JP5549911B2 - Insect diapause egg inducer, insect diapause egg production method and pest control method - Google Patents

Description

  The present invention relates to an insect diapause egg inducer, an insect diapause egg production method using the same, and a pest control method.

  Silkworms are industrial animals and are used for silk production. In silk production, it is extremely important to produce silkworm eggs efficiently and to protect the eggs safely. And dormancy hormone is actively used for protection and stable supply of silkworm eggs. In other words, by providing dormancy to eggs, it is possible to retain eggs at an early age and supply larvae artificially at any time.

  On the other hand, some insects are pests in agriculture, forestry and medical hygiene. Since pest damage mainly occurs during their growth and development, they can be controlled by actively resting the pests. In this aspect, dormant hormone is expected to exhibit an effective action. Thus, the search for dormant hormones has been desired in terms of protection and safe supply of silkworm eggs and pest control.

  Under such circumstances, the present inventors have so far isolated a dormancy hormone that induces egg dormancy in silkworms, revealing a peptide amide structure consisting of 24 amino acids, and also for chemical synthesis. Successful (Patent Document 1, Non-Patent Document 1).

  Also, various dormant hormone fragments were synthesized, and the minimum active expression unit was a C-terminal tripeptide amide (PRLa), and a pentapeptide consisting of phenylalanine, glycine, proline, arginine, and leucine was amidated. Peptide amide (hereinafter referred to as FGPLa) has been shown to be 20 times more active than the minimally active expression unit. Furthermore, the synthetic | combination method of these derivatives is also developed (nonpatent literature 2).

  However, in order to express the dormancy-inducing ability in silkworms using the above peptide amides and the like, it was necessary to administer them to the pupa 3-4 days after hatching. That is, even when the dormant egg inducer is injected into the pupa before (larvae stage) or after this period, the dormancy induction rate is reduced or does not show any activity.

  Furthermore, the present inventors also synthesized derivatives (C16-PRLa, C16-FGPRLa) and the like in which a fatty acid (C16) is bonded to the N-terminus, such as the tripeptide amide (PRLa) and pentapeptide amide (FGPRLa). It has been confirmed that the dormant egg inducing ability of C16-FGPLa is particularly excellent (Non-patent Document 3).

  However, for example, C16-FGPLa has also been confirmed to have the ability to induce dormant eggs by injecting pupae 3-4 days after hatching, but it was administered to puppies before this (larva stage) or after this stage. It has never been studied.

On the other hand, the present inventors have developed a novel peptide amide (DILRG) having an amino acid sequence, aspartic acid-isoleucine-leucine-arginine-glycine (DILRG), having a C-terminal amidation, and a molecular weight of 570.959. -NH ₂₎ found, the peptide amides, the present inventors have named "Yamamarin".

The present inventors also have the ability to induce silkworm diapause eggs for a peptide amide derivative (C16-DILRG-NH ₂ ) in which a fatty acid (C16) is bound to the N-terminus of this peptide amide (DILRG-NH ₂ ). This is reported (Non-patent Document 4).

However, the peptide amide (DILRG-NH ₂ ) and its derivative (C16-DILRG-NH ₂ ) are also administered by injection to the pupa 3-4 days after conversion, like the above FGPRLa and its fatty acid derivative. It is considered necessary, and it has not been studied at all for administration to the pupa before (the larval stage) or after this period. In addition, DILRG-NH ₂ also has a problem that the dormant egg-inducing activity is weaker than the above FGPLa derived from the dormant egg-inducing hormone.

  Therefore, in order to express the activity of a known dormant egg inducer, there is a severe restriction that it must be administered by injection at a predetermined time, and thus, for gene preservation and insect control of egg dormant insects. There was a limit to application.

