Method for cultivating trichogramma ostriniae in vitro

Abstract

The invention discloses a method for cultivating trichogramma ostriniae in vitro, and belongs to the field of insect feeding. The artificial host ovum comprises a sealed hemisphere prepared from a plastic penetrating material, an oviposition attractant coated on the outer convex surface of the hemisphere and an internal artificial culture solution, the artificial culture solution is a tussah egg solution. According to the method, the tussah egg liquid is used for cultivating the trichogramma ostriniae, and the effect of reducing the breeding cost can be achieved. The body length of the trichogramma ostriniae cultivated by the method is increased by about 21.4% compared with that of trichogramma ostriniae bred by rice moth eggs; the average brood amount is increased by about 24.9%; the parasitic rate is obviously higher than that of trichogramma ostriniae bred by rice moth eggs. The biological control effect on the trichogramma ostriniae is remarkably improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of insect rearing, in particular to a method for in vitro culturing of Trichogramma ostriniae. BACKGROUND

[0002] Ostrinia furnacalis is one of the most destructive pests in the corn industry in China, with a wide distribution and causing significant threats to corn yield and quality. Among the biological control methods, Trichogramma ostriniae is the most important Trichogramma species for controlling O. furnacalis in China, with high parasitism fitness, wide suitable egg age range, and strong parasitism ability. However, unlike Trichogramma dendrolimi, which can utilize Antheraea pernyi eggs for factory-scale reproduction, T. ostriniae cannot currently be efficiently mass-produced based on A. pernyi eggs, resulting in high reproduction costs and low production efficiency. Therefore, T. dendrolimi is still mainly used as the Trichogramma species for controlling O. furnacalis in most regions of China.

[0003] Studies have shown that parasitoid size often increases with the increase in host egg size, and larger Trichogramma individuals usually have better environmental adaptability and parasitism performance. Currently, A. pernyi eggs are commonly used as intermediate hosts for large-scale reproduction of Trichogramma in China, with advantages such as low cost, high reproduction efficiency, and easy transportation. Compared to T. dendrolimi reproduced using Corcyracephalonica eggs, T. dendrolimi reproduced based on A. pernyi eggs has larger body size, higher emergence rate, and higher female ratio, and also exhibits good parasitism ability on multiple pest eggs. However, A. pernyi eggs are not suitable for reproduction of T. ostriniae, thereby limiting the practical application of this species. T. ostriniae is more suitable for reproduction using small eggs (such as C. cephalonica eggs), but this method is costly in terms of manpower and material resources. Therefore, finding a new type of intermediate host to replace small eggs and improving the reproduction of T. ostriniae has become a technical problem that needs to be solved urgently.

[0004] In the large-scale breeding of Trichogramma wasps, the supply and cost of host eggs restrict their widespread adoption. Therefore, researchers have conducted studies on using in vitro culture technology to replace insect eggs for large-scale breeding. Since the successful in vitro culture of *Trichogramma pretiosum* using hemolymph from *Helicoverpa armigera* larvae in 1975, various artificial egg solution formulas with insect hemolymph as the main component have been developed. Lü Xin et al. optimized the in vitro culture system for *Trichogramma pretiosum* by adding a certain proportion of Ganoderma lucidum polysaccharide and chitosan oligosaccharide solution, reducing the proportion of *Trichogramma pretiosum* pupa hemolymph in the artificial egg solution to 30%. Wang Suqin et al. developed an artificial egg formula consisting of 30% *Trichogramma pretiosum* pupa hemolymph, 26% milk, 14% egg yolk, and 30% Nissl mixed saline solution, enabling *Trichogramma pretiosum* to complete its entire developmental cycle within artificial eggs. However, due to the difficulty in obtaining large quantities of insect hemolymph, recent research has gradually shifted towards artificial feeds that do not contain insect substances. Previous studies have shown that artificial feeds containing no insect matter, such as chicken embryo extract, egg yolk, milk, and yeast hydrolysate, can support the development of Trichogramma pine borer wasps from egg to adult, but the emergence rate remains low. Currently, artificial culture media suitable for Trichogramma wasps still largely rely on insect hemolymph or complex artificial feeds, while a highly efficient artificial breeding method for Trichogramma corn borer wasps that does not contain insect hemolymph has not yet been achieved. Summary of the Invention

[0005] The purpose of this invention is to provide a method for in vitro culture of Trichogramma corn borer to solve the problems existing in the prior art. This invention provides an artificial host egg for in vitro culture of Trichogramma corn borer that can reduce the cost of industrial production of Trichogramma corn borer and improve the parasitic ability of Trichogramma corn borer. Using the method of this invention, larger and more parasitic Trichogramma corn borer can be cultured.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides an artificial host egg for in vitro culture of Trichogramma corn borer wasp. The artificial host egg comprises a sealed hemisphere made of a plastic permeable material, an oviposition attractant coated on the convex surface of the hemisphere, and an artificial culture medium inside; the artificial culture medium is tussah silkworm egg fluid.

