A method for non-invasive infection of Wolbachia to trichogramma by using artificial host

Abstract

This invention belongs to the field of biotechnology, specifically relating to a method for non-invasively infecting Trichogramma wasps using artificial hosts. Wolbachia The method involves first preparing artificial host egg cards, then infecting... Wolbachia Biological materials were inserted into artificial host egg cards, and finally, uninfected eggs were inserted. Wolbachia The parasitism of Trichogramma wasps is sufficient. Compared to microinjection, this method is more convenient and efficient; moreover, the open system with operable artificial hosts allows for the artificial inoculation of Trichogramma wasps with eggs from other insect sources besides parasitoid wasps. Wolbachia It provided a possibility.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for non-invasively infecting Wolbachia with artificial hosts using Trichogramma wasps. Background Technology

[0002] Wolbachia, belonging to the α subphylum of Proteobacteria, is an intracellular symbiotic bacterium widely distributed in arthropods. It plays a role in regulating host reproduction, including inducing cytoplasmic incompatibility, parthenogenesis, male killing, and feminization. Wolbachia shows broad application prospects in biological pest control. Because some Wolbachia strains can induce cytoplasmic incompatibility, releasing artificially transfected male insects with Wolbachia can result in sterility when mating with uninfected wild females, thus suppressing pest populations. Simultaneously, Wolbachia can reduce viral replication in the host and has been used to inhibit the transmission of insect-borne viruses. For example, artificially infecting Aedes albopictus with the Wolbachia wPip strain of Culex pipiens can suppress the wild population of Aedes albopictus and inhibit its transmission of mosquito-borne viruses such as dengue fever and Zika virus. Similarly, artificial transfection of the Wolbachia wStri strain, which infects the gray planthopper, into the brown planthopper can be used to suppress wild populations of the brown planthopper and significantly inhibit its transmission of rice tooth dwarf virus. Furthermore, Wolbachia can induce parthenogenesis in parasitic wasps and other insects, showing significant potential for improving the female ratio and pest control in parasitic wasps.

[0003] To date, the most common method for artificially infecting Wolbachia is through microinjection, as illustrated by patents CN202110991337.3 (a method for artificially transfecting the exogenous symbiotic bacterium Wolbachia into citrus psyllids), CN201110230149.5 (a rapid and effective method for transfecting mosquitoes with Wolbachia), and PCT / CN2017 / 085275 (Brown planthopper and production method therefor). All these methods achieve the transfection of the symbiotic bacterium Wolbachia through microinjection. First, Wolbachia is directly aspirated or extracted from the embryo or ovary of the donor insect infected with it, and then microinjected into the embryo or nymph stage of the recipient insect. However, microinjection is time-consuming, labor-intensive, difficult to perform, and has a low success rate, presenting significant technical challenges.

[0004] Trichogramma, an egg parasitic wasp belonging to the family Trichogrammae in the order Hymenoptera, is the most widely used and successful natural enemy of pests worldwide to date. Trichogramma wasps have been widely used to control major agricultural and forestry pests such as corn, rice, cotton, fruits and vegetables, and trees, achieving significant economic and ecological benefits. Among Trichogramma wasps, Wolbachia can induce parthenogenesis, and this has been reported in at least 16 species. Since only female wasps can parasitize and control pests, parthenogenesis has significant potential applications in Trichogramma wasp pest control. Previous reports indicated that Wolbachia infection of Trichogramma wasps could be achieved through microinjection. However, due to the tiny size of Trichogramma wasps (only about 0.5 mm), the survival rate of microinjection is low, posing certain technical challenges. Other reports indicate that Wolbachia can spread within and between Trichogramma wasp species by sharing the same pest host. Compared to microinjection, this method is more convenient and efficient. However, when parasitizing the same pest host, Wolbachia infection is limited to the egg parasitoids sharing the same host, and cannot transfer Wolbachia from other insects to Trichogramma wasps, such as the Wolbachia that induces parthenogenesis in Encarsia form.

[0005] There have been no reports of Trichogramma wasps using artificial hosts to non-invasively infect Wolbachia. Summary of the Invention

[0006] To address the shortcomings of existing methods, the present invention aims to provide a method for non-invasively infecting Wolbachia with Trichogramma wasps using an artificial host. Compared to microinjection, the method of the present invention is more convenient and efficient; furthermore, the open system of the artificial host allows for the artificial infection of Wolbachia from other insect sources besides egg parasitoids by Trichogramma wasps.

[0007] The technical solution adopted by this invention to solve its technical problem is:

[0008] This invention first protects a method for non-invasively infecting Wolbachia with Trichogramma wasps using artificial hosts. The method involves first preparing an artificial host egg card, then inoculating the artificial host egg card with biological material infected with Wolbachia, and finally inoculating the wasps that are not infected with Wolbachia with the wasps.

[0009] As a preferred technical solution of this application, the biological material is Trichogramma wasp or cell line.

[0010] As a preferred technical solution of this application, the method for non-invasively infecting Wolbachia with Trichogramma wasps using artificial hosts specifically includes the following steps:

[0011] (1) Preparation of artificial host egg card: Take a plastic permeable material, press out a depression; add artificial egg fluid to the depression, heat seal, and make an artificial host egg card;

[0012] (2) Trichogramma parasitizing artificial egg cards: Place the artificial host egg card in the beekeeping tube, and inoculate Trichogramma infected with Wolbachia at a ratio of approximately 10:1 to 100:1. After sufficient parasitism, remove the Trichogramma on the surface of the artificial host egg card; then, inoculate Trichogramma uninfected with Wolbachia; take out the artificial egg card that has been parasitized by Trichogramma and place it in a sterilized sterile culture dish, and then place it in a constant temperature and humidity incubator for culture until the Trichogramma emerges.

[0013] As a preferred technical solution in this application, the artificial egg fluid is either commercially purchased or prepared in-house.

[0014] As a preferred technical solution of this application, the artificial egg fluid contains one or a mixture of multiple of the following: a culture medium of insect-derived substances and a salt ion solution.

[0015] Preferably, the culture medium for the insect-derived material mainly consists of tussah silkworm pupa hemolymph, egg yolk, milk powder solution, Nathanheimer salt solution, trehalose, sterile water, and penicillin-streptomycin antibiotic solution. The mass-to-volume ratio of the tussah silkworm pupa hemolymph, egg yolk, milk powder solution, Nathanheimer salt solution, trehalose, sterile water, and penicillin-streptomycin antibiotic solution is 3-5 mL: 2-4 mL: 0.5-2 mL: 0.5-2 mL: 0.05-0.2 g: 0.05-0.2 g: 100-200 μl.

[0016] More preferably, the artificial egg solution is prepared according to the following ratio: 3 ml of silkworm pupa hemolymph, 2.5 ml of egg yolk, 1 ml of 10% milk powder solution, 1 ml of Nathanheimer salt solution, 0.1 g of trehalose, 0.1 g of sterile water, and 180 μl of penicillin-streptomycin antibiotic solution.

[0017] Preferably, the Nathanheimer salt solution is selected from one or more of KCl, NaCl, MgSO4, CaCl2, NaHCO3, and MgCl2, or a mixture thereof.

[0018] More preferably, the Nathanheimer salt solution is composed of NaCl, KCl, CaCl2, NaHCO3 and H2O, with a corresponding mass ratio of 7.5:0.1:0.2:0.2:1000.

[0019] More preferably, the Nathanheimer salt solution is prepared using 7.5g NaCl, 0.1g KCl, 20.2g CaCl2, 0.2g NaHCO3, and 1000ml distilled water.

[0020] As a preferred technical solution of this application, in step (1), the plastic permeable material is wax, Parafilm film or plastic film, preferably, the plastic film is polyethylene plastic film or polypropylene plastic film.

[0021] The plastic penetrability of the material in this application needs to be controlled between 1-100μm. If the material thickness is too thin, the strength is insufficient; if the material thickness is too thick, it cannot penetrate. The thickness is controlled between 10-30μm, and more preferably, the thickness is 20μm.

[0022] As a preferred technical solution of this application, in step (2), the culture conditions in the constant temperature and humidity incubator are: 27±1℃, RH 80±5%, and photoperiod 14L:10D.

[0023] As a preferred technical solution of this application, the parasitism time of Trichogramma wasps infected with Wolbachia and those not infected with Wolbachia is 2 to 5 hours, preferably 3 hours.

[0024] As a preferred technical solution of this application, in step (2), the petri dish is sterilized with 75% alcohol.

[0025] As a preferred technical solution of this application, in step (2), a moist cotton ball is used to keep the petri dish moist.

[0026] Beneficial effects

[0027] The present invention provides a method for non-invasively infecting Wolbachia with artificial hosts using artificial hosts, which has the following advantages compared with the prior art:

[0028] (1) The method of the present invention is more convenient and efficient than microinjection.

[0029] (2) The open system with operable artificial hosts makes it possible for Trichogramma wasps to artificially infect Wolbachia from other insect sources besides egg parasitoids.

[0030] (3) This invention patent provides a technical means for Trichogramma wasps to efficiently and conveniently transfect Wolbachia, and provides a systematic support for analyzing the scientific mechanism of Wolbachia in horizontal transmission among Trichogramma wasps. It has important application prospects in improving the female ratio and pest control of Trichogramma wasps. Attached Figure Description

[0031] Figure 1 The results are PCR detection results of the pine caterpillar Trichogramma wasp transfected with Wolbachia in Example 1.

[0032] Figure 2 The results are PCR detection results of *Trichogramma pulmonata* transfected with *Wolbachia* in Comparative Example 1. Detailed Implementation

[0033] The present invention will be further described in detail below with reference to the embodiments. Reagents or instruments used without a specified manufacturer are considered to be conventional products that can be purchased on the market.

[0034] Example 1

[0035] In this embodiment, an artificial host was used to transfect Wolbachia from T. pretiosum into T. dendrolimi, a pine caterpillar wasp.

[0036] (1) Preparation of artificial host egg cards

[0037] Take a 20μm thick polyethylene film and use a glass grinding rod to press out hemispherical indentations (2-3mm in diameter and 2-3mm in height) onto it. Apply a 10% polyvinyl alcohol aqueous solution evenly to the raised outer surface of the film with a soft brush and let it air dry in a clean bench. Then, add 4μl of artificial egg solution to the indentation in the polyethylene film. The artificial egg solution is prepared according to the following ratio: 3ml of silkworm pupa hemolymph, 2.5ml of egg yolk, 1ml of 10% milk powder solution, 1ml of Nathanheimer's salt solution, 0.1g of trehalose, 0.1g of sterile water, and 180μl of penicillin-streptomycin antibiotic solution. The Nathanheimer's salt solution is prepared using 7.5g of NaCl, 0.1g of KCl, 20.2g of CaCl2, 0.2g of NaHCO3, and 1000ml of distilled water. Take another flat polyethylene film, open all four sides, and heat-seal it to create an artificial host egg card.

[0038] (2) Trichogramma wasp parasitizing artificial egg cards

[0039] Artificial host egg cards were placed in beekeeping tubes, and infected with a *Trichogramma short-tubed* strain infected with *Trichogramma simonii* at a ratio of approximately 100:1. After 3 hours of full parasitism, the egg cards were removed, and the *Trichogramma simonii* wasps on the surface of the artificial egg cards were brushed off with a soft brush. Then, *Trichogramma pine caterpillar* wasps uninfected with *Trichogramma simonii* were introduced for 3 hours of parasitism. The artificial egg cards, which had been parasitized by *Trichogramma simonii* and *Trichogramma pine caterpillar* respectively, were removed, their surfaces were wiped with 75% alcohol, and then placed in sterile petri dishes. These dishes were then placed in a constant temperature and humidity incubator (27±1℃, RH 80±5%, photoperiod 14L:10D) until the *Trichogramma simonii* emerged as wasps.

[0040] (3) DNA extraction from Trichogramma wasps

[0041] The newly emerged Trichogramma wasps from artificial hosts were stunned using CO2, and female Trichogramma wasps were selected under a stereomicroscope based on their body color differences. Each female wasp was placed in a grinding buffer (30 μl STE buffer + 2 μl proteinase K per wasp, freshly prepared) and thoroughly ground. The grinding buffer was then incubated at 56°C for 2 hours, followed by inactivation of proteinase K at 95°C for 7 minutes to obtain a crude extract of Trichogramma wasp DNA.

[0042] (4) PCR detection in Wolbachia

[0043] Using DNA from the pine caterpillar wasp *Trichogramma pulcherrima* as a template, the *Wolbachia* *wsp* gene was amplified using WSP-FP and WSP-RP primers. The WSP-FP sequence was 5'-TGGTCCAATAAGTGATGAAGAAAC-3', and the WSP-RP sequence was 5'-AAAAATTAAACGCTACTCCA-3'. The PCR program was set to activate at 94℃ for 5 min, followed by 35 cycles of 94℃ for 30 s, 53℃ for 30 s, 72℃ for 1 min, and then extension at 72℃ for 7 min. The PCR products were then subjected to gel electrophoresis at 150V for 20 min, and the bands were observed using a UV gel imaging system.

[0044] The results are as follows Figure 1 As shown, after *Trichogramma short-tubed* and *Trichogramma pineneatus* co-parasitized the artificial host, the female *Trichogramma pineneatus* in lanes 18 and 19 were infected with *Wolbachia*. This indicates that *Wolbachia* horizontally infects *Trichogramma short-tubed* via the artificial host, with an infection rate of 10% (two out of twenty *Trichogramma pineneatus* were infected).

[0045] Example 2

[0046] In this embodiment, an artificial host was used to transfect Wolbachia from the Trichogramma deion into the Trichogramma kayakai.

[0047] (1) Preparation of artificial host egg cards

[0048] Following the method of Example 1, the difference is that a 30 μm thick polypropylene film is used, and the artificial egg solution is prepared according to the following ratio: 3 ml of silkworm pupa hemolymph, 2 ml of egg yolk, 0.5 ml of 10% milk powder solution, 0.8 ml of Nathanheimer's salt solution, 0.05 g of trehalose, 0.1 g of sterile water, and 150 μl of penicillin-streptomycin antibiotic solution. Another flat polypropylene film is then taken, and its four sides are opened and heat-sealed to prepare an artificial host egg card.

[0049] (2) Trichogramma wasp parasitizing artificial egg cards

[0050] Artificial host egg cards were placed in beekeeping tubes, and T. deion strain infected with Wolbachia wasps were introduced at a wasp-to-egg ratio of approximately 50:1. After 4 hours of full parasitism, the egg cards were removed, and the surface of the artificial egg cards was brushed clean with a soft brush. Then, uninfected T. deion wasps were introduced for another 4 hours of parasitism. The parasitized artificial egg cards were removed, their surface was wiped with 75% alcohol, and then placed in sterile petri dishes. These dishes were then placed in a constant temperature and humidity incubator (27±1℃, RH 80±5%, photoperiod 14L:10D) until the T. deion wasps emerged.

[0051] Example 3

[0052] This embodiment uses an artificial host to transfect Wolbachia from Trichogramma short-tubed wasp into Trichogramma flat-bellied wasp.

[0053] (1) Preparation of artificial host egg cards

[0054] Following the method described in Example 1, a 50 μm thick parafilm was taken, and a hemispherical indentation (2–3 mm in diameter and 2–3 mm in height) was pressed into it using a glass abrasive rod. A 10% polyvinyl alcohol aqueous solution was evenly coated onto the outer surface of the protrusion using a soft brush, and the film was then placed in a clean bench to air dry. Subsequently, 4 μl of artificial egg solution was added to the indentation in the parafilm. A separate flat polyethylene film was then taken, and its four edges were opened and heat-sealed to create an artificial host egg card. The artificial egg solution is composed of silkworm pupa hemolymph, egg yolk, milk powder solution, Nathanheimer's salt solution, trehalose, sterile water, and penicillin-streptomycin solution in a mass-to-volume ratio of 3-5 mL: 2-4 mL: 0.5-2 mL: 0.5-2 mL: 0.05-0.2 g: 0.05-0.2 g: 100-200 μl; the Nathanheimer's salt solution consists of NaCl, KCl, CaCl2, NaHCO3, and H2O in a mass ratio of 7.5: 0.1: 0.2: 0.2: 1000.

[0055] The formula can be adjusted according to actual conditions. For example, 3mL of silkworm pupa hemolymph, 2mL of egg yolk, 0.5ml of 10% milk powder solution, 0.5ml of Nathanheimer's salt solution, 0.1g of trehalose, 0.1g of sterile water, and 100μl of penicillin-streptomycin antibiotic solution; or 4mL of silkworm pupa hemolymph, 4mL of egg yolk, 1ml of 10% milk powder solution, 2ml of Nathanheimer's salt solution, 0.05g of trehalose, 0.2g of sterile water, and 200μl of penicillin-streptomycin antibiotic solution; or 5mL of silkworm pupa hemolymph, 2.5mL of egg yolk, 2ml of 10% milk powder solution, 0.8ml of Nathanheimer's salt solution, 0.2g of trehalose, 0.05g of sterile water, and 200μl of penicillin-streptomycin antibiotic solution.

[0056] (2) Trichogramma wasp parasitizing artificial egg cards

[0057] Artificial host egg cards were placed in beekeeping tubes, and infected with a *Trichogramma short-tubed* strain infected with *Wolbachia* at a ratio of approximately 10:1. After 5 hours of full parasitism, the egg cards were removed, and the *Trichogramma short-tubed* wasps on the surface of the artificial egg cards were brushed off with a soft brush. Then, *Pterygodium squarrosum*, which was not infected with *Wolbachia*, was introduced for 5 hours of parasitism. The parasitized artificial egg cards were then removed, their surfaces were wiped with 75% alcohol, and they were placed in sterile petri dishes and incubated in a constant temperature and humidity incubator (27±1℃, RH 80±5%, photoperiod 14L:10D) until the *Pterygodium squarrosum* emerged as wasps.

[0058] Example 4

[0059] In this embodiment, Wolbachia cells were added to an artificial host to infect the pine caterpillar Trichogramma wasp.

[0060] (1) Preparation of artificial host egg cards

[0061] The Aa23 cell line infected with the Wolbachia wAlbB strain was cultured at 26°C until approximately 70% confluence. The cell culture medium was a mixture of equal proportions of Mitsuhashi-Maramorosch medium and VP12 medium, containing 15% fetal bovine serum. After resuspending the adherent Aa23 cells by pipetting, 1 ml of the cell-containing medium was centrifuged at 1000g for 1 min. The supernatant was removed, and the Wolbachia-infected cell pellet was added to the artificial egg solution from Example 1 to prepare artificial host egg cards.

[0062] (2) Parasitic transfection and PCR detection

[0063] Following the steps in Example 1, uninfected Trichogramma pine caterpillar wasps were inoculated onto artificial host egg cards of Wolbachia. After the wasps emerged, PCR testing was performed to determine whether the wasps were infected with Wolbachia.

[0064] Comparative Example 1 uses rice moth eggs as a natural host

[0065] Rice moth eggs are fixed onto blank egg cards with glue to create rice moth egg cards. After anesthetizing *Trichogramma short-tubed* wasps with CO2 or cryotherapy, the egg cards are placed inside and allowed to parasitize for half an hour. The wasps are then anesthetized again with CO2. The egg cards are removed, and adult *Trichogramma short-tubed* wasps attached to the cards are brushed off with a soft brush. After confirming no residue remains, the egg cards are placed into anesthetized *Trichogramma short-tubed* wasp rearing tubes and parasitized again for half an hour. The parasitized egg cards are then removed and placed in new culture tubes in a constant temperature and humidity incubator for cultivation.

[0066] The results are as follows Figure 2As shown, Wolbachia can be horizontally transmitted from the body of *Trichogramma short-tubed* to *Trichogramma pineinae* via the eggs of the natural host *Rhizoctonia solani*, but the transmission rate is only 5% (one in twenty *Trichogramma pineinae* is infected).

[0067] The scope of protection of this invention is not limited to the above embodiments. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in this invention and are protected by the appended claims.

Claims

1. A method for non-invasive infection of Wolbachia to Trichogramma by using artificial host, characterized in that, First, artificial host egg cards were prepared. Then, the Aedes albopictus Aa23 cell line, a biological material infected with Wolbachia, was cultured at 26°C until it reached approximately 70% confluence. The adherent Aa23 cells were resuspended by pipetting, centrifuged to remove the supernatant, and the Wolbachia-infected cell pellet was added to artificial egg solution to form an artificial egg solution mixture. This mixture was then used to prepare artificial host egg cards. Uninfected Trichogramma pine caterpillars were inoculated into the artificial host egg cards. After full parasitism, the artificial host egg cards were removed and placed in a constant temperature and humidity incubator for culture. