesizer (Shimadzu Co., Ltd) and purified by reversed phase (ULTRON VX-ODS column) HPLC. To ensure that the desired structures were obtained, the synthesized peptides were analyzed by a matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry. Intracellular

2 52 An et al. disulfide bond formation between Cys 7 and Cys 19 of Antya-ParP was also confirmed by mass spectrometry. Bioassays Female pupae destined to lay either non-diapausing or diapausing eggs were used for the bioassays. Their suboesopghageal ganglion (SG) was extirpated, either alone or along with the brain, within 10 h after the larval-pupal ecdysis. This operation was undertaken to remove the source of diapause hormone (Bommo-DH), which is in SG (Yamashita and Hasegawa, 1985; Yamashita, 1996). The pupae deprived of SG were anesthetized on ice for 30 min and then each injected with 12 μl of distilled water containing a synthetic peptide. Emerged females were mated with untreated males and the oviposited eggs were scored in respect to the presence or absence of diapause. For detailed morphological examinations of the embryos, the eggs were fixed in Carnoy s fluid overnight and stained with 0.03% carbolthionin. They were destained with a graded series of ethanol, cleared in xylene and finally mounted in (Fisher Scientific, USA). In some cases, the embryonic diapause was terminated by soaking pre-chilled (about 23 days at 5 C) diapausing eggs in diluted hydrochloric acid (specific gravity of 1.095) for 6 min at 47 C (Yamashita and Suzuki, 1991). RESULTS AND DISCUSSION Embryonic diapause of B. mori is induced by Bommo- DH, which is secreted from SG of maternal pupae. Bommo-DH was isolated and its sequence of 24 amino acid residues verified by the cdna analysis (Fig. 1) (Imai et al., 1991; Sato et al., 1993). The action of Bommo-DH is targeted to the eggs developing in pupal ovaries. Upon oviposition, the affected eggs initiate development but embryogenesis is arrested at late gastrula stage (Fig. 2D). Such eggs are easily recognized by their dark coloration. So far, only Bommo-DH has been identified as an inducer of insect embryonic diapause. In the non-diapause egg producers, no Bommo-DH is secreted due to a superseded brain action on the SG. Such insects lay, when they emerge as adults, only the non-diapausing yellow eggs (Fig. 2A). Injections of Antya-ParP into such pupae, however, convert them to the diapause egg producers. The deposited eggs turn to be brown, indicating the developmental arrest of embryogenesis at late gastrula stage (Fig. 2B and E; Table 1). To recognize if Antya-ParP acts directly on the ovaries or only by affecting the release of Bommo-DH, the peptide was injected into pupae deprived of SG. The pupae lacking SG laid exclusively non-diapausing eggs, irrespectively of the original destiny as determined during their embryonic development. When such pupae were injected with Antya- Fig. 1. Amino acid sequences. Antya-ParP, paralytic peptide of A. yamamai (Seino et al., 1998). Bommo-DH, diapause hormone of B. mori (Imai et al., 1991). ParP, however, they emerged as diapause egg-producing adults. The development of their eggs was halted in the late gastrula stage and the eggs turned brown (Fig. 2C and F; Table 1). Extirpation of brain along with SG had no influence on the induction of diapausing eggs production by Antya-ParP (Table 1). These results demonstrate that Antya-ParP does not act via endogenous Bommo-DH but mimics its action at the level of target tissues. To elucidate whether embryos arrested at the late gastrula stage due to Antya-ParP effect have entered a bona fide diapause, we examined if this developmental arrest is terminated by the same cues as is the natural diapause. The embryonic diapause of B. mori, which evolved as an adaptation for overwintering, ends after exposure to low temperature for a period of about 60 days. It can be switched after just few days of chilling by dipping the eggs into diluted hydrochloric acid. This treatment is very popular with the silkworm breeders and has been used for more than hundred years (Yamashita and Suzuki, 1991). We tried to terminate embryonic diapause induced by Antya-ParP with both chilling and hydrochloric acid application, and both these treatments proved effective. About 80 percent of eggs broke their diapause and hatched (Fig. 2G, detailed data not shown). We conclude that the embryonic arrest caused by Antya-ParP is indistinguishable from the inherent embryonic diapause. The following investigations were designed to compare the mode of action of Antya-ParP with that of Bommo- DH. It has been known that Bommo-DH can affect only those ovarian follicles that undergo vitellogenesis (Ikeda et al., 1993). Since vitellogenesis occurs in different follicles at different times, the injected Bommo-DH, which is present in the body only for a limited period of time, induces diapause only in a certain proportion of the eggs. We now found that the highest proportion of eggs is induced to diapause when either Antya-ParP or Bommo-DH is injected in about middle of the pupal-adult transformation (Fig. 3). This result indicates that Antya-ParP has a similar mode of action as Bommo-DH. The dose-response diapause-induction curves of Antya- ParP and Bommo-DH, however, were dissimilar (Fig. 4). Bommo-DH caused a dose-dependent increase in a range of nmol/pupa. The activity was saturated at doses larger than 0.38 nmol/pupa, and lowered at doses larger than 3.8 nmol/pupa. Antya-ParP also induced a dose-dependent rise in the percentage of diapause eggs in a range of 3- nmol/pupa. It gave the value of about

3 Mimicry of Diapause Hormone by Paralytic Peptide 53 Fig. 2. Eggs, diapausing embryos and hatched larvae of the bivoltine Daizo, B. mori. A-C, the eggs were oviposited by insects destined to lay non-diapause eggs. Within 10 h after the pupal ecdysis, some of pupae were deprived of the suboesophageal ganglion (SG). At h after the pupal ecdysis, pupae were injected with 12 µl distilled water (A), solution of Antya-ParP (B) and solution of Antya-ParP into SG-removed pupae (C). D-F, diapausing embryos stained with carbolthionin: natural diapause (D), induced diapause (E, corresponding to B; F, corresponding to C). G, the hatched larvae. the embryonic diapause in B and C was terminated by chilling and hydrochloric acid, respectively. Table 1. Effect of Antya-ParP on the induction of diapause eggs in egg producers extirpated brain and/or suboesophageal ganglion (SG) Type of pupae injected ND1 ND-SG2 D-SG3 D-Br-SG4 Concentration (nmoles/pupa) No. pupae injected (oviposited moth) No. batch of mixed eggs Percentage of diapause eggs 10 (10) 10 ( 8) 10 ( 8) 10 ( 6) ± ± ± ± 11.5 The pupae, bivoltine Daizo of B. mori were extirpated brain and/or SG within 10 h, and then injected with Antya-ParP at h after the pupal ecdysis. 1, pupae of non-diapause egg producers; 2, pupae of non-diapause egg producers extirpated SG; 3, pupae of diapause egg producers extirpated SG; 4, pupae of diapause egg producers extirpated brain and SG. 60% at 150 nmol/pupa, a dose about 400 times more than the Bommo-DH concentration (0.38 nmol/pupa) that produced a peak. The difference between effective doses of Antya-ParP and Bommo-DH suggest that only the latter compound is the inherent diapause inducer in B. mori and that Antya-ParP just mimics Bommo-DH action. In the decade since the finding of the first member of the ENF peptide family, such peptides have been found in eleven lepidopteran species including B. mori (Ha et al., 1999). Our previous study showed that five members of ENF family have the effect on inducing diapause eggs in B. mori (Song et al., 2001). Although the B. mori ENF peptide (Bommo-ParP) was lower than Antya-ParP in effect, a 60 nmol/pupa dose of Bommo-ParP induced 27.8% of diapause eggs in the Daizo strain of B. mori (data not shown). This value was different from that of 0.1% in our previous study (Song et al., 2001). In addition, AntyaParP-NH2 was at the same level in inducing diapause eggs as Antya-ParP-OH in B. mori (details will be published elsewhere). These results suggest that the carboxyl amidation is not essential for inducing diapause eggs in B. mori, different from the case of Bommo-DH (Imai et al., 1998). Three-dimensional structures of four members of this peptide family have been proposed on the bases of NMR analyses (Aizawa et al., 1999; Volkman et al., 1999; Yu et al., 1999; Miura et al., 2002). The defense against some kind of stress is as common denominator of the various effects these peptides cause. The plasmatocyte spreading and paralysis occur as parts of immune response, and the growth blocking is induced by parasitoid infection (Strand et al., 2000; Nakahara et al., 2003). Our finding that a member of ENF family can in the silkworm convert non-

4 54 An et al. Fig. 3. Induction of embryonic diapause by Antya-ParP and Bommo-DH injected into pupae (non-diapause egg producers) of different age. The percentage of diapausing eggs was established 15 days after oviposition in at least 10 egg batches. et al., 1988; Kingan et al., 1995), but this sex peptide does elicit pheromonostatic activity in Helicoverpa armigera (Fan et al., 1999). Thus, similar conformation of these two peptides may provide for their binding to identical receptors. This assumption can also be applied to Antya- ParP and Bommo-DH that have no amino acid sequence similarity (Fig. 1). Since the cdna encoding Bommo-DH receptor is cloned from developing ovaries of B. mori (Homma et al., 2006), the same receptor may be related to the mechanism of Antya-ParP action. Irrespectively of the mechanism of the mimicking action of Antya-ParP, its effect on diapause induction can be exploited in studies on the diapause control and possibly also in the design of novel insect growth regulators. Many insects enter diapause and intervention of this process might be used for controling some insect pests. ACKNOWLEDGMENTS We thank Prof. F. Sehnal of the Institute of Entomology, Czech Academy of Sciences, for the critical reading of the manuscript. This work was supported by grants from the Ministry of Education, Science, Sports and Culture of Japan (No ) and from the Research for the Future Program of the Japan Society for the Promotion of Science (JSPS-RFTF99L01203). REFERENCES Fig. 4. Dose-response curves for diapause induction by Antya-ParP and Bommo-DH. The peptides were injected into the non-diapause egg producers at h after the larval-pupal ecdysis. The percentage of diapausing eggs was established 15 days after oviposition in at least 10 egg batches. diapause egg producers to the diapause egg producers is difficult to interpret in this context. There is no homology between the primary sequences of the Bommo-DH and ENF peptide families, but this does not exclude that one can mimic the action of the other one. There are examples that structurally unrelated peptides can exert similar functions. For example, the locustatachykinins, which stimulate visceral muscle contractions, can elicit sex pheromone production in B. mori female moths, albeit much less efficiently than the intrinsic pheromone biosynthesis activating neuropeptide, PBAN (Fónagy et al., 1992). It is assumed that the cross-activity is due to similar C-termini that are FXPRLamide in PBAN and FXGVRamide in locustatachykinins. On the other hand, a pheromone suppressive male factor, which has been isolated from the moth Helicovepra zea, does not bear any sequence homology to the sex peptide of Drosophila melanogaster (Chen Aizawa, T., Fujitani, N., Hayakawa, Y., Ohnishi, A., Ohkubo, T., Kumaki, Y., Kawano, K., Hikichi, K., and Nitta, K. (1999) Solution structure of an insect growth factor, growth-blocking peptide. J. Biol. Chem. 274, Chen, P.S., Stumm-Zollinger, E., Aigaki, T., Balmer, J., Bienz, M., and Bohlen, P. (1988) A male accessory gland peptide that regulates reproductive behavior of female D. melanogaster. Cell 54, Clark, K.D., Pech, L.L., and Strand, M.R. (1997) Isolation and identification of a plasmatocyte-spreading peptide from the hemolymph of the lepidopteran insect Pseudoplusia includens. J. Biol. Chem. 272, Fan, Y., Rafaeli, A., Gileadi, C., Kubli, E., and Applebaum, S.W. (1999) Drosophila melanogaster sex peptide stimulates juvenile hormone synthesis and depresses sex pheromone production in Helicoverpa armigera. J. Insect Physiol. 45, Fónagy, A., Matsumoto, S., Schoofs, L., De Loof, A., and Mitsui, T. (1992) In vivo and in vitro pheromonotropic activity of two locustatachykinin peptides in Bombyx mori. Biosci. Biotech. Biochem. 56, Furuya, K., Hackett, M., Cirelli, M.A., Schegg, K.M., Wang, H., Shabanowitz, J., Hunt, D.F., and Schooley, D.A. (1999) A cardioactive peptide from the southern armyworm, Spodoptera eridania. Peptides 20, Ha, S.D., Nagata, S., Suzuki, A., and Kataoka, H. (1999) Isolation and structure determination of a paralytic peptide from hemolymph of the silkworm, Bombyx mori. Peptides