[0008] Preferably, the plastic permeable material includes a polymer film; the polymer film includes a sealing film.

[0009] Preferably, the method for preparing the tussah silkworm egg liquid includes the following steps: taking newly emerged female tussah silkworm moths, cutting open their abdomens to remove the eggs, washing and rinsing to remove impurities and immature eggs, soaking and disinfecting, drying, selecting plump tussah silkworm eggs, and sucking out the egg liquid.

[0010] Preferably, the oviposition attractant is a polyvinyl alcohol solution; the mass percentage concentration of the polyvinyl alcohol solution is 10% to 30%.

[0011] Preferably, the mass percentage concentration of the polyvinyl alcohol solution is 20%.

[0012] Preferably, the artificial host egg is a sealed hemisphere with a diameter of 2-3.5 mm, a height of 2-3.5 mm, and containing 2 μL or 7 μL of artificial culture solution.

[0013] Preferably, the artificial host egg is a sealed hemisphere with a diameter of 2 mm, a height of 2 mm, and containing 2 μL of artificial culture solution.

[0014] The application also provides a method for in-vitro culturing of Trichogramma ostriniae.

[0015] The application also provides an application of the method for in-vitro culturing of Trichogramma ostriniae in the breeding of natural enemies.

[0016] The application also provides an application of the artificial host egg in the in-vitro culturing of Trichogramma ostriniae. Trichogramma ostriniae that have been hatched for 24 hours are placed in a container with a lid, with the hemispherical convex surface facing the collection box, the container lid is buckled, and parasitization is performed for 24 hours. Development is performed in a light incubator with a temperature of 25±1℃, a relative humidity of 70±5%, and a light cycle of 16:8, and hatching is completed.

[0017] The application discloses the following technical effects:

[0018] The application first constructs an artificial host egg system based on egg liquid of Antheraea pernyi and successfully realizes in-vitro breeding of Trichogramma ostriniae. Experimental data (Tables 1-3) show that the biological performance of the system is improved. In terms of morphological development, the average body length of Trichogramma ostriniae cultured by the artificial host egg is 638.67 μm, which is significantly increased by 21.4% compared with the control group of eggs of Galleria mellonella; in terms of reproductive potential, ovarian dissection shows that the average number of eggs carried is increased to 65.43 per female, which is increased by 24.9% compared with the control group; and in terms of parasitic efficiency, the average number of eggs of Galleria mellonella parasitized by a single female is 87.27, which is increased by 45.1% compared with the control group. The application breaks through the limitation that traditional egg liquid of Antheraea pernyi is not suitable for breeding of Trichogramma ostriniae, realizes synchronous improvement of biological efficiency of Trichogramma ostriniae and reduction of production cost through biological material innovation and process optimization, and provides a new and efficient tool for biological control of corn borer. The core innovation point of the application is to combine the low-cost advantage of egg liquid of Antheraea pernyi with the controllability of artificial host eggs, and the technical bottleneck of large-scale breeding of the bee is solved. BRIEF DESCRIPTION OF DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present application, and for those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0020] Figure 1 Comparison chart of average body length of Trichogramma ostriniae bred in corn earworm eggs of artificial host of Example 1 and Trichogramma ostriniae bred in corn earworm eggs of Comparative Example 1. DETAILED DESCRIPTION

[0021] The various illustrative embodiments of the present application will now be described in detail below. The detailed description is made with reference to the drawings, in which like reference numerals are used to refer to like elements throughout. The detailed description is made with reference to the drawings, in which like reference numerals are used to refer to like elements throughout. The detailed description is made with reference to the drawings, in which like reference numerals are used to refer to like elements throughout.

[0022] It should be understood that the terms used in the present application merely describe particular embodiments and are not intended to limit the present application. In addition, for the numerical ranges in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range between any stated value or stated range, and any other stated value or intermediate value in the stated range, is also encompassed within the present application. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All publications mentioned in this specification are herein incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any reference in this specification is not an admission that it is prior art.

[0024] Many modifications and variations of this application specification can be made in light of the above teachings without departing from the spirit or scope of the application. Other implementations of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only.

[0025] As used herein, the terms "comprise", "comprising", "including", "include", "contain", "containing", "have", "having" or variants thereof are open-ended, and include the stated item or items and equivalents thereof.