Patent Publication 5-86095

Proc. Japan Acad., Vol. 67 Ser B (No. 6), p98-101, 1991 Biosci. Biotech. Biochem., Vol. 62 (No. 10), p1875-1879, 1998 Japan Comparative Endocrine Society News 103, 15-22 (2001) A palmitonyl conjugate of an insect pentapeptide causes growth arrest in mammalian cells and mimics the action of diapause hormone, Journal of Insect Biotechnology and Sericology 76,63-69 (2007)

  An object of the present invention is to provide a dormant egg inducer having an extended drug administration time for dormancy induction, and to provide a dormant egg inducer that can be administered by a method other than injection.

  The present inventors obtained new knowledge from the study of the ability to induce dormant eggs by FGPPRLa isoprenoid carboxylic acid modified products and amino acid substitution products, and have completed the dormant egg inducers of the present invention.

In order to solve the above problems, the present invention provides the following dormant egg inducers and the like.
<1> An insect diapause egg inducer containing a peptide amide derivative, wherein the peptide amide derivative comprises an amide group at the C-terminal and an isoprenoid carboxylic acid at the N-terminal of a pentapeptide consisting of phenylalanine, glycine, proline, arginine, and leucine. dormant eggs inducer of insects that have a residue.
<2> The first dormant egg inducer, wherein the isoprenoid carboxylic acid is farnesic acid.
<3> The first dormant egg inducer, wherein the isoprenoid carboxylic acid is geranilic acid.
<4> A method for producing dormant eggs, wherein the second dormant egg inducer is injected into larvae.
<5> A method for producing a dormant egg, wherein the dormant egg inducer according to any one of the first to third methods is transdermally administered to a pupa.
<6> The method for producing a dormant egg according to the fourth or the fifth aspect, wherein the administration target of the dormant egg inducer is a silkworm.
<7> A method for controlling pests, wherein the dormant egg inducer according to any one of the first to third is allowed to act on pest larvae and willow.

  The insect diapause egg-inducing agent of the present invention maintains its activity for a long time in vivo, and can extend the administration time by injection from the pupa stage to the larva stage on the fourth day.

  In addition, dormant eggs can be induced by transdermal administration such as application or spraying on willows, so that it is possible to obtain dormant eggs very easily without the need for advanced techniques.

  Therefore, the insect diapause egg inducer of the present invention can be improved in the preservation method of precious genes of insects represented by pluripotent silkworms and applied to insect control.

Percentage of dormant eggs produced when the prescribed amount of Far-FGPRLa dissolved in 10 μL of 0.1% Brij35 solution was injected into 3 days old polymorphic silkworm larvae (total dormant eggs) It is a figure which shows (occurrence rate%). Error bars indicate standard deviation. From this figure, it can be seen that almost 30 laying eggs can be dormant by administering 30 μg of drug per head. It is a figure which shows the dormant egg incidence rate when C16, Rho-KFGPRLa is injected and administered to the polymorphic silkworm larvae on the third day of age. In the case of this sample, almost the same result as that of Far-FGPRLa was obtained. It is a figure which shows the egg-laying diapause rate at the time of apply | coating 5-10 microliters of sample solutions which melt | dissolved the predetermined amount of Far-FGPRLa in 2-propanol to the pupa of the polymorphism silkworm immediately after hatching. This figure shows that almost all laying eggs can be made dormant by administration of 30 μg per head. In addition, it can be seen that about 20% of the laying eggs can be brought to dormancy by administration of 20 μg, and 15% of the laying eggs can be put to sleep by administration of 10 μg. It is a figure which shows the egg-laying diapause rate at the time of apply | coating 5-10 microliters sample solution which melt | dissolved Ger-FGPRLa of a predetermined amount in 2-propanol to the pupa of the polymorphous silkworm immediately after hatching. From this figure, it can be seen that about 80% of laying eggs can be dormant by administration of 20 μg per head. In addition, it can be seen that about 10% of the laying eggs can be brought to dormancy by administration of 10 μg, and 15% of the laying eggs can be put to dormancy by administration of 5 μg. It is a figure which shows the egg-laying diapause rate at the time of apply | coating 5-10 microliters of sample solutions which melt | dissolved predetermined amount C16-FGPRLa in 2-propanol to the pupa of the polymorphous silkworm immediately after hatching. From this figure, it can be seen that 15% of laying eggs can be diapause by administration of 30 μg per head, and by administration of 15 μg, 20 μg and 10 μg. It is a figure which shows the transparency improvement by PEG conversion. Rho, PEG-KFGPRLa and C16, Rho-KFGPRLa 20ug was applied to the abdomen of pupae immediately after conversion, and the fluorescence based on the rhodamine group was observed 3 days after application. Was promoted. It is a figure which shows the dormant egg-laying rate at the time of apply | coating Rho, PEG-KFGPRLa, and C16, PEG-KFGPRLa to the pupa immediately after hatching. The dormant egg production rate was found to be inferior to that of Far-FGPRLa or Ger-FGPRLa.

  The insect diapause egg inducer of the present invention contains a peptide amide derivative having the following characteristics as an active ingredient.

  That is, the peptide amide derivative has an amide group at the C-terminal and a fat-soluble residue at the N-terminal of a peptide consisting of 2 to 6 amino acids. The second amino acid from the C-terminal is composed of any one of arginine, lysine, and histidine. The fact that the second amino acid from the C-terminal has a positive charge is greatly related to the dormant egg-inducing activity of the present invention. In addition, the structure of the amino acid of another position is not specifically limited.

  And the said fat-soluble residue couple | bonded with the N terminal of a peptide is a fat-soluble residue derived from isoprenoid carboxylic acid represented by a farnesyl acid residue or a geranilic acid residue, However, even if it is long chain or short chain from these good. The presence of these fat-soluble residues is involved in the percutaneous permeability of the peptide derivative and is also important for the dormant egg-inducing activity.

  Specific examples of the peptide derivative having the above-described characteristics include the following peptide derivatives.

  This peptide derivative has an amide group at the C-terminal of a pentapeptide consisting of phenylalanine, glycine, proline, arginine, and leucine. Furthermore, R in the above chemical formula means a fat-soluble residue derived from the isoprenoid carboxylic acid shown below.

  Here, “m” in the chemical formula 2 can be set to about m = 1 to 4.

  Further, it is considered that lysine is further bound to the N-terminus of the peptide amide. In this case, since the side chain has an amino group, a double-modified product can be formed. That is, when a double-modified product is formed, residues having different characteristics can be modified at the N-terminus in addition to modification with a fat-soluble residue. For example, a substance that promotes percutaneous absorption of the peptide derivative can be bound to one of the amino groups. Examples of the substance include polyethylene glycol (PEG).

  The peptide amide derivative having the above characteristics can be prepared by a peptide synthesizer or other known methods. Known enzymatic methods and chemical methods can be applied to derivatize peptides.

  And it can be set as the dormant egg inducer of this invention by dissolving the said peptide derivative in a solvent. As the solvent, water, a surfactant solution such as Brij35, an organic solvent such as 2-propanol or ethanol, or a mixed solvent thereof with water can be used depending on the administration method.

  Furthermore, in addition to the above peptide derivatives, a substance that does not inhibit the dormant egg inducing action can be appropriately added to the solvent. For example, antibiotics, excipients, binders, disintegrants, dispersants, viscosity agents, reabsorption accelerators, surfactants, solubilizers, preservatives, preservatives, emulsifiers, isotonic agents, stabilizers In addition, one or more of buffering agents, pH adjusting agents, and the like can be blended.

  And according to the dormant egg inducer of the present invention containing the above peptide derivative, the activity can be maintained for a long time in vivo. Therefore, it is possible to extend the administration period by injection from the pupa stage to the larva stage on the fourth day of hatching. That is, it can be administered by injection to various larvae and can induce dormant eggs. When injection administration is performed on larvae, it is preferable to administer to larvae.