5 Mimicry of Diapause Hormone by Paralytic Peptide 55 20, Hayakawa, Y. (1990) Juvenile hormone esterase activity repressive factor in the plasma of parasitized insect larvae. J. Biol. Chem. 265, Hayakawa, Y. (1991) Structure of a growth-blocking peptide present in parasitized insect hemolymph. J. Biol. Chem. 266, Homma, T., Watanabe, K., Tsurumaru, S., Kataoka, H., Imai, K., Kamba, M., Niimi, T., Yamashita, O., and Yaginuma, T. (2006) G protein-coupled receptor for diapause hormone, an inducer of Bombyx embryonic diapause. Biochem. Biophys. Res. Commun. 344, Ikeda, M., Su, Z.H., Saito, H., Imai, K., Sato, Y., Isobe, M., and Yamashita, O. (1993) Induction of embryonic diapause and stimulation of ovary trehalase activity in the silkworm, Bombyx mori, by synthetic diapause hormone. J. Insect Physiol. 39, Imai, K., Konno, T., Nakazawa, Y., Komiya, T., Isobe, M., Koga, K., Goto, T., Yaginuma, T., Sakakibara, K., Hasegawa, K., and Yamashita, O. (1991) Isolation and structure of diapause hormone of the silkworm, Bombyx mori. Proc. Jpn. Acad. Ser. B. 67, Imai, K., Nomura, T., Katsuzaki, H., and Komiya, T. (1998) Minimum structure of diapause hormone required for biological activity. Biosci. Biotechnol. Biochem. 62, Kingan, T.G., Bodnar, W.M., Raina, A.K., Shabanowitz, J., and Hunt, D.F. (1995) The loss of female sex pheromone after mating in the corn earworm moth Helicoverpa zea: identification of a male pheromonostatic peptide. Proc. Natl. Acad. Sci. USA. 92, Menn, J.J., Kelly, T.J., and Masler, E.P. (1991) Insect neuropeptides. p.260, American Chemical Society, Washington DC. Miura, K., Kamimura, M., Aizawa, T., Kiuchi, M., Hayakawa, Y., Mizuguchi, M., and Kawano, K. (2002) Solution structure of paralytic peptide of silkworm, Bombyx mori. Peptides 23, Nakahara, Y., Kanamori, Y., Kiuchi, M., and Kamimura, M. (2003) Effects of silkworm paralytic peptide on in vitro hematopoiesis and plasmatocyte spreading. Arch. Insect Biochem. Physiol. 52, Sato, Y., Oguchi, M., Menjo, N., Imai, K., Saito, H., Ikeda, M., Isobe, M., and Yamashita, O. (1993) Precursor polyprotein for multiple neuropeptides secreted from the suboesophageal ganglion of the silkworm Bombyx mori: Characterization of the cdna encoding the diapause hormone precursor and identification of additional peptides. Proc. Natl. Acad. Sci. USA. 90, Seino, A., Sato, Y., Yamashita, T., Sato, Y., and Suzuki, K. (1998) Identification of a novel member of the paralytic peptide family in the silkmoth Antheraea yamamai. J. Seric. Sci. Jpn. 67, Skinner, W.S., Dennis, P.A., Li, J.P., Summerfelt, R.M., Carney, R.L., and Quistad, G.B. (1991) Isolation and identification of paralytic peptides from hemolymph of the lepidopteran insects Manduca sexta, Spodoptera exigua, and Heliothis virescens. J. Biol. Chem. 266, Song, H.S., An, Y., Yamahita, T., and Suzuki, K. (2001) Antheraea yamamai paralytic peptide induces egg diapause as well as larval paralysis in Bombyx mori: the primary sequence-activity correlations. J. Insect Biotechnol. Sericol. 70, Strand, M.R., Hayakawa, Y., and Clark, K.D. (2000) Plasmatocyte spreading peptide (PSP1) and growth blocking peptide (GBP) are multifunctional homologs. J. Insect Physiol. 46, Volkman, B.F., Anderson, M.E., Clark, K.D., Hayakawa, Y., Strand, M.R., and Markley, J.L. (1999) Structure of the insect cytokine peptide plasmatocyte-spreading peptide 1 from Pseudoplusia includens. J. Biol. Chem. 274, Yamashita, O. (1996) Diapause hormone of the silkworm, Bombyx mori: structure, gene expression and function. J. Insect Physiol. 42, Yamashita, O., and Hasegawa, K. (1985) Embryonic diapause. In Comprehensive Insect Physiology, Biochemistry and Pharmacology (Kerkut, G.A. and Gilbert, L.I., eds), Vol. 1, pp , Pergamon Press, London. Yamashita, O., and Suzuki, K. (1991) Roles of morphogenetic hormone in embryonic diapause. In Morphogenetic Hormones in Arthropods (Gupta, A.P., ed.), 3, pp Rutgers Univ. Press, New Brunswick. Yu, X.Q., Prakash, O., and Kanost, M.R. (1999) Structure of a paralytic peptide from an insect, Manduca sexta. J. Peptide Res. 54,

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