[0026] Example 1

[0027] The artificial host eggs containing 2 μL of egg liquid of the silkworm moth were used to cultivate the Trichogramma ostriniae in vitro, and the method was as follows:

[0028] A piece of 10.2 cm wide, 17.7 cm long, 127 μm thick sealing film was taken, and 30 hemispherical protrusions with a diameter of 2 mm and a height of about 2 mm were pressed out on a 96-well PCR plate by pressing a double-circle washing eye needle with a diameter of 2 mm. A polyvinyl alcohol solution with a concentration of 20% was coated on the surface of each protrusion. The egg liquid of the silkworm moth was obtained by dissecting the abdomen of the newly hatched female silkworm moth, repeatedly washing and removing impurities and immature eggs with water, immersing in 0.1% new jieer disinfectant for 10 min, drying, selecting the silkworm eggs with full appearance, irradiating under ultraviolet light for 30 min in a clean bench, and then pricking the silkworm eggs and sucking out the egg liquid with a pipette tip. 2 μL of the egg liquid of the silkworm moth was injected into each hemisphere, and a small piece of sealing film was used to cover and seal each hemisphere. The test tube containing the Trichogramma ostriniae that had hatched for about 24 hours was placed in a fresh-keeping box (length 13.6 cm, width 9.6 cm, height 5 cm) with a cover, and the hemispherical protrusion was placed towards the collection box. The cover of the fresh-keeping box was buckled, and the parasitization was performed for 24 h. The development was carried out in a light incubator with a temperature of (25±1) ℃, a relative humidity of (70±5) %, and a light cycle of 16:8. After 5 days of development, the number of parasitized artificial host eggs was counted, and the number of Trichogramma ostriniae in each artificial host egg was counted. 30 adult bees developed in the artificial host eggs were randomly selected, and the body length was measured.

[0029] Results statistics: After 5 days of development, the number of parasitized artificial host eggs was 20 out of 30 artificial host eggs; the number of Trichogramma ostriniae in the artificial host eggs was 664.

[0030] Example 2

[0031] The artificial host eggs containing 7 μL of egg liquid of the silkworm moth were used to cultivate the Trichogramma ostriniae in vitro, and the method was as follows:

[0032] Take a piece of 10.2 cm wide, 17.7 cm long, 127 μm thick sealing film, press 30 hemispherical protrusions with a diameter of 3.5 mm and a height of about 3.5 mm on a 96-well PCR plate with a 3.5 mm diameter round rod-shaped brush. Apply a polyvinyl alcohol solution with a concentration of 20% on the surface of each protrusion. The egg liquid of the oak silkworm egg is taken from the newly hatched female oak silkworm moth, and the egg is taken by laparotomy. Wash repeatedly with clean water to remove impurities and immature eggs, soak in 0.1% new jieer disinfectant for 10 minutes, dry, select full- shaped oak silkworm eggs, and irradiate under ultraviolet light for 30 minutes in a clean bench. The oak silkworm egg is pierced and the oak silkworm egg liquid is sucked out with a pipette tip. 7 μL of oak silkworm egg liquid is injected into each hemisphere. Cut a small piece of sealing film to cover and seal each hemisphere. Place the test tube containing the corn borer trichogramma that has been hatched for about 24 hours into a covered fresh-keeping box (length 13.6 cm, width 9.6 cm, height 5 cm), with the hemispherical protrusion facing the collection box. Put on the fresh-keeping box cover, parasitize for 24 hours, and develop in a light incubator with a temperature of (25±1) ℃, a relative humidity of (70±5) %, and a light cycle of 16:8. After 5 days, count the number of parasitized artificial host eggs and the number of corn borer trichogramma in each artificial host egg.

[0033] Results statistics: After 5 days of development, the number of parasitized artificial host eggs: 14 artificial host eggs containing 7 μL of oak silkworm egg liquid were parasitized; the number of corn borer trichogramma parasitized in artificial host eggs: a total of 409 corn borer trichogramma were parasitized.

[0034] According to the statistical results of example 1 and example 2, the corn borer trichogramma of example 1 with better in vitro cultivation effect is selected as the further research object. Randomly select 30 head artificial host eggs developed into adult bees, measure the body length, and count the average body length, the results are shown in table 1. Dissect the ovary of corn borer trichogramma, count the average egg load of corn borer trichogramma bred in artificial host eggs, the results are shown in table 2.

[0035] Comparative example 1

[0036] The corn borer trichogramma is bred with rice moth eggs, and the method is as follows: the artificial host eggs in example 1 are replaced with natural rice moth eggs, and the other culture conditions remain the same as in example 1. Randomly select 30 rice moth eggs bred into adult bees, measure the body length. The results are shown in table 1. Dissect the ovary of corn borer trichogramma, count the average egg load of corn borer trichogramma bred in rice moth eggs, the results are shown in table 2.

