CN113994846B - Application of juvenile hormone analogue methoprene in promotion of hepialus armoricanus larva rigor - Google Patents
Application of juvenile hormone analogue methoprene in promotion of hepialus armoricanus larva rigor Download PDFInfo
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- CN113994846B CN113994846B CN202111094672.XA CN202111094672A CN113994846B CN 113994846 B CN113994846 B CN 113994846B CN 202111094672 A CN202111094672 A CN 202111094672A CN 113994846 B CN113994846 B CN 113994846B
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- 229950003442 methoprene Drugs 0.000 title claims abstract description 21
- 241000330896 Thitarodes armoricanus Species 0.000 title claims abstract description 15
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- 238000011282 treatment Methods 0.000 description 4
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- CRDAMVZIKSXKFV-FBXUGWQNSA-N (2-cis,6-cis)-farnesol Chemical compound CC(C)=CCC\C(C)=C/CC\C(C)=C/CO CRDAMVZIKSXKFV-FBXUGWQNSA-N 0.000 description 1
- 239000000260 (2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-ol Substances 0.000 description 1
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- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 description 1
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- 239000003910 polypeptide antibiotic agent Substances 0.000 description 1
- NTTOTNSKUYCDAV-UHFFFAOYSA-N potassium hydride Chemical compound [KH] NTTOTNSKUYCDAV-UHFFFAOYSA-N 0.000 description 1
- 229910000105 potassium hydride Inorganic materials 0.000 description 1
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- JZRWCGZRTZMZEH-UHFFFAOYSA-N thiamine Chemical compound CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N JZRWCGZRTZMZEH-UHFFFAOYSA-N 0.000 description 1
- CRDAMVZIKSXKFV-UHFFFAOYSA-N trans-Farnesol Natural products CC(C)=CCCC(C)=CCCC(C)=CCO CRDAMVZIKSXKFV-UHFFFAOYSA-N 0.000 description 1
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G18/00—Cultivation of mushrooms
-
- A01K67/033—
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- Life Sciences & Earth Sciences (AREA)
- Mycology (AREA)
- Environmental Sciences (AREA)
- Farming Of Fish And Shellfish (AREA)
- Mushroom Cultivation (AREA)
Abstract
The invention discloses application of a juvenile hormone analogue methoprene in promoting hepialus armoricanus larva rigidization. Aiming at the problems that the hardening process of the injected and infected hepialus armoricanus larvae is time-consuming, so that the artificial culture period of the cordyceps sinensis is long, the efficiency is low and the like, the hepialus armoricanus larvae are infected by adding the juvenile hormone analogue methoprene and the cordyceps sinensis blastospores together through injection, the hardening rate (61.85 +/-3.37%) of the hepialus armoricanus larvae is improved and is remarkably higher than the hardening rate of the larvae only injected with the blastospores (48.15 +/-2.69%), and the fact that the cordyceps sinensis blastospores and methoprene are injected simultaneously can remarkably improve the hardening rate of the larvae.
Description
The technical field is as follows:
the invention belongs to the technical field of artificial cultivation of cordyceps sinensis, and particularly relates to application of a juvenile hormone analogue methoprene to promotion of stiff ghost moth larvae.
Background art:
the cordyceps sinensis is an entomogenous fungi complex formed by infecting hepialus larvae with cordyceps sinensis, and is a unique and valuable biological resource in Qinghai-Tibet plateau. With the increase of the acceptance of the cordyceps sinensis by consumers and the sharp increase of consumption, the yield of the wild cordyceps sinensis cannot meet the market demand. Through dozens of researches, the artificial cultivation of the cordyceps sinensis has made a major breakthrough, including the artificial cultivation of cordyceps sinensis sporocarp, the low-altitude scale artificial breeding of host insect bat moth and the obtaining of the cordyceps sinensis comparable to wild by infecting the bat moth larvae with the cordyceps sinensis, and the protection of plateau ecological environment and precious resources and the development of biological economy are greatly promoted. The high-efficiency production of the cordyceps sinensis is restricted by factors such as degeneration of fungi and high mortality rate of pathogenic bacteria of host larvae, low and slow infection and rigidification rate, and the factor influencing rigidification rate (namely rigidification ratio formed after gemma spores are converted into hyphae) is mainly characterized in that the efficiency of converting the gemma spores into the hyphae of the cordyceps sinensis in host insects is high.
The fungus binary type refers to the capability of some fungi that the thallus form is transformed between yeast type and hypha type under the influence of environmental factors. Cordyceps sinensis is a typical two-state fungus (blastospore-hypha), and under the induction of specific factors, more blastospores can be produced through budding growth, or slender mycelia (pseudohypha) and hyphae can be formed through apical growth. N-acetylglucosamine, proline, farnesol, tyrosol, methyl farnesyl ester, and ecdysone can promote the conversion of Cordyceps spore into hypha in vitro to different degrees. However, it has not been determined which compounds promote the rigor-ing of hepialus larvae in vivo.
Insect ecdysone (20E) and juvenile hormone participate in controlling insect metamorphosis process, influence many physiological activities such as reproduction and diapause, and are very important regulation factors of insect growth, and meanwhile, the insect hormone is also an important regulation factor of an immune system. The juvenile hormone can inhibit Phenol Oxidase (PO) and embedding function of Tenebrio molitor (Tenebrio molitor) at the same time, and also can inhibit immune related genes (such as antibacterial peptide) of Aedes aegypti female. After the cordyceps sinensis enters blood cavities of hepialus larvae, insect hormone is one of stress pressures to be borne by the cordyceps sinensis. However, it is reported whether the simultaneous injection of juvenile hormone and fungi affects the rigidness of the hepialus armoricanus larvae.
The invention content is as follows:
based on the problems, the invention aims to provide the application of the juvenile hormone analogue methoprene in promoting the rigidization of the hepialus armoricanus larvae, thereby solving the problems that the injection infection of the hepialus armoricanus rigidization is time-consuming and the period of artificially culturing cordyceps sinensis is long.
Experiments show that the hepialus larva can be infected by injecting cordyceps sinensis blastospore and juvenile hormone analogue methoprene to infect the hepialus larva, so that the infected hepialus larva can be rigidified.
Therefore, the invention can apply the juvenile hormone analogue methoprene to promote the rigor of the infected hepialus armoricanus larvae. For example, the compound can be applied to the preparation of the preparation for promoting the stiff ghost moth larvae.
The second purpose of the invention is to provide a method for promoting the rigidification of the bat moth larvae, which is characterized in that the bat moth larvae are infected by injecting cordyceps sinensis blastospores and juvenile hormone analogue methoprene simultaneously, and then the rigidification of the infected bat moth larvae is promoted in the culture process.
The cordyceps sinensis germ spores are obtained by filtering cordyceps sinensis culture solution. The inoculation dosage of blastospore is not higher than 10 per larva 7 And (4) respectively. Further preferably, the blastospore is inoculated at a dose of 1.2X 10 per larva 4 And (4) respectively.
The juvenile hormone analogue methoprene which is inoculated into the hepialus armoricanus larvae in a dosage of 20 micrograms per larva.
The instar of the hepialus armoricanus larvae is 6 years old.
Aiming at the problems that the period of artificial culture of the cordyceps sinensis is long and the efficiency is low due to the fact that the hardening process of the injected and infected hepialus armoricanus larvae is time-consuming, the hepialus armoricanus larvae are infected by adding the juvenile hormone analogue methoprene and the cordyceps sinensis blastospores together through injection, the hardening rate (61.85 +/-3.37%) of the hepialus armoricanus larvae is improved to be remarkably higher than the hardening rate of the larvae only injected with the blastospores (48.15 +/-2.69%), and the fact that the hardening rate of the larvae can be remarkably improved by simultaneously injecting the cordyceps sinensis blastospores and methoprene is demonstrated.
Description of the drawings:
FIG. 1 shows the rate of the Hepialus cuniculus larva rigescens rigidness at 8-10 deg.C for 120 days after being injected with KD, KD +20E and KD + M. Different letters above the bars indicate significant differences at the 5% level between treatments.
The specific implementation mode is as follows:
the following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example 1:
cordyceps strain Ophiocerdyceps sinensis is isolated from wild Cordyceps sinensis (No. KD) of Sichuan Kangding by tissue isolation method, and is stored at-80 deg.C after molecular identification. Cordyceps sinensis liquid medium (PMG): 200g of potato cooking juice, 20g of maltose, 10g of peptone and diphosphoric acid3g of potassium hydride, 1.5g of magnesium sulfate and vitamin B 1 0.02g and 5g of grinding fluid of fresh galleria mellonella larvae, mixing, fixing the volume to 1L by using distilled water, subpackaging into 250mL triangular bottles with each bottle being 150mL, carrying out autoclaving at 121 ℃ for 30min, and cooling for use. Placing Cordyceps sinensis block (about 0.5 cm) on clean bench 3 ) Inoculating into the liquid culture medium, culturing on a shaker at 120rpm and 9-13 deg.C for 45 days, and collecting blastospores. The method for collecting the blastospores comprises the following steps: filtering the liquid culture medium with 3 layers of sterile lens wiping paper, collecting filtrate, centrifuging in 50mL sterilized centrifuge tube at 8000rpm and 10 deg.C for 15min, discarding supernatant, resuspending with sterile phosphate buffer (PBS; pH = 7.0), centrifuging again to discard supernatant, diluting collected blastospores with sterile PBS to 6 × 10 6 one/mL is used for injecting 6 th larva of Hepialus xylostella.
The larvae of Hepialus xylostella are obtained by artificial breeding, and the larvae of 6 th instar are used for injection infection.
The juvenile hormone analogue Methoprene (Methoprene, M) (Sigma) was prepared as a solution at a concentration of 10 ug/. Mu.L using 35% DMSO as a solvent, and used after filtration through a 0.22 μ M bacterial filter (Millipore).
The following treatments and controls were performed using a microinjector (Narishige, japan): KD, injection of blastospores, injection concentration 3X 10 6 Per mL; KD +20E, the final concentration of the mixture of geminispore and ecdysone is 3X 10 6 one/mL, the latter concentration is 5 μ g/μ L; KD + M, the final concentration of the mixture of geminispore and methoprene is 3 multiplied by 10 6 seed/mL, the latter concentration being 5. Mu.g/. Mu.L; 20E, injecting ecdysone with the concentration of 5 mug/muL; m, injecting methoprene into the mixture, wherein the concentration of the methoprene is 5 mug/muL; h, no injection. Injecting into artificially-fed 6-instar larvae of Hepialus xylostella; the injection volume for each larva was 4. Mu.L. Culturing the injected hepialus larva in 8-10 deg.C room; 900 larvae were set up for each treatment.
The results showed that the larvae did not become rigid in the 20E, M, H groups because no blastospores were injected. While larvae injected with three treatment groups of KD, KD +20E, and KD + M began to catalepsy at 90 days, with catalepsy rates exceeding 40% at 120 days (fig. 1). The larva rigidness rate (61.85 +/-3.37%) of KD + M injection is obviously higher than that of KD (48.15 +/-2.69%) and KD +20E (41.48 +/-4.00%) injection, which shows that the larva rigidness rate can be obviously improved by simultaneously injecting cordyceps sinensis blastospores and methoprene.
Claims (1)
1. A method for promoting the stiff of bat moth larvae is characterized in that cordyceps sinensis blastospores and methoprene are infected with the bat moth larvae together; the cordyceps sinensis blastospore is obtained by filtering cordyceps sinensis culture solution, and the inoculation dosage of the blastospore is 1.2 multiplied by 10 for each larva 4 And each larva of the methoprene is inoculated with 20 mug, and the instar of the hepialus armoricanus is 6 years old.
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| CN102283022A (en) * | 2011-07-22 | 2011-12-21 | 重庆市中药研究院 | Method for isolating Cordyceps sinensis Sacc. asexual-stage spawn |
| CN104381011B (en) * | 2014-09-30 | 2017-06-06 | 中山大学 | A kind of preparation of aweto strain material that activity is infected with height and the method for infecting hook Hepialus larva |
| CN108094047B (en) * | 2017-12-29 | 2020-08-14 | 谢远泰 | Strain suitable for infecting parent strain with hepialus armoricanus larvae and cordyceps militaris cultivation method |
| CN109618811B (en) * | 2018-12-25 | 2021-03-23 | 长兴制药股份有限公司 | Industrialized artificial cultivation method for cordyceps sinensis |
| CN109952912B (en) * | 2019-03-08 | 2021-06-15 | 广东省科学院动物研究所 | Method for obtaining cordyceps sinensis by infecting host insect hepialus larva with hirsutella sinensis |
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