  Moreover, since the dormant egg inducer of the present invention is excellent in percutaneous permeability, it can be transdermally administered to insects having egg dormancy, such as Chadokuga pupa. The method of transdermal administration can be appropriately selected. For example, the diapause egg inducer of the present invention can be applied directly to the pupa, or can be administered transdermally by spraying the diapause egg inducer. Transdermal administration to pupa does not require advanced techniques and is extremely simple.

  Moreover, although the kind of insect used as object is not limited, Since a silkworm is highly industrially useful, it is suitable.

  For example, in the case of silkworms, the dose of the peptide derivative is preferably in the range of 0.01 μg to 500 μg, more preferably in the range of 1 μg to 100 μg per head. Other insects can be appropriately adjusted in consideration of the size of the insects.

  As described above, conventionally, dormant eggs were induced by injection administration for pupae, but the diapause egg inducer of the present invention induces dormant eggs by injection administration to larvae or transdermal administration to willow. As a result, insect dormant eggs can be produced efficiently and the eggs can be protected safely.

  For example, when preserving eggs (genetic resources) such as egg dormant polymorphic silkworms and other egg dormant insects that do not normally dormancy, the dormant egg inducer of the present invention is administered to larvae by injection, or passed to pupae. It is sufficient to administer the skin and preserve the dormant eggs delivered. During the storage period, it can normally be stored at room temperature and does not bother any hand. When next-generation larvae, willows, and adults are needed, they can be awakened from dormancy by returning to room temperature after low-temperature treatment or by applying acid treatment according to the usual hatching method from dormant eggs. According to the dormant egg inducer of the present invention, egg protection and safe supply are realized.

  Furthermore, if the insect dormant egg inducer of the present invention is allowed to act on pests, the pests can be made to dormant. Therefore, the present invention also provides a method for controlling pests using the dormant egg inducer.

  As a specific embodiment, for example, the dormant egg inducer may be applied to or sprayed on the cocoon immediately after hatching. As a result, the egg to be laid becomes a dormant egg, so that the growth of the pest is surely inhibited.

  Furthermore, the dormant egg inducer of the present invention is extremely useful because it has less influence on humans and the surrounding environment than conventional organophosphorus pesticides (insecticides).

  In addition, embodiment of the said pest control method is only an illustration, and can be implemented in a suitable aspect according to the kind and environment of a pest.

  Examples of the present invention will be described below, but the present invention is not limited to the following examples.

<1> Synthesis of Far-FGPRLa and Ger-FGPRLa Peptide, phenylalanyl-glycyl-prolyl-arginyl-leucine-NH by a usual method using a peptide synthesizer (PSSM-8, manufactured by Shimadzu Corporation) ₂ (FGPRLa) was synthesized. Subsequently, a free peptide was obtained by cleaving reaction. This is dissolved in DMF and reacted with farnesic acid or geranilic acid under basic conditions using WSCD as a dehydrating agent to obtain Far-FGPRLa and Ger-FGPRLa having a fat-soluble residue at the N-terminus of FGPRLa. It was. The farnesic acid used was synthesized according to the method of S. Yashwant et al. (I. Org. Chem., 52, 1568-1576 1978) and used as a mixture of geometric isomers. Geranilic acid was purchased from Research Chemicals and used without purification.

  The product was purified by connecting a reverse phase column Develosil-ODS HG-5 (20 mm × 250 mm, manufactured by Nomura Chemical Co., Ltd.) to an HPLC system (Gulliver Corporation JASCO). Elution is performed at a flow rate of 4 mL / min using an acetonitrile concentration gradient (0 to 100% from 0 to 120 minutes) in the presence of 0.1% trifluoroacetic acid (TFA) to elute the active fraction. It was. Absorbance was measured at 220 nm. The peptide was mixed with an equal amount of matrix (saturated with 40% acetonitrile / 0.1% TFAα-CHCA) on a sample plate, dried, and then MALDI-TOF MS (Discovery, manufactured by Shimadzu Corporation) or HPLC. -The structure was confirmed by MS (2020 type system, Shimadzu Corporation).

<2> Synthetic example of double modified product (Synthesis of C16, PEG-KFGPRLa)
Peptides C16 and PEG-KFGPRLa double-modified with palmitic acid and PEG were synthesized.

  Specifically, first, a peptide resin was synthesized with a peptide synthesizer as shown in <1> above. In addition, lysine (K) was introduced at the N-terminus for double modification.

  Then, the obtained peptide resin was suspended in DMF, and palmitoyl chloride and pyridine were added and reacted. Then, it was subjected to a cleaving reaction to obtain crude palmitoyl KFGPRLa.

  The obtained crude palmitoyl KFGPRLa was reacted with activated PEG (PEG-OSu) to synthesize a double label. Purification was performed by connecting a reverse phase column Develosil-ODS HG-5 (20 mm × 250 mm, manufactured by Nomura Chemical Co., Ltd.) to an HPLC system (Gulliver Corporation JASCO). Elution is performed at a flow rate of 4 mL / min using an acetonitrile concentration gradient (0 to 100% from 0 to 120 minutes) in the presence of 0.1% trifluoroacetic acid (TFA) to elute the active fraction. It was. Absorbance was measured at 220 nm. The peptide was mixed with an equal amount of matrix (saturated with 40% acetonitrile / 0.1% TFAα-CHCA) on a sample plate, dried, and then MALDI-TOF MS (Discovery, manufactured by Shimadzu Corporation) or HPLC. -The structure was confirmed by MS (2020 type system, Shimadzu Corporation).

  The rhodamine group of Rho and PEG-FGPRLa synthesized by the same method is the pharmacokinetics, that is, whether the dormancy inducer injected into larvae is maintained in pupae and adults and transferred to eggs. It was used as a fluorescent label for examining whether it was taken into the body when applied.

<3> Dormancy-inducing activity test (1) Test insects N4 species of multi-spotted silkworms that do not sleep during normal breeding were reared on artificial feed.

(2) Preparation of dormant egg inducer (i) Solution for injection administration Each sample obtained in <1> and <2> above is dissolved in water or 0.005% to 5% Brij solution (surfactant solvent). A solution of 0.05-50 μg per 10 μL was prepared.
(Ii) Solution for transdermal administration Each sample obtained in the above <1> and <2> was dissolved in water or 0.005% to 5% Brij solution to prepare 0.05-50 μg solution per 10 μL.

  When 2-propyl alcohol or ethyl alcohol was used for dissolution, the sample solution having the above-mentioned concentration was prepared by dissolving it.

(3) Medication (i) Injection administration to larvae A predetermined amount of the dormant egg inducer was injected into the larvae's back vein using a microsyringe. After dosing, the conditions were returned to normal breeding conditions.
(Ii) Transdermal administration The dormant egg inducer was administered to the abdominal surface of the pupa on the first day using a pipetman. In this case, small droplets were formed and applied so that the tip of the Pipetman did not touch the willow directly.

  The test silkworm coated with the test sample was placed on the dosing table to prevent the sample solution from being lost to the incubator. When the sample was an organic solvent solution or a mixed solvent solution of an organic solvent and water, it was air-dried.

(4) Determination of dormancy-inducing activity Test silkworms to which a dormant egg inducer is administered are normally raised, and mated and spawned after emergence. After the laying eggs were allowed to stand for 2 weeks and the non-dormant eggs hatched, the number of dormant eggs and the number of non-dormant eggs were measured to determine the incidence of dormant eggs.

(5) Results (i) Administration of injection to larvae A graph showing the dormant egg induction rate when Far-FGPRLa was administered by injection to multi-spotted silkworm larvae on the third day of age is shown in FIG. The dosage is 30, 20, 10 μg per head.

  From this result, it became clear that 83% laying eggs were dormant at 30 μg, 15% at 20 μg, and 3% at 10 μg.

  In addition, a graph of the incidence of dormant eggs when C16, Rho-KFGPRLa is administered by injection is shown in FIG.

  On the other hand, when Ger-FGPRLa was similarly formulated, no activity was shown even after administration of 30 mg (not shown).

  From the above results, Far-FGPRLa in which the N-terminus is modified with a farnesyl acid residue can induce dormancy by injection administration to larvae of polymorphic silkworms, and low in the case of Ger-FGPRLa with low fat solubility. It became clear that it was active.

(Ii) Transdermal administration to pupae immediately after hatching FIG. 3 shows the dormant egg induction rate when a predetermined amount of Far-FGPRLa was applied as a 2-PrOH solution to pupae immediately after hatching.

  As a result, as shown in FIG. 3, it was found that 82% of the laying eggs were dormant at 30 μg administration, 40% at 20 μg, and 15% at 10 μg. Similar results were obtained when applied as a solution other than 2-PrOH.

  In addition, when Ger-FGPRLa is similarly formulated, as shown in FIG. 4, 80% by 20 μg administration, approximately half by 10 μg administration, and 10% by 5 μg administration can lay 10% of laying eggs. It became clear. It was confirmed that Ger-FGPRLa exerts particularly excellent dormant egg-inducing activity in transdermal administration to pupa.

  For comparison, FIG. 5 shows the incidence of dormant eggs when C16-FGPRLa is applied to pupa. As shown in FIG. 5, it was found that dormant eggs could be induced by application of C16-FGPRLa, but the intensity of activity was considerably weaker than that of Far-FGPRLa or Ger-FGPRLa.

  From this result, it was found that isoprenoid carboxylic acid modification is extremely effective in the case of transdermal administration.

<4> Other peptide derivatives The following findings were obtained as a result of the dormancy-inducing activity test for other peptide derivatives prepared by the same method as described above.

  (1) The peptide derivative in which PEG was introduced at the N-terminus of the peptide, Rho, PEG-KFGPRLa, promoted percutaneous absorption to pupa (FIG. 6). However, the dormancy-inducing activity was 5 to 10 times lower than that of Far-FGPRLa and Ger-FGPRLa (FIG. 7). As shown in FIG. 3 and FIG. 4, it was confirmed that the dormant egg-inducing activity of Far-FGPRLa and Ger-FGPRLa was particularly excellent in transdermal administration to pupa.

  (2) The dormant egg-inducing activity of C16, PEG-KFGPRLa was confirmed by transdermal administration to pupa, but in this case as well, the dormant egg-inducing activity was 3 to 4 times that of Far-FGPRLa and Ger-FGPRLa It was weak (Figure 7).

  This test also confirmed that Far-FGPRLa and Ger-FGPRLa are particularly superior in dormant egg-inducing activity in transdermal administration to pupa.

  (3) The dormancy-inducing activity similar to Far-FGPRLa and Ger-FGPRLa was also confirmed in the peptide derivatives in which arginine (second from the C-terminal) in Far-FGPRLa and Ger-FGPRLa was substituted with lysine and histidine (Fig. Not shown).

Claims (7)

An insect diapause egg inducer comprising a peptide amide derivative, wherein the peptide amide derivative comprises an amide group at the C-terminal and an isoprenoid carboxylic acid residue at the N-terminal of a pentapeptide comprising phenylalanine, glycine, proline, arginine, and leucine diapausing eggs inducer of insects characterized by have a.   The dormant egg inducer according to claim 1, wherein the isoprenoid carboxylic acid is farnesyl acid.   The dormant egg inducer according to claim 1, wherein the isoprenoid carboxylic acid is geranilic acid.   A method for producing dormant eggs, comprising administering the dormant egg inducer of claim 2 to larvae by injection.   A method for producing dormant eggs, wherein the dormant egg inducer according to any one of claims 1 to 3 is transdermally administered to a pupa.   The method for producing a dormant egg according to claim 4 or 5, wherein the administration target of the dormant egg inducer is a silkworm.   A method for controlling pests, which comprises causing the dormant egg inducer of any one of claims 1 to 3 to act on pest larvae and willow.