[0037] Table 1 Measurement results of body length of artificial host eggs and rice moth eggs bred into adult bees

[0038] Table 2 Average egg load of corn borer trichogramma bred in artificial host eggs and rice moth eggs

[0039] As shown in Table 1, the average body length of corn borer Trichogramma was 638.67 μm, which was significantly higher than that of the egg rearing control group, with an absolute increase of 112.67 μm and a relative increase of 21.4% (P<0.05). Figure 1 Ovary dissection data (Table 2) further showed that the average number of eggs per female in the artificial host egg group was 65.43, which was 24.9% higher than that of the control group. The above results showed that the artificial host egg system of tussah egg liquid could simultaneously promote the morphological development and reproductive potential of Trichogramma.

[0040] Example 3

[0041] Single female Trichogramma was introduced into a finger tube within 12 h of initial emergence, and an egg card made of about 200 eggs of the rice moth was provided, and then placed in an incubator for parasitization. The parasitic number of Trichogramma was investigated, and the Trichogramma reared on rice moth eggs was used as a control, with 30 replicates in each group. The results are shown in Table 3.

[0042] Table 3 Parasitic number of Trichogramma reared on artificial host eggs and rice moth eggs

[0043] Table 3 data showed that, compared with the traditional rice moth egg host, the Trichogramma reared on artificial host eggs of tussah egg liquid showed a significant advantage in parasitic ability. The average parasitic amount of individuals in the experimental group was 87.27 rice moth eggs, which was 45.1% higher than that of the control group. This result showed that the artificial host egg system could significantly improve the biological efficiency of Trichogramma.

[0044] Further analysis showed that when the volume parameter of the artificial host egg was set to 2 μL, not only could a higher parasitic rate per egg be achieved, but also the production efficiency of large-scale rearing was significantly improved. The research results provide an important experimental basis for optimizing the artificial rearing technology system of Trichogramma.

[0045] The above-described embodiments are only preferred modes of the present application and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. An artificial host egg of Trichogramma ostriniae reared in vitro, characterized in that, The artificial host egg comprises a sealed hemisphere made of a plastic penetrable material, an oviposition attractant coated on the outer convex surface of the hemisphere, and an internal artificial culture solution; the artificial culture solution is a egg solution of a tussock moth egg.

2. The artificial host egg of claim 1, wherein The plastic penetrable material comprises a polymer material film; the polymer material film comprises a sealing film.

3. The artificial host egg of claim 1, wherein The preparation method of the tussock moth egg solution comprises the following steps: taking newly emerged female tussock moths, opening the abdomen to take eggs, cleaning and removing impurities and immature eggs, soaking and disinfecting, drying, selecting full-featured tussock moth eggs, and sucking out the egg solution.

4. The artificial host egg of claim 1, wherein the artificial host egg is a chicken egg. The oviposition attractant is a polyvinyl alcohol solution; the mass percentage concentration of the polyvinyl alcohol solution is 10%-30%.

5. The artificial host egg of claim 4, wherein the at least one of the plurality of artificial host egg components is a yolk component. The mass percentage concentration of the polyvinyl alcohol solution is 20%.

6. The artificial host egg of claim 1, wherein the artificial host egg is a chicken egg. The artificial host egg comprises a sealed hemisphere with a diameter of 2-3.5 mm, a height of 2-3.5 mm, and 2 μL or 7 μL of the artificial culture solution.

7. The artificial host egg of claim 1, wherein the artificial host egg is a chicken egg. The artificial host egg is a sealed hemisphere with a diameter of 2 mm, a height of 2 mm, and 2 μL of the artificial culture solution.

8. A method of rearing in vitro Trichogramma nubilale (Hagen) (Hymenoptera: Trichogrammatidae), characterized by, It comprises the step of culturing corn borer Trichogramma in vitro by using the artificial host egg according to any one of claims 1-7.

9. The use of the method for culturing corn borer Trichogramma in vitro according to claim 8 in the breeding of natural enemies.

10. Use of the artificial host egg according to any one of claims 1 to 7 for in vitro rearing of Trichogramma ostriniae, characterized in that, The newly emerged corn borer Trichogramma is placed in a container with a cover, the convex surface of the hemisphere faces the collection box, the cover of the container is buckled, parasitization is performed for 24 h, and development is performed in a light incubator with a temperature of 25±1 ℃, a relative humidity of 70±5%, and a light cycle of 16:8, and the newly emerged corn borer Trichogramma is obtained.

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