JP3475239B2 - Insect dormant egg inducer and method for producing dormant egg - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a dormant egg inducer for insects and a dormant egg producing method thereof, and more particularly to a dormant egg inducer for silkworm and a dormant egg producing method for silkworm.

[0002]

2. Description of the Related Art More than 200 kinds of antibacterial peptides derived from insects and known as so-called biological defense substances have been isolated and their structures have been determined.
In the present specification, since a protein which is a physiologically active substance having a dormancy-inducing function of wild silkworms and silkworms is targeted, a substance having a function of inducing dormancy of insects will be mainly described below to understand the background of the invention. Make it easy.

Conventionally, insect paralytic peptides have been known. The term paralytic peptide is used as a general term for active peptides that cause paralytic behavior by injecting the active fraction from the outside of the body to contract all or part of the body. In recent years, in these known peptides, in addition to the paralytic activity, the activity involved in the biological defense system has been found, so that it has become considered to be multifunctional in the living body, and is attracting attention among insect peptides. It has become so.

Since the same substance may be used in different terms such as "paralytic peptide" and "diapause controlling substance" in the present specification, these terms will be defined below.

Conventionally, 10 kinds of insect-derived paralytic peptide families have been known. Recently, a silkworm paralytic peptide (Seino,
A. et al., J. Seric. Sci. Jpn., 67 , 473-478, 1998.)
Has been newly added to this family due to its primary structure amino acid sequence and paralytic bioactivity (Strand, MR
et al., J. Insect Physiol., 46 , 817-824, 2000). Therefore, in the present specification, the paralytic peptide derived from the silkworm, Bombyx mori, is described as one of the family of paralytic peptide derived from insects. However, in order to avoid confusion, Seino et al. Have isolated and identified a peptide and have already revealed that it has a paralytic function.
This peptide is sometimes called a “diapause control substance” instead of the “celestial silkworm paralytic peptide”.

Among the physiologically active peptides of insects, the ones called the paralytic peptide family include, for example: That is, a plasma cell spreading peptide (Clark K. D et al., J. Biol.
Chem., 272 , 23440-23447, 1997), a growth inhibitory peptide from armyworm (Hayakawa, Y., J. Biol. Chem., 266 ,
7982-7984, 1991), a growth-inhibiting peptide from Spodoptera litura and Spodoptera litura (Hayakawa and Ohnishi, Biochem.
nd Biophys. Res. Commun., 250 , 194-199, 1998), a paralytic peptide (Skinner WS et al., J. Biol. Che from Tobacco sp.
m., 266 , 12873-12877, 1991), a paralytic peptide from one species of ouaba (Skinner WS et al., Biochem. Physio
l., 104C , 133-135, 1993), a cardiac contractile peptide from one species of Spodoptera litura (Furuya et al., Peptides, 20 , 53-61, 19).
99), and paralytic peptides from silkworms (Ha et al.,
Peptides, 20, 561-568, 1999) are known.

Among the insect paralytic peptide families known so far, one having the largest paralytic function is a diapause controlling substance which is a paralytic peptide derived from silkworm having the amino acid sequence shown in SEQ ID NO: 1. . Therefore, in this specification, the peptide shown in SEQ ID NO: 1 may be abbreviated as a dormancy control substance for silkworm.

In the 19th century, a so-called "hydrochloric acid hatching method" was established in which hydrochloric acid was used to hatch silkworm eggs as a practical hatching technique for sericulture, but the details of the hatching mechanism have not been reached until now. Not revealed. Moreover, although the artificial hatching method of the silkworm has been developed, its molecular mechanism has not been elucidated at all. The development of the bioactive protein of the silkworm, which is being advanced by the present inventors, is not only a clue for elucidating the artificial hatching mechanism in insects, but also the long-term low-temperature mechanism analysis in which the two sides are integrated as described below. It will also open the way to.

[0009] For many dormant organisms that live in temperate regions as well as insects, the long-term cold acclimation mechanism is inextricably linked with the elucidation of the artificial hatching mechanism in the organisms, which is an important life history. It is an environmental signal for regulation. The vernalization mechanism of plant seeds can be said to be one example. Regarding short-term cold acclimation and long-term cold acclimation, cold stimulation from the molecular level has been actively studied recently. Various cDNAs have come to be determined in the plant kingdom, and from the deduced amino acid sequence, RNA binding protein,
It is believed that elongation factors or antifreeze proteins have important functions. However, the genes and proteins that critically explain the biological reaction due to long-term cold acclimation and dormancy / wakefulness have not been discovered.

[0010]

By the way, in major raw silk-producing countries such as India, Vietnam, and Thailand, mulberry can be cultivated all year round, and silkworm cultivars are bred in non-diapause strains that do not sleep in eggs. Therefore, silkworms are being bred one after another all year round. However, no matter how far south, the growth of mulberry is bad from January to March, and it is difficult to set the period for disinfecting the silkworm chamber.
The production volume is low in March and the quality is deteriorated, which causes bad influence on the next silkworm rearing.
In order to overcome this vicious cycle, the silkworm breeding must be stopped once, but the silkworms in these countries are non-diapause and cannot sleep. This has become a major problem in the silkworm industry where silkworms are raised and raw silk is produced.
There has been a strong demand for a technique for diapausing silkworm varieties in these countries. However, a new substance that is an insect-derived physiologically active substance that can control the diapause of silkworms and, as a result, can produce dormant eggs has a short history, and there is little knowledge about the new substance, which is still satisfactory. No substance has been obtained, and the development of a useful dormant egg inducer is desired.

As described above, the functions of substances that induce diapause / growth / metamorphosis have been gradually clarified.
The present inventors consider that the paralytic peptide of Bombyx mori, which has already been isolated and structurally determined, is also an important peptide in Lepidoptera insects in analyzing the mechanism of dormancy, development and metamorphosis. Some of the so-called insect paralytic peptide families that have been discovered so far are deeply involved in the biological defense system, such as the plasma cell diffusion peptide of Spodoptera litura, and the growth inhibitory peptide of armyworm. , Some are considered to be direct promoters of larval growth retardation and pupal diapause. However, a substance that induces dormant eggs of Bombyx mori has not been found yet, and development of a useful substance for inducing dormant eggs of Bombyx mori is desired.

Further, as described above, as a physiologically active substance for controlling the diapause / paralytic behavior of insects,
Plasma cell diffusion peptide from one species, growth inhibitory peptide from armyworm, growth inhibitory peptide from Spodoptera litura and Spodoptera litura, paralytic peptide from one species of tobacco Stumella / Tobacco / Yotoga, paralytic peptide from one of uwawa, There is a cardiac contractile peptide of Spodoptera frugiperda and a paralytic peptide from silkworm. However, for these conventional peptides, since the effect of producing dormant eggs in silkworms has not been reported at all, other than silkworm dormant hormone, regarding peptides that produce dormant eggs, is a physiologically active substance of insect origin, It is strongly desired to develop a physiologically active substance that efficiently produces dormant eggs while maintaining the normal state without disturbing the physiological state of insects.

Next, the problem will be explained in relation to the silkworm liability. Silkworms, which have been bred for sericulture in Japan so far, are called hybrids in which a dimorphic / multicellular line and a high-yielding genetic characteristic are introduced. Expressively, it is univalent. Generally,
The polymorphic lines are less capable of high-yielding than the unisexual lines. However, since the dimorphic and polymorphic strains also possess excellent genetic characteristics such as disease resistance and high temperature resistance, the characteristics have been introduced as a progenitor species into today's silkworm hybrids.

On the other hand, the silkworm cultivars in countries such as India, Thailand and Vietnam, which are now world raw silk producing countries mainly in Southeast Asia, have a pleiotropic genetic characteristic. These silkworms are excellent in disease resistance and high temperature resistance because they are variegated lineages, but on the contrary, they are poor in high-yield and require repeated breeding year-round even when they are inappropriately raised. There's a problem. In order to overcome the weaknesses of the silkworm strains in these countries, rather than introducing a selective breeding method that requires a long time, the development of an insect growth control agent that causes dormant varieties of breeding strains at any time Has been strongly desired. Therefore, in the 21st century, the main production of raw silk is expected to shift from China / Brazil to the regions mainly in Southeast Asia. If this is done, it will be an important technology at the world level of raw silk.

The present invention provides a dormant egg inducer for insects (eg, silkworm) and a dormant egg thereof, which has the function of producing dormant eggs of insects (eg, silkworm) and does not disturb the physiological state of the living body. The challenge is to provide a production method.

[0016]

[Means for Solving the Problems] The inventors of the present invention filed a prior application of the same applicant (Japanese Patent Application No. 11-152273; Japanese Patent No. 3023790).
In, in the pre-larval dormancy type silkworm, gypsy moth, moth butterfly, Obikareha, Kashiwamaimai and other widespread insects of more than 40 species, mainly lepidopteran insects and orthoptera insects (as these pre-larva dormant insects , Yochirou Umeya, Overwintering Phenomena of Insects Seen from Overwintering Eggs of Silkworms, Silkworm Experiment Station Report, 12: 393-481 (1946) and Umeya Y., Studi
es on embryonic hibernation and diapause in insect
s, Proc. Jpn. Acad., 26, 1-9 (1950))
The invention relating to a physiologically active substance having a diapause controlling activity that specifically and efficiently acts on erythrocyte was clarified. The present inventors have subsequently found that this physiologically active substance has an efficient regulatory activity for living cells, for example, an effective growth inhibitory activity for cancer cells. That is, the gene Any-RF in the prior invention, the amino acid sequence shown in SEQ ID NO: 1 of the sequence listing, has Asp-Ile-Leu-Arg-Gly, the C-terminal is amidated, the molecular weight is 570.959. It is a protein that encodes a protein, and the present inventors have found that this physiologically active substance has an activity of effectively suppressing the growth of cancer cells, and by extension has an activity of efficiently controlling living cells, the patent application (Japanese Patent Application No. 2000-81012).

The inventors of the present invention set the subject of the previous application to a physiologically active substance which maintains the dormancy state of silkworms and further has a cell-regulating activity. It was found that the dormancy induction was imitated. This is not a function for maintaining a dormant state described by the present inventors in the invention of the prior application, but a function similar to a dormant hormone. Under such a technical background, the present inventors have completed the present invention with a substance that induces the production of dormant eggs of Bombyx mori isolated and purified from larvae of the silkworm, Bombyx mori, as an active ingredient.

The dormant egg inducer for insects (particularly, silkworm) of the present invention comprises the amino acid sequence Glu-Asn shown in SEQ ID NO: 1 in the sequence listing.
-Phe-Ala-Gly-Gly-Cys-Ala-Thr-Gly-Phe-Met-Arg-Thr-A
It has la-Asp-Gly-Arg-Cys-Lys-Pro-Thr-Phe-, the C-terminal is free-oxidized, and the peptide having a molecular weight of 2435.73 is contained as an active ingredient. This active ingredient, for example, removes blood cells from the body fluid of lepidopteran insects including celestial silkworms, makes it a final concentration of 25% with ethanol, collects the centrifugal supernatant as a crude extract, freeze-drys it, and then drys it into a cartridge.
It can be obtained as an isolated and purified product by eluting with C18Sep-Pak Vac and repeating the HPLC operation three times. Alternatively, it can also be prepared according to a known method using a known peptide synthesizer.

The above-mentioned active ingredient peptide is an insect paralytic peptide derived from the body fluid of the silkworm larva.

The dormant egg inducer for insects of the present invention contains either plasma cell spreading peptide I (Psi PSP I) or armyworm growth inhibitory peptide (Pss GBP) as an active ingredient. is there.

Furthermore, the dormant egg production method of the present invention comprises administering the peptide (150 to 300 nmoles / pupa) to the silkworm non-diapause egg producing pupae at a stage of 72 to 96 hours after pupation.
The pupae are grown and mated females that have emerged are mated with males and then laid to obtain dormant eggs. Thus, by applying the peptide to the pupae at the stage of 72-96 hours, which is the pupal yolk-forming stage, the induction rate of the dormant eggs obtained was 60%.
As described above, the incidence of unfertilized eggs can be lowered below the 2.5-3% level. If the time exceeds 96 hours, the number of stages in the yolk-forming stage decreases, and the dormant egg induction rate also decreases.

[0022]

BEST MODE FOR CARRYING OUT THE INVENTION In the above-mentioned prior invention, the present inventors have revealed that a physiologically active substance isolated from an insect has a function of maintaining the diapause of the insect. Prior to describing the details of the present invention in detail, the outline of the prior invention is first repeatedly described in order to facilitate understanding of the present invention.

First, the industrial features of general wild silkworms, such as Tenji silkworms and tussah silkworms, will be described. The Japanese silkworm (formally Japanese name: Yamamayu, scientific name: Antheraea yamamai Guerin-Meneville) has its origin in Japan and has a long history with breeding records since the Edo period. Recently, artificial feed has been developed to facilitate the breeding of larvae. Since it is now bred and it is generally bred at the farm stage, there is a lot of information on breeding and it is easy to obtain. Silkworm larvae grow exclusively by eating mulberry leaves, while moss worms eat leaves such as kunugi, oak, oak, and abemaki. Whereas sericulture farmers breed domestic silkworm larvae, wild mosquito larvae grow naturally. The hatching rate of the silkworm silkworm species is low, and the ratio of cocoon thread to silk thread (thread walk) is extremely small. In addition, since it is difficult to take the cocoon thread, it has significance as a rare value. It is said that the price of 1 kg of silkworm silk is 200,000 yen or 300,000 yen, which is why it is so rare that it is called a silk diamond. Silk fabrics with an increased blending ratio of tussah silk threads, which are wild silkworms, can suppress thread slippage and improve slipping resistance of seams. In addition, wild silkworm silk fabrics are preferred because they are resistant to friction. Therefore, in the future, there is great expectation for the development of fields that utilize silkworm-producing large-scale insect silkworm, and along with that, academic research for isolating bioactive substances with various functions derived from silkworm and determining the structure of bioactive substances. Is becoming more and more important.

From these viewpoints, the present inventors have conducted research on physiologically active substances derived from silkworm, and found that, like the invention of the prior application, from a dormant pre-mosquito larva, the amino acid sequence was 5 and the C-terminal was We succeeded in determining the isolated structure of a novel dormancy regulator that is free-oxidized. As for the physiological activity of this substance, it was clarified that it has a function for maintaining the dormant state of pre-mosquito larvae, and further, a new physiological activity function of suppressing the growth of rat hepatoma cells. Was also revealed. From this, the dormancy control substance derived from the silkworm has a wide universality and can be an excellent lead compound for application development as a growth control agent for living cells.

Further, the present inventors have reported the results regarding paralytic peptides of the silkworm, Bombyx mori (Seino et al., 1998).
That is, when the body fluid extract of the larvae of the larvae of the silkworm, Bombyx mori, was injected into the pre-larvae, paralytic activity was observed at the injected site, and it was confirmed that the same activity could be confirmed in the body fluid extract of the 5th instar larva. . In a subsequent study, active factors were isolated from body fluid extracts of 5th instar larvae and their primary structure determined.
NH ₂ -Gl, a 23-residue peptide with a determined primary structure
u-Asn-Phe-Ala-Gly-Gly-Cys-Ala-Thr-Gly-Phe-Met-Arg-
Thr-Ala-Asp-Gly-Arg-Cys-Lys-Pro-Thr-Phe-COOH shows high homology with Lepidoptera paralytic peptide, plasma cell diffusion peptide, and growth inhibitory peptide, as described below. Therefore, it was concluded that it is a kind of paralytic peptide. Since the peptide in the present invention is likely to be universally present in lepidopteran insects, the activity was investigated by injecting the synthetic peptide having the above amino acid structure into the 5th instar larva of Bombyx mori (Taizou), and the same effect was obtained. Admitted.

Therefore, when a peptide (Seino et al., 1998, described above) that was isolated as a paralytic peptide from the body fluid of the silkworm larvae and was able to be structurally determined was injected into the non-diapause pupae of the silkworm, a dormant egg was produced. Was produced. The term "non-diapause pupae" refers to the pupae of a female parent whose pupae become adults and whose pups hatch after mating in about 10 days.
On the contrary, the pupae of a female parent that does not hatch and lays an egg that diapauses in the egg in a state in which growth is arrested in the late stage of gastrulation in the egg is referred to below as `` diapause pupae ''. ". The production of dormant eggs in this manner means that the above-mentioned Pseudomonas aeruginosa paralytic peptides have been isolated and structurally determined, or the insect paralytic peptide family whose presence was predicted from the cDNA. This indicates that there are novel physiological activities that are completely different from the reported physiological activities. This is because, as described above, in addition to the fact that the bioactive functions of the insect paralytic peptide family are diverse, the hormone system that induces egg dormancy in the silkworm has already been established. As described below, it is induced by a hormone composed of an amino acid sequence having no similarity to conventional paralytic peptides.

The paralytic behavior of the silkworm is induced by the silkworm paralytic peptide and the celestial silkworm paralytic peptide. But,
The silkworm egg dormancy phenomenon is quite different from this paralytic behavior and is explained by the silkworm's unique hormonal system. In the case of silkworm egg dormancy, dormant hormones are secreted from the subesophageal ganglia of the female pupae, and if they act on the ovaries, the laid eggs will stop developing at the late gastrulation stage and start dormancy. This dormant hormone has been reported to be a peptide with a molecular weight of 2645 consisting of 24 amino acids and having an N-terminal amidation (Imai, K. et al., Proc. Ja.
pan Acad., 67 (B) , 98-101, 1991.). When 1.5 ng of this peptide hormone was injected into a non-diapause pupae (in this case, a non-diapause line called N4 was used), 20% of the diapause eggs were produced (Imai et al., 1991, supra). From the mechanism of trehalase activation and diapause induction mechanism of silkworm diapause hormone, there is no doubt that this hormone is a peptide for true diapause induction of silkworm.

On the other hand, it is also possible to produce dormant eggs from the non-diapause pupae of silkworms by using a completely different chemical synthetic substance without using a known dormant hormone. For example,
Chrysalis ouabain that specifically inhibits Na ⁺ -K ⁺ -ATPase 1 ~
10 ~ 2 for non-diapause egg pupae on day 3 (using N4 and Taizo line)
It is known that injection of 0 nmoles through the skin produces about 70% of dormant eggs. If the subesophageal ganglia, which are the secretory organs of dormant hormones, are not extracted in advance, dormant eggs will not be produced.This phenomenon causes ouabain to act on the central nervous system, causing the secretion of dormant hormones from the subesophageal ganglia. It is considered to be the result of promotion.

[0029] The present inventors also injected into a non-diapausing pupae of a silkworm, a "silkworm paralytic peptide" derived from a silkworm, which is a physiologically active substance having a completely different function and structure from the diapause hormone of the silkworm. It was found that the dormant eggs of Escherichia coli can be produced, and the method for producing dormant eggs of the present invention was developed.
It was also confirmed that dormant eggs of silkworms can be produced by removing the esophageal ganglion, which is a dormant hormone secretory center, from the silkworm body in advance. This means that unlike silkworm paralytic peptides, ouabain
It demonstrates that it is a physiologically active substance having the same function as the silkworm diapause hormone. It is also pointed out that the paralytic peptide family of insects has various functions, and it will be extremely useful for the development of new growth regulators that control the development, metamorphosis, dormancy and reproduction of pests and beneficial insects in the future. This suggests the possibility of becoming an important lead compound. That is, the diapause hormone of the silkworm acts only on egg diapause induction of the silkworm, but the paralytic peptide of the silkworm induces paralytic behavior, blood cell diffusion function, larval growth retardation function, asystole function, and It also shows that it has a new function of producing dormant eggs in the silkworm found in 1.

From the above, the physiologically active substance of the present invention,
Alternatively, it can be seen that the synthetic helminthic paralytic peptide is useful for technological development for stably producing dormant eggs of silkworm. Using this peptide as a lead compound is also useful for developing insect growth regulators, and the phenomenon of diapause, which is universal in many organisms, is elucidated from the higher-order structure of proteins and the molecular mechanism with receptors. It is also effective for Thus, it is possible to say that the celestial silkworm paralytic peptide is a useful insect peptide.

The helminthic paralytic peptide and other known insect paralytic peptide families used in the following examples have the following amino acid sequences.

[0032] In the above amino acid sequence, the helminthic paralytic peptide Any Pa
In the case of an amino acid sequence different from rP, the amino acid in that portion is underlined.

The amino acid sequence of silkworm diapause hormone (Bom DH) is as follows.

[0034] As described above, in the case of helminthic silkworm paralytic peptide (Any ParP), the 12th amino acid from the N-terminus is methionine,
Replacing methionine with lysine is a silkworm paralytic peptide (Bom ParP) and a tobacco sparrow paralytic peptide II (Mas ParPII). Bombyx paralytic peptide (Bom
ParP) also replaces the fourth amino acid with alanine (for Any ParP) to valine. Furthermore, the amino acid sequence of silkworm diapause hormone (Bom DH) has little homology to the insect paralytic peptide family, except that the 19th amino acid from the N-terminus is the only cysteine.

[0035] The above-mentioned silkworm paralytic peptide (abbreviated to Scientific Parse and Abbreviation of Physiological Function as "Any ParP" and may be used hereinafter), Silkworm Paralytic Peptide (Bom ParP), Tobacco Smomega Peptide II (Mas ParPII ), Armyworm 1
Pseudoplasma cell diffusion peptide I (Psi PSPI), armyworm growth inhibitory peptide (Pss GBP), and one heart contractile peptide of Spodoptera litura (Spe CAP) were synthesized according to a standard method and used in the following examples.

The paralytic peptide in the present invention can be applied by a known administration method as follows. The non-diapause pupae of the dimorphic strain Taizo were used at the stage of 80 to 85 hours after pupation. The reason is that it is optimal for the experiment to select the optimal sensing stage existing in the pupal ovary, which is the target organ of the physiologically active substance. Celestial silkworm paralytic peptide 15
0 nmoles was dissolved in 30 μl of sterilized distilled water, and this was injected into the dorsal surface of the second and third joints of the abdomen to give the experimental group. In the subesophageal ganglion, which is a secretory organ of silkworm diapause hormone, dormant hormones are synthesized and accumulated even in non-diapause pupae, so for safety, this organ should be pupated within 24 hours after pupation. The previously extracted one was similarly injected and used as an experimental section. Since the helminthic paralytic peptide is always dissolved in sterile distilled water before use, 30 μl of sterile distilled water was injected into the dorsal surface of the second and third joints in the abdomen to serve as a control. The injection site is not limited to this site, but injection into the back vein is not preferable in order to avoid a large amount of bleeding. Any site other than the dorsal vein may be used. Usually, 150 to 300 nmoles of the helminthic silkworm-peptic peptide dissolved in 30 μl of sterile distilled water per animal may be injected. By doing so, the induction rate of dormant eggs can be increased to 60% or more, and the incidence of unfertilized eggs can be suppressed to the level of 2.5 to 3%. If the dose of the helminthic paralytic peptide is less than 150 nmoles, the induction rate of dormant eggs is low, and 300 nmol
If it exceeds es, there is a problem that the unfertilized egg rate increases.

The dormant egg inducer of the present invention can be administered to silkworms by any means other than the above injection, as long as it can be administered in vivo. For example, a method of encapsulating a dormant egg inducer in a powder state, implanting this in a pupa, and eluting it in the body may be used.

The characteristics of the silkworm dormant peptide are described below by comparing the silkworm diapause hormone with the paralytic peptide derived from the silkworm of the present invention. Comparing the activity of producing dormant eggs of silkworm with silkworm diapause hormone and helminthic paralytic peptide (hereinafter abbreviated as dormant egg production activity of silkworm) in terms of peptide concentration, the silkworm dormant peptide was used to induce diapause of silkworm. , Dormant eggs are not produced unless a high concentration of about 790 times higher than that of the silkworm diapause hormone is administered. For example, the concentration to obtain about 50% dormant eggs with synthetic silkworm diapause hormone is
An injection of 0.19 nmoles (corresponding to approximately 0.5 μg) is sufficient for one non-diapause pupae, whereas at least 150 nmoles (corresponding to approximately 366 μg) is required for the helminthic paralytic peptide. The higher concentration of helminthic paralytic peptide required for the induction of dormant eggs in the silkworm is only due to the difference between the activity of the true dormant hormone and the activity of the otherwise bioactive peptide. . In this case, the silkworm dormancy hormone produced 9.3% of unfertilized eggs, while the silkworm paralytic peptide produced only 2.5% of unfertilized eggs.

When the dose of the dormant hormone for the non-diapause pupae of the silkworm was increased, the number of unfertilized eggs was increased, and the concentration was as high as that of the celestial silkworm paralytic peptide (38 nmol / head).
es, equivalent to about 100 μg), the dormancy induction rate is 42
%, And 43% of unfertilized eggs occur. This means that since it is a true dormant hormone in silkworm, in vivo toxicity is exhibited when it is used at a high concentration. Even if the celestial silkworm paralytic peptide is administered to a living body at a high concentration, the level of unfertilized egg development is extremely low, and even at a high concentration, it does not induce paralytic behavior for silkworm pupae, and other physiological conditions (eg, , Including eclosion behavior, mating behavior, etc.), but almost completely affects only dormant egg production.

Further, in terms of classification, silkworm diapause hormones are classified into the FXPRLamide family, but helminth silkworm paralytic peptides are classified into the insect paralytic peptide family. Therefore, the celestial silkworm paralytic peptide having completely different amino acid sequence homology mimics the physiological activity of the dormant hormone that converts the life history of silkworms from non-diapause to dormant. The opposite phenomenon, that is, the silkworm diapause hormone, does not induce paralytic behavior of the silkworm.

The substance which is the active ingredient of the present invention is itself useful as a paralytic peptide of silkworm,
In the future, it will be important as a lead compound for the development of new drugs including bioprotective agents. The helminthic paralytic peptide in the present invention is a biological defense substance for celestial silkworm, and is a peptide having a novel function in the living world because it is effective in producing dormant eggs of silkworms.

[0042]

EXAMPLES The present invention will be described in more detail with reference to Examples and Comparative Examples. The invention is not limited by these examples. Before specifically explaining the examples, an outline of insects used, bioassay method, synthesis of active substance (physiologically active substance), and elucidation of characteristics of the active substance will be described.

Silkworms used in the following examples: 2
Taizo (silkworm variety name) eggs, which are volatile silkworms, were stored at 15 ° C in a so-called "total darkness" where no light entered the storage environment. The silkworm larvae that have hatched in such an environment are raised at 25 ° C for 12 hours in a "light environment", and the subsequent 12 hours are called a "dark environment". Were fed to obtain non-diapause pupae. After the pupae female has emerged,
When mated with males and then laying eggs, almost 100% become non-diapause eggs and all eggs hatch within 10 days after spawning. on the other hand,
Large embryonated silkworm eggs are raised at 25 ° C in a bright environment.
If the hatched larvae are bred under the same conditions as described above in an environment such as so-called “under the light environment” preservation, dormant egg-laying pupae can be obtained. If this pupae-grown female mates with a male after emergence and then lays eggs, almost 100% dormant eggs are obtained. That is, after egg laying, the embryo development in the egg is stopped in the late gastrulation stage and continues to be dormant. Furthermore, in addition to the above method of obtaining non-diapause egg-pupae by strictly controlling the storage state after egg-laying by using large-sized eggs, a line called N4, which is originally a non-diapause egg-producing line, was also used.

Regarding bioassay method: The activity of the sample was examined by injecting silkworm paralytic peptide or the like into each stage of the above-mentioned Daizo non-dormant pupae and dormant pupae of silkworm. Some were also injected into N4 non-diapause pupae.

Regarding the synthesis of physiologically active substances: Among those known as a group of insect paralytic peptides, the celestial silkworm paralytic peptide (Seino et al., 1998) and the silkworm paralytic peptide (Ha et al., Above). 1999), Tobacco Schimega Paralytic Peptide II (Skinner et al., 1991, supra), and one plasma cell spreading peptide of Spodoptera litura (Clark et a, supra).
l., 1997), the armyworm growth inhibitory peptide (see above Hayakaw
a, 1991), a cardiac contractile peptide of Yoto (see Furuyae, supra).
Tal., 1999), each peptide having a primary structure that completely matches the reported primary structure was synthesized using a peptide synthesizer (PSSM-8: manufactured by Shimadzu Corporation). For comparison of dormant egg production activity,
A dormant hormone (Imai et al., 1991), which originally functions in silkworms as an egg dormant inducer in vivo, was also synthesized using the above apparatus according to its primary structure. For the purity of each peptide, see HPLC and MALDI-TOFMS.
(Matrix-Assisted Laser Desorption Ionization-Time-o
f-Flight Mass Spectrometer, VoyagerPerSeptive Biosystems
The purity of each is 90%.
That was all. (Example 1) Analysis of new physiologically active function of celestial silkworm paralytic peptide: after pupation of non-diapause pupae of the dimorphic strain Taizo 80-80
I used a 5-hour stage. 150 nmoles of the helminthic paralytic peptide was dissolved in 30 μl of sterilized distilled water, and this was injected into the dorsal surface of the second and third joints of the abdomen to give an experimental group. In the subesophageal ganglion, which is a secretory organ of silkworm diapause hormone, dormant hormones are synthesized and accumulated even in non-diapause pupae, so for safety, this organ should be pupated within 24 hours after pupation. Regarding those that had been extracted in advance, those that were similarly injected were used as experimental sections. Furthermore, 30 μl of sterilized distilled water was injected into the dorsal surface of the second and third joints in the abdomen to serve as a control. A photograph was taken of the coloration of the eggs laid by the silkworms in each of the experimental and control areas. The obtained results are shown in FIG.

As is clear from FIG. 1, in the control group in which non-diapausing pupae were injected with sterile distilled water, all the eggs produced (about 250 to 400 eggs) were yellowish white, and within 10 days. It was a non-diapause egg that hatched (Fig. 1 (A)). However, in the experimental group where the non-diapause egg-producing pupae were injected with the silkworm paralytic peptide, a colored egg (a pale brown to
A dark brown color was produced (Fig. 1 (B)). In addition, even in the experimental group in which the subesophageal ganglion was excised from the non-diapausing egg-producing pupae within 24 hours after pupation, and the helminth silkworm paralytic peptide was injected 85 hours after pupation, colored eggs (light brown to dark (Brown) was produced (Fig. 1 (C)). From the above results, it is clear that the isolated and structurally determined helminthic cerebral palsy peptide has a novel physiologically active function, which is completely unexpected from the conventional wisdom of diapausing silkworm dormant eggs. Next, in Example 2, this point will be examined in more detail. (Example 2) Verification of whether silkworm colored eggs are true dormant eggs: Whether the silkworm colored eggs induced by the silkworm paralytic peptide described in Example 1 were truly true dormant eggs was examined as follows. I decided to verify it by the method.

From the colored eggs induced in Example 1, eggs that did not hatch even after 10 days from the spawning were treated with Carnoy's fixative (volume ratio: ethanol 6: chloroform 3: glacial acetic acid 1) to give 24
I fixed the time. The eggs were washed in the order of 100%, 95%, 90%, 80% ethanol-water system, and the thus dehydrated eggs were finally put in 80% ethanol to remove the eggshell in ethanol. Eggs from which eggshells were removed were stained with a thionine stain solution for 2 hours and then dehydrated in the order of 80%, 90%, 95%, 100% ethanol. The thionine stain solution was prepared as follows. A stock solution obtained by dissolving 1 g of thionine and 5 g of phenol in 100 ml of distilled water was diluted 30 times with 80% ethanol to obtain a thionine staining solution. The state of embryo development was observed under a microscope field with respect to an egg that had been stained by a conventional method, which had been transparentized with benzene and subjected to encapsulation. As a control group, dormant eggs produced from Taizo's original dormant egg-producing pupae were fixedly stained and observed by the same method as above. The obtained results are shown in FIG.

As shown in FIG. 2 (A), the dormant eggs laid from the original pupae of the dormant eggs had embryonic development stopped in the late gastrulation stage. Colored eggs obtained by injecting helminthic silkworm paralytic peptide into non-diapausing pupae, and colored eggs obtained by pre-extracting the subesophageal ganglion from non-diapause egg-pupae pupae by injecting helminth-silvering peptide However, embryonic development stopped at the late stage of the gastrulation embryo like the original dormant egg (Figs. 2 (B) and (C)). To further confirm this point, it is known that the original dormant eggs can be broken by a method called refrigerated stearic acid.Therefore, this method is used to break dormant eggs and The hatching rate was examined.

To facilitate understanding of the phenomenon of silkworm dormancy break, general knowledge of the embryo development stage will be explained. Generally, the embryonic development stages of silkworms are 1) fertilization stage, 2) blastoderm stage,
3) Embryonic zone formation stage, 4) Gastrulation stage, 5) Long-term, 6) Protrusion formation stage, 7) Inversion stage, 8) Bristle formation stage, 9) Blue season (also called prelarval stage), 10 ) It is composed of various stages of development such as the period just before hatching. In particular, it has been conventionally explained that embryonic development in silkworm dormant eggs occurs at the array stage, but since array is a convenient term for expressing the shape of the embryo, embryonic growth arrest in silkworm dormant eggs is ,
Developmentally, it occurs at the late stage of the gastrulation stage, and is therefore referred to as "late gastrulation stage" in the present specification.

As described above, the dormant eggs of Bombyx mori obtained by injecting the helminthic silkworm-paralytic peptide stop embryonic development in the late gastrulation stage in the external morphology like the true dormant eggs. Therefore, it is not possible to determine physiologically whether or not it is in the dormant state. However, if the dormancy is broken by artificially refrigerating and pickling, the dormant egg can be judged to be true dormancy. Therefore, 69 original dormant eggs 48 hours after spawning and 135 dormant eggs produced by the silkworm paralytic peptide were prepared, and after cryopreserving at 5 ° C for 20 days, hydrochloric acid with a specific gravity of 1.10 kept at 47 ° C. Those eggs treated with hot water for 6 minutes in an aqueous solution were observed at 25 ° C for 10 days to examine the hatching rate of dormant eggs by refrigerated soaking. The results obtained are shown in Table 1. (Table 1) Hatching rate of dormant eggs by refrigerated soaking

As shown in Table 1, the hatching rate of the original dormant eggs was 87%, and the hatching rate of the dormant eggs produced by the induction treatment with the silkworm paralytic peptide was 89.6%.

Thus, it is clear that the dormant eggs of Bombyx mori produced by the silkworm paralytic peptide are true dormant eggs both externally and physiologically, like the dormant eggs induced by the silkworm diapause hormone. . (Example 3) Relationship between pupal stage and paralytic peptide concentration on dormant egg production: From the results of Examples 1 and 2, the celestial silkworm paralytic peptide is called dormant egg production of silkworm as well as dormancy hormone of silkworm. It was confirmed that it has a completely new function. Therefore, in order to compare this new function with the original function of the silkworm diapause hormone, the relationship between the diapausing effect of helminthic paralytic peptide and the pupal stage, and the relationship between the concentration of the helminthic silkworm paralytic peptide and diapause diapause. I decided to consider about. As a control, the diapause hormone of silkworm was always used.

First, about 6 to 10 hours to 144 hours after pupation of the non-diapause pupae of dimorphic taizo, 150 nmoles of helminth-parasitic peptide were injected per head, and silkworm diapause hormone was added. The induction rate of dormant eggs when 0.38 nmoles was injected per animal was examined. The obtained results are shown in FIG.

As shown in FIG. 3, in the case of the silkworm paralytic peptide, the effective stage was 72 to 96 hours after pupation and 51.0 to 60.1% of dormant eggs were produced. . On the other hand, in the case of the silkworm diapause hormone used as a control, those from 72 to 96 hours after pupation were also effective, and 50.6 to 51.7% of dormant eggs were produced. Next, 80 to 85 hours after pupation, the pupae were used as experimental groups when the helminthic peptide concentration was 3 to 300 nmoles per animal, and the silkworm diapause hormone was 0.038 to 1
When 38 nmoles were injected as a control, the induction rate of dormant eggs by the action of silkworm diapause hormone and celestial silkworm paralytic peptide at different concentrations was examined. The obtained results are shown in FIG.

As shown in FIG. 4, with the silkworm paralytic peptide, the production of dormant eggs increased almost linearly with increasing concentration. The highest concentration of 300 nmoles injected yielded 72.4% dormant eggs. On the other hand, with the silkworm dormant hormone, the dormant egg production effect increased linearly at low concentrations, reaching almost the maximum value of 65.6% at 0.38 nmoles, maintaining a constant level above that level, and decreasing from 3.8 nmoles concentration. Beginning, 38
The dormant egg production effect was 41.5% at nmoles concentration.

The results of Example 3 suggest a new elucidation of the mechanism of the relationship between the pupal stage and the dormant egg production effect of the helminthic paralytic peptide. That is, it was possible to verify that, like the original silkworm diapause hormone, the susceptibility stage exists in the middle pupa stage, but in relation to the concentration, in order to produce dormant eggs equivalent to the diapause hormone, the helminth paralytic peptide It was found that the concentration was about 350 to 790 times higher. (Example 4) Benefits of silkworms on dormant egg production: From the results of Example 3 described above, the effect of silkworm dormant egg production of silkworm dormant peptide is equivalent to that of silkworm dormant hormone at high concentration. Became clear. Therefore,
To investigate functional safety, we investigated the relationship between the levels of helminthic silkworm paralytic peptide and silkworm diapause hormone and unfertilized egg development. The obtained results are shown in Table 2. (Table 2) Relationship between the levels of helminthic palliative peptides and silkworm diapause hormone and unfertilized egg development

As shown in Table 2, in the silkworm diapause hormone, the higher the concentration, the higher the incidence of unfertilized eggs and the disturbed physiological function. However, in the case of the silkworm paralytic peptide, , Such a phenomenon is not confirmed at all. Therefore, it is considered that using the celestial silkworm paralytic peptide as the skeleton of the lead compound of the insect growth regulator has higher functional safety and lower risk of causing disturbance other than the intended physiological reaction.

Next, by comparing the physiological functions of the insect paralytic peptides that have been isolated and structurally determined up to now with the celestial silkworm paralytic peptide that is the active ingredient of the present invention, the celestial silkworm paralytic peptide of the present invention was identified. It was evaluated whether or not it exerts an excellent effect in producing the dormant eggs of the silkworm.

That is, the silkworm paralytic peptide, tobacco splendid paralytic peptide II, one kind of plasma cell spreading peptide of the armyworm, armyworm growth inhibitory peptide,
Five insect paralytic peptide families of one cardiac contractile peptide of Spodoptera litura were synthesized by a known peptide synthesizer, and the silkworm dormant egg production effect by these peptides was determined by the same method as in Example 1 and Example 3. Table 3 shows the obtained results. (Table 3) Dormancy of silkworm dormant eggs produced by the insect paralytic peptide family (Taizou)

As is clear from Table 3, in the silkworm paralytic peptide, 80 to 85 after pupation, as in the case of the silkworm paralytic peptide.
Injecting 60 nmoles into pupae of time showed a very low dormant egg production rate of 0.1 ± 0.2%. On the other hand, the growth inhibition peptide with a high dormant egg production rate was 45.6 ± 7.8%, which was considerably lower than the 63.9 ± 9.3% of the helminth silkworm paralytic peptide. The rate of unfertilized egg development was suppressed to a level of 2.0 to 4.0% for all peptides.

From the above results, as the dormant egg-producing effect of silkworms, among the insect paralytic peptide families that have been discovered so far, the celestial silkworm paralytic peptide used in the present invention, whose isolated structure has been determined from the silkworm, is used. It turned out to be the most effective. This indicates that the primary structure of the Pseudomonas aeruginosa peptide is valuable as a skeleton of the lead compound in the development of new insect growth regulators in the future. (Example 5) Verification of dormant egg production effect by silkworm strain varieties: In the above Examples 1 to 4, non-diapause egg pupae of the dimorphic silkworm, "Taizo", were used to induce dormant eggs of the silkworm of the present invention. The results are shown when the dormant egg inducer, which is the active ingredient of the agent, was injected. It was confirmed that the active ingredient of the present invention is effective against unspecified silkworms as well, using polymorphic silkworms, not dimorphic silkworms, as follows.

The dosing conditions for the dormant egg inducer were exactly the same as those shown in Table 3 of Example 4, and the silkworm pupae to be tested were N4 of the polymorphic strain used in the bioassay of the silkworm dormant hormone. We examined the effect on the inducibility of dormant eggs by using the pupa of the pupa by administering a total of the following six peptide families including the helminthic helminthic peptide. The dose of each paralytic peptide was set at 60 nmoles per animal, and the dormant egg production effect of the silkworm was examined. The results obtained are shown in Table 4. (Table 4) Effect of silkworm dormant egg production by insect paralytic peptide family (N4)

As is clear from Table 4, the N4 strain also had the highest effect of producing silkworm diapausing eggs by the silkworm paralytic peptide, confirming the same tendency as the "Taizuku" strain described in Table 3. From this, it is clear that the novel function of the helminthic paralytic peptide is exerted without being influenced by the difference between strains of the silkworm.

As is clear from the above examples, since the helminthic paralytic peptide has an excellent diapause egg-inducing property to silkworms, if the insect species dormant at the same late stage of gastrulation embryo as silkworms, silkworms are not Of course, it can be applied to mulberry, mulberry squirrel, kusan, himeshiromonduga, etc., and can also be used to control the living environment from useful insects to harmful insects.

[0065]

EFFECTS OF THE INVENTION The dormant egg inducer of the present invention has an amino acid sequence shown in SEQ ID NO: 1 of the Sequence Listing, is free-oxidized at the C-terminus, and contains a peptide having a molecular weight of 2435.73 as an active ingredient. For example, since it has a physiological activity that efficiently induces dormant eggs of the silkworm, it has a function of producing dormant eggs by acting on insects such as silkworms, and a simple administration method for producing dormant eggs can be expected. It is also possible to elucidate the mechanism of insect dormancy mechanism by using the novel physiologically active substance of the present invention. Furthermore, by using the diapause control substance of the present invention in other types of organisms confirmed to diapause, it is possible to elucidate the life mechanism of "low energy metabolism" which can be said to be the essence of diapause of these organisms. Is also effective. From the perspective of the future, the dormant egg inducer of the present invention can be a promoter for long-term life support, and finally a lead compound for the development of a longevity promoter. Thus, the dormant egg inducer of the present invention can be expected to play an important role in each of the fields of biology, life science, pharmacy, and agriculture, and the peptide of the present invention is a novel dormant egg inducer having a dormant egg inducing function. Can be a medicine.

Comparing the dormant egg production efficiency of silkworms by the dormant egg inducer of the present invention and the conventional silkworm dormant hormone, the helminthic palliative peptide requires a higher dose than that of the silkworm dormant hormone When the dormant hormone concentration of silkworms administered to silkworms is increased in order to increase the efficiency of producing eggs, the number of unfertilized eggs increases, and in vivo toxicity is expressed. Even at a considerably high dose, the level of unfertilized egg development is extremely low, and there is the advantage that no phenomenon that disturbs the overall physiological activity of silkworms is observed.

FXPRLamide in the case of silkworm diapause hormone
The helminthic paralytic peptide is classified into the insect paralytic peptide family. Therefore, the celestial silkworm paralytic peptide whose amino acid sequence homology is completely different from that of the silkworm diapause hormone mimics the physiological activity of the diapause hormone that converts the life history of the silkworm from non-diapause to dormant. However, the opposite phenomenon, that is, the dormant hormone of the silkworm, does not induce the paralytic behavior of the silkworm.
This suggests important discoveries both academically and practically from the perspective of "protein function and structure," which is a major issue in the post-genome of the 21st century.

