CN112119980A - Low-temperature propagation method for in vitro culture of trichogramma - Google Patents
Low-temperature propagation method for in vitro culture of trichogramma Download PDFInfo
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- CN112119980A CN112119980A CN202010894430.8A CN202010894430A CN112119980A CN 112119980 A CN112119980 A CN 112119980A CN 202010894430 A CN202010894430 A CN 202010894430A CN 112119980 A CN112119980 A CN 112119980A
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- trichogramma
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- temperature
- eggs
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- 241000256618 Trichogramma Species 0.000 title claims abstract description 49
- 238000000034 method Methods 0.000 title claims abstract description 18
- 238000000338 in vitro Methods 0.000 title claims abstract description 17
- 235000013601 eggs Nutrition 0.000 claims abstract description 87
- 238000009395 breeding Methods 0.000 claims abstract description 20
- 230000001488 breeding effect Effects 0.000 claims abstract description 16
- 230000032669 eclosion Effects 0.000 claims abstract description 9
- 238000004519 manufacturing process Methods 0.000 claims abstract description 8
- 238000012258 culturing Methods 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 5
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims description 3
- 239000004743 Polypropylene Substances 0.000 claims description 3
- 239000004372 Polyvinyl alcohol Substances 0.000 claims description 3
- 241000382353 Pupa Species 0.000 claims description 3
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 3
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 3
- 239000007864 aqueous solution Substances 0.000 claims description 3
- 239000002131 composite material Substances 0.000 claims description 3
- 239000011521 glass Substances 0.000 claims description 3
- 210000000087 hemolymph Anatomy 0.000 claims description 3
- 229920001155 polypropylene Polymers 0.000 claims description 3
- 229920002451 polyvinyl alcohol Polymers 0.000 claims description 3
- 239000012266 salt solution Substances 0.000 claims description 3
- 238000007789 sealing Methods 0.000 claims description 3
- 235000020183 skimmed milk Nutrition 0.000 claims description 3
- 239000008223 sterile water Substances 0.000 claims description 3
- 230000001954 sterilising effect Effects 0.000 claims description 3
- 238000004659 sterilization and disinfection Methods 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 239000012530 fluid Substances 0.000 claims 1
- 241000186214 Trichogramma dendrolimi Species 0.000 abstract description 17
- 241000257303 Hymenoptera Species 0.000 abstract description 2
- 230000000694 effects Effects 0.000 abstract description 2
- 238000002474 experimental method Methods 0.000 description 4
- 102000002322 Egg Proteins Human genes 0.000 description 3
- 108010000912 Egg Proteins Proteins 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 241000121220 Tricholoma matsutake Species 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 230000002035 prolonged effect Effects 0.000 description 3
- 241000238631 Hexapoda Species 0.000 description 2
- 238000010162 Tukey test Methods 0.000 description 2
- 241000607479 Yersinia pestis Species 0.000 description 2
- 230000006978 adaptation Effects 0.000 description 2
- 210000002969 egg yolk Anatomy 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 210000004681 ovum Anatomy 0.000 description 2
- 230000003071 parasitic effect Effects 0.000 description 2
- 230000019617 pupation Effects 0.000 description 2
- 241000255789 Bombyx mori Species 0.000 description 1
- 241000223238 Trichophyton Species 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 235000013345 egg yolk Nutrition 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000001678 irradiating effect Effects 0.000 description 1
- 239000002655 kraft paper Substances 0.000 description 1
- 239000004816 latex Substances 0.000 description 1
- 229920000126 latex Polymers 0.000 description 1
- 235000013348 organic food Nutrition 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 230000001902 propagating effect Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Zoology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Farming Of Fish And Shellfish (AREA)
Abstract
The invention provides a low-temperature propagation method for in vitro culture of trichogramma. The method comprises the following steps: and (3) manufacturing an artificial egg card, placing the artificial egg card in a biological incubator, then parasitizing the trichogramma in the artificial egg, and placing the egg card with the trichogramma at 16-23 ℃ for culturing until the imago eclosion occurs. The trichogramma cultured by the method has longer delay time than that of natural trichogramma (trichogramma dendrolimi parasitizing tussah eggs). The method is more convenient for field application, prolongs the shelf life and reduces the cost on the basis of not influencing the quality and the application effect of the breeding bees.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a low-temperature propagation method for in vitro culture of trichogramma.
Background
The Trichogramma is an important egg parasitic natural enemy insect, has rich resources, wide distribution, large application area and many pest control objects, and plays an important role in biological pest control in grain safety production, pollution-free food production and organic food production. The inoculation and propagation of trichogramma is the key of biological control, and the most widely applied method at present is to carry out massive indoor propagation by utilizing tussah eggs, so that a great amount of manpower and material resources are consumed, the mass production application is limited by factors such as time, regions, transportation and the like, the production cost is high, and the problem of insufficient supply exists. Therefore, the low-temperature breeding method for breeding the trichogramma in vitro has important significance in prolonging the shelf life and reducing the breeding cost.
Disclosure of Invention
The invention aims to provide a low-temperature breeding method for breeding trichogramma in vitro, which can select different low-temperature adjusting breeding time according to practical application requirements when breeding the trichogramma in large quantity by utilizing artificial eggs, thereby prolonging the shelf life and reducing the breeding cost.
In order to achieve the purpose, the invention adopts the following technical scheme:
a low-temperature breeding method for in vitro breeding trichogramma is characterized in that trichogramma is parasitized in artificial eggs and cultivated at the temperature of 16-23 ℃.
Specifically, the low-temperature propagation method for in vitro culture of trichogramma includes the following steps: and (3) manufacturing an artificial egg card, placing the artificial egg card in a biological incubator, then parasitizing the trichogramma in the artificial egg, and placing the egg card with the trichogramma at 16-23 ℃ for culturing until the imago eclosion occurs.
Further, the egg liquid of the artificial egg comprises: 3mL of tussah pupa hemolymph, 0.1g of trehalose, 2.5mL of eggs (taking egg yolk), 1mL of 10 wt% skimmed milk, 1mL of NS salt solution and 1.5mL of sterile water.
The preparation steps of the artificial egg card are as follows: a polyethylene-polypropylene composite film with the thickness of 30 mu m is adopted, 20 hemispherical artificial eggs (the diameter is 2-3mm and the height is 2-3mm) are pressed on the film with the size of 8 x 7cm through a special mould and a warm glass rod, and the film is irradiated under ultraviolet light for 30min for sterilization. Under aseptic condition, 4 μ L trichogramma culture solution is injected into each ovum to make the hemispherical convex surface outside, and each edge of the film is sealed by a sealing machine. After the egg card is prepared, a 10% polyvinyl alcohol aqueous solution is coated on the surface of the egg card by using a soft brush pen to induce trichogramma to lay eggs. Each temperature treatment corresponded to 1 artificial egg card, which was repeated 3 times per treatment.
The artificial egg card is placed in a biological incubator at the temperature of 26-28 ℃, the relative humidity of air is 70-80%, and the photoperiod is L: D: 16:8 h; the trichogramma parasitizing in the artificial eggs is to culture the trichogramma parasitizing in the artificial eggs for 24 hours, and then place the egg cards with the trichogramma in a biological incubator with the temperature of 16-23 ℃, the relative air humidity of 70-80% and the photoperiod L: D: 16:8 hours until the imago eclosion.
The trichogramma parasitizing in the artificial eggs is that the trichogramma parasitizes in the artificial eggs according to the bee-egg ratio of 8: 1.
Compared with the prior art, the invention has the advantages that: the invention provides a low-temperature propagation method for in vitro cultivation of Trichogramma, which is characterized in that Trichogramma dendrolimi (Trichogramma dendrolimi) is taken as an object, artificial eggs are taken as host materials, the Trichogramma dendrolimi is parasitized in the artificial eggs, cultivation is carried out at the temperature of 16-23 ℃ until imagoes eclosion occurs, and the result shows that compared with a control group in which the Trichogramma dendrolimi parasitizes in the tussah eggs, the Trichogramma cultivated by the method has longer delay time than the natural Trichogramma parasitizing the tussah eggs. The method is more convenient for field application, prolongs the shelf life and reduces the cost on the basis of not influencing the quality and the application effect of the breeding bees.
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example 1
1. Experimental Material
Insects: trichogramma dendrolimi (Trichogramma dendrolimi).
Host: artificial eggs and tussah eggs.
The formula of the egg liquid of the artificial egg comprises the following components: 3mL of tussah pupa hemolymph, 0.1g of trehalose, 2.5mL of eggs (yolk is taken), 1mL of 10 wt% skimmed milk, 1mL of NS salt solution and 1.5mL of sterile water, and the components are uniformly mixed to obtain the egg liquid of the artificial eggs.
The NS saline solution is a physiological saline solution (0.9% aqueous sodium chloride solution).
2. The experimental method comprises the following steps: the experiment set 4 low temperature, 23 + -1 deg.C, 20 + -1 deg.C, 16 + -1 deg.C and 13 + -1 deg.C, control temperature is 27 + -1 deg.C. The artificial egg card is prepared by using the tussah egg card and the optimized trichogramma culture solution. Wherein, (1) the tussah egg card preparation method: sticking 20 tussah eggs on kraft paper coated with white latex to prepare a tussah egg card; each temperature treatment corresponds to 1 tussah egg card, and each treatment is repeated for 3 times. (2) The preparation method of the artificial egg card comprises the following steps: adopting a polyethylene-polypropylene composite film with the thickness of 30 mu m, pressing 20 hemispherical artificial eggs (the diameter is 2-3mm and the height is 2-3mm) on the film with the size of 8 multiplied by 7cm by a special mould and a warm glass rod, and irradiating the film for 30min under ultraviolet light for sterilization; under aseptic condition, 4 μ L trichogramma culture solution is injected into each ovum to make the hemispherical convex surface outside, and each edge of the film is sealed by a sealing machine. After the egg card is prepared, a 10% polyvinyl alcohol aqueous solution is coated on the surface of the egg card by using a soft brush pen to induce the trichogramma dendrolimi to lay eggs. Each temperature treatment corresponded to 1 artificial egg card, which was repeated 3 times per treatment. Placing the egg cards in a biological incubator with the temperature of 27 +/-1 ℃, the humidity of 75 +/-5% RH and the photoperiod of 16:8h (L: D), allowing trichogramma dendrolimi to parasitize on artificial eggs and tussah eggs according to the ratio of 8:1 (namely 8 trichogramma dendrolimi trichodermalis connected to 1 parasitic egg), after 24 hours of bee connection, respectively placing the egg cards connected with the trichogramma dendrolimi into 5 setting temperatures, and developing in the biological incubator with the relative humidity of air of 70-80% and the photoperiod of L: D of 16:8h until the imagoes eclosion. The breeding quality of the vitro cultured trichogramma dendrolimi in different low temperatures is inspected from biological parameters such as pupation rate, eclosion rate, female proportion, normal bee quantity and the like. The experiment was set up in 3 replicates.
3. Results of the experiment
Table 1 shows the breeding quality parameters of the trichogramma dendrolimi bred by the artificial eggs at the set temperature. As shown in Table 1, the development time of the Trichophyton matsutake bred in artificial eggs at 13 deg.C, 16 deg.C, 20 deg.C and 23 deg.C was prolonged by 21 days, 17 days, 8 days and 5 days, respectively, as compared with the optimum temperature (27 deg.C). The pupation rate, the eclosion rate, the female proportion and the normal bee quantity difference between 16 ℃, 20 ℃, 23 ℃ and 27 ℃ are not obvious. The development quality of the artificial bee at the temperature of 13 ℃ is lower than that of other test temperatures. The artificial in vitro cultured trichogramma can be propagated at the low temperature of 16-23 ℃ without affecting the quality.
TABLE 1 reproduction of Tricholoma matsutake in vitro at different temperatures
The same capital letters after standard error of the same column mean indicate that the difference was not significant (Tukey's test: P >0.05).
Table 2 shows the breeding quality parameters of trichogramma dendrolimi bred from tussah eggs at the set temperature. As shown in Table 2, the quality of the trichogramma dendrolimi bred by the tussah silkworm eggs is equivalent to that of the artificial eggs, but the development time is respectively prolonged by 20 days, 15 days, 6 days and 3 days under the conditions of 13 ℃, 16 ℃, 20 ℃ and 23 ℃, and is shorter than that of the artificial egg culture. It can be seen that the trichogramma cultured by the method of the present invention has a longer delay time than that of the natural world (trichogramma dendrolimi parasitizing the tussah egg) compared to the control group parasitizing the trichogramma dendrolimi to the tussah egg. Therefore, when the artificial eggs are used for propagating the trichogramma in large quantities, different low temperatures can be selected to adjust the culture time according to the actual application requirements, the shelf life is prolonged, and the propagation cost is reduced.
TABLE 2 reproduction of Tricholoma matsutake (L.) Kuntze parasitizing tussah eggs at different temperatures
The same capital letters after standard error of the same column mean indicate that the difference was not significant (Tukey's test: P >0.05).
The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should be considered within the scope of the invention.
Claims (6)
1. A low-temperature breeding method for in vitro breeding trichogramma is characterized in that the trichogramma is parasitized in artificial eggs and cultivated at the temperature of 16-23 ℃.
2. The low-temperature propagation method for breeding trichogramma in vitro as claimed in claim 1, comprising the steps of: and (3) manufacturing an artificial egg card, placing the artificial egg card in a biological incubator, then parasitizing the trichogramma in the artificial egg, and placing the egg card with the trichogramma at 16-23 ℃ for culturing until the imago eclosion occurs.
3. The method for low-temperature reproduction of trichogramma in vitro culture according to claim 1 or 2, wherein the egg fluid of the artificial egg is composed of: 3mL of tussah pupa hemolymph, 0.1g of trehalose, 2.5mL of eggs, 1mL of 10 wt% skimmed milk, 1mL of NS salt solution and 1.5mL of sterile water.
4. The low-temperature propagation method for in vitro breeding trichogramma as claimed in claim 2, wherein the artificial egg card is prepared by the steps of: a polyethylene-polypropylene composite film with the thickness of 30 mu m is adopted, 20 hemispherical artificial eggs with the diameter of 2-3mm and the height of 2-3mm are pressed on the film with the size of 8 x 7cm through a special mould and a warm glass rod, the film is placed under ultraviolet light for irradiation for 30min for sterilization, 4 mu L of trichogramma culture solution is injected into each egg under the aseptic condition, the hemispherical convex surface is exposed, each side of the film is sealed by a sealing machine, after an egg card is manufactured, 10% polyvinyl alcohol aqueous solution is coated on the surface of the egg card by a soft brush pen to induce the trichogramma to lay eggs, each temperature treatment corresponds to 1 artificial egg card, and each treatment is repeated for 3 times.
5. The low-temperature propagation method for in vitro culture of trichogramma as claimed in claim 2, wherein the temperature of the artificial egg card in the biological incubator is 26-28 ℃, the relative humidity of air is 70-80%, and the photoperiod is L: D: 16:8 h; the trichogramma parasitizing in the artificial eggs is to culture the trichogramma parasitizing in the artificial eggs for 24 hours, and then place the egg cards with the trichogramma in a biological incubator with the temperature of 16-23 ℃, the relative air humidity of 70-80% and the photoperiod L: D: 16:8 hours until the imago eclosion.
6. The method for low-temperature breeding of trichogramma in vitro as claimed in claim 2, wherein the trichogramma parasitizing in the artificial eggs is carried out by parasitizing the trichogramma in the artificial eggs at a ratio of 8: 1.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202010894430.8A CN112119980A (en) | 2020-08-31 | 2020-08-31 | Low-temperature propagation method for in vitro culture of trichogramma |
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| CN202010894430.8A CN112119980A (en) | 2020-08-31 | 2020-08-31 | Low-temperature propagation method for in vitro culture of trichogramma |
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| CN112119980A true CN112119980A (en) | 2020-12-25 |
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| CN202010894430.8A Pending CN112119980A (en) | 2020-08-31 | 2020-08-31 | Low-temperature propagation method for in vitro culture of trichogramma |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113598044A (en) * | 2021-08-24 | 2021-11-05 | 华中农业大学 | Method for pollinating early spring crops by domesticated insects at low temperature |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4418647A (en) * | 1982-05-20 | 1983-12-06 | The United States Of America As Represented By The Secretary Of Agriculture | Artificial host egg for rearing trichogramma |
| CN101438691A (en) * | 2007-11-23 | 2009-05-27 | 北京市农林科学院 | Mass propagation method of Oophagous trichogrammae from artificial ovum |
| KR20110005356A (en) * | 2009-07-10 | 2011-01-18 | 안동대학교 산학협력단 | A short-term storage technique of an egg parasitoid, trichogramma sp. using developmental quiescence induced by low temperature |
| CN102823550A (en) * | 2012-09-19 | 2012-12-19 | 吉林农业大学 | Light environment control method for breeding trichogramma dendrolimi with oak silkworm eggs |
| CN103859210A (en) * | 2014-02-20 | 2014-06-18 | 广东省昆虫研究所 | Artificial medium of trichogramma |
-
2020
- 2020-08-31 CN CN202010894430.8A patent/CN112119980A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4418647A (en) * | 1982-05-20 | 1983-12-06 | The United States Of America As Represented By The Secretary Of Agriculture | Artificial host egg for rearing trichogramma |
| CN101438691A (en) * | 2007-11-23 | 2009-05-27 | 北京市农林科学院 | Mass propagation method of Oophagous trichogrammae from artificial ovum |
| KR20110005356A (en) * | 2009-07-10 | 2011-01-18 | 안동대학교 산학협력단 | A short-term storage technique of an egg parasitoid, trichogramma sp. using developmental quiescence induced by low temperature |
| CN102823550A (en) * | 2012-09-19 | 2012-12-19 | 吉林农业大学 | Light environment control method for breeding trichogramma dendrolimi with oak silkworm eggs |
| CN103859210A (en) * | 2014-02-20 | 2014-06-18 | 广东省昆虫研究所 | Artificial medium of trichogramma |
Non-Patent Citations (2)
| Title |
|---|
| 山东省农业科学院植保所等: "《赤眼蜂》", 1 January 1978 * |
| 魏艳敏: "《生物农药及其应用技术问答》", 31 March 2007 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113598044A (en) * | 2021-08-24 | 2021-11-05 | 华中农业大学 | Method for pollinating early spring crops by domesticated insects at low temperature |
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Application publication date: 20201225 |