From the above, according to the silkworm dormant egg inducer of the present invention, not only the dormant egg production of silkworm is stabilized by the large-scale synthesis of a silkworm paralytic silkworm in the future, but also this peptide is used as a lead compound. It becomes possible to develop a new insect growth regulator. Furthermore, in pursuing the phenomenon of "diapause", which is commonly found in many organisms, from the relationship between the specificity of the higher-order structure of proteins and the molecular mechanism of receptors, helminthic silkworm peptides are excellent insect peptides. Thought to be possible.

The helminthic paralytic peptide can be used as follows. Compared with the conventionally known insect paralytic peptide family, the celestial silkworm paralytic peptide of the present invention,
It has the most excellent function in the production of dormant eggs of silkworms.
Although its mechanism of action mimics the silkworm diapause hormone, even if a high-concentration silkworm paralytic peptide is administered to silkworms, unlike conventional diapause hormones, it rarely produces unfertilized eggs. Therefore, by using the primary structure of the celestial silkworm paralytic peptide as a lead compound, it becomes possible to basically elucidate the mechanism of the induction of dormant eggs in silkworms, and as an application, it is a method for the occasional preservation of silkworm non-diapause strains around the world. Can be a basic technology for the establishment of Since the celestial silkworm paralytic peptide of the present invention has a dormant egg-inducing property, if it is an insect species that diapauses at the same late stage of the same gastrointestinal embryo as silkworm, not only celestial silkworms, but also mulberry, mosquito balsamum, kusan, himeshiromonduga, etc. Not only can it be applied to, but it can also be used to control the living environment from useful insects to harmful insects.

As described above, the dormant egg inducer of the present invention has an extremely low unfertilized egg development rate than the silkworm dormant hormone and does not disturb the physiological state of silkworms. Non-diapause strains can be effectively diapaused to silkworms in major raw silk-producing countries such as Thailand.

The dormant egg inducer of the present invention is not limited to dimorphic silkworms, but can be applied to polymorphic silkworms bred in Southeast Asia such as India, Thailand and Vietnam. Therefore, the dormant egg inducer for making the polymorphic line variety dormant at any time is also important at the sericulture and industrial level in the world of producing raw silk.

As described above, according to the present invention, the paralytic peptide that controls induction of dormant eggs derived from silkworm, silkworm, can be used very effectively for the production of dormant eggs in silkworms. Become the lead compound.

[0073]

[Sequence list]                           SEQUENCE LISTING <110> Inoue, Hajime; Director General of National Institute of Sericultu ral and Entomological Science Ministry of Agriculture, Forestry and Fish erries <120> Agent for deriving diapausing eggs of insects and method of produc ing diapausing eggs <130> K000326 <160> 1 <210> 1 <211> 23 <212> PRT <213> Antheraea yamamai Guerin-Meneville <400> 1 Glu Asn Phe Ala Gly Gly Cys Ala Thr Gly Phe Met Arg Thr Ala Asp   1 5 10 15 Gly Arg Cys Lys Pro Thr Phe             20

[Brief description of drawings]

FIG. 1 (A) is a photograph (control section) in which distilled water was injected into a non-diapause egg-laying pupa to photograph the coloring state of eggs laid by silkworms. (B) A photograph (experimental section) showing the coloration of eggs laid by silkworms by injecting a helminth silkworm paralytic peptide into a non-diapause egg pupae (experimental section). (C) A photograph (experimental section) in which the color of eggs laid by silkworms was photographed by injecting a helminth-parasitic peptide into an excised esophageal ganglion from a non-diapausing pupae (experimental section).

FIG. 2 (A) is a photomicrograph of the developmental state of dormant embryos obtained from the original dormant egg pupae (control). (B) Injecting a helminth-silent paralytic peptide into a non-diapause egg-laying pupa,
Micrograph of the developmental state of induced embryos of dormant eggs (experimental section). (C) A micrograph (experimental section) of the developmental state of embryos of dormant eggs induced by injecting a helminth silkworm paralytic peptide into an isolated esophageal ganglion from a non-diapausing pupae. (D) A photograph of hatched larvae obtained when the dormant eggs obtained from the experimental section of (B) above were subjected to cold soaking treatment.

FIG. 3 is a graph showing the induction rate of each dormant egg upon administration of a helminthic silkworm paralytic peptide and a silkworm diapause hormone.

FIG. 4 is a graph showing the induction rate of each dormant egg when a silkworm diapause hormone and a celestial silkworm paralytic peptide having different concentrations were administered.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁷ Identification code FI C07K 14/435 C07K 14/435 (72) Inventor Song Hongsei 2-6-20 Aoyama, Morioka-shi, Iwate Municipal Appartment No. 4 No. 402 (56) Reference JP-A-5-86095 (JP, A) JP-A-2000-342254 (JP, A) JP-A-2001-261572 (JP, A) JP-A-5-227967 (JP, A) Kaihei 11-255799 (JP, A) (58) Fields surveyed (Int.Cl. ⁷ , DB name) A01N 63/00 A01K 67/00 A01N 25/00 A01N 37/00 C07K 14/435

Claims (5)

(57) [Claims] 1. An amino acid sequence represented by SEQ ID NO: 1 in the sequence listing, Glu-Asn-Phe-Ala-Gly-Gly-Cys-Ala-Thr-Gly-Phe-Me.
It has t-Arg-Thr-Ala-Asp-Gly-Arg-Cys-Lys-Pro-Thr-Phe-, is characterized by containing a peptide having a C-terminal free oxidation and a molecular weight of 2435.73 as an active ingredient. An insect dormant egg inducer. 2. The dormant egg inducer for insects according to claim 1, wherein the insect is a silkworm. 3. The insect dormant egg inducer according to claim 1, wherein the peptide is an insect paralytic peptide derived from the body fluid of the silkworm, Bombyx mori. 4. A dormant egg inducer for insects, which comprises, as an active ingredient, one of the plasma cell spreading peptide I (Psi PSP I) and the millet inhibition peptide (Pss GBP) of Spodoptera litura. 5. The peptide of any one of claims 1 to 3 is administered to the non-diapause pupae of silkworms at a stage of 72 to 96 hours after pupation, and the pupae are administered with the peptide of 150 to 300 nmoles per pupa. A method for producing dormant eggs, which comprises breeding a female, mating a female that has emerged, mating with a male, and then laying the eggs to obtain dormant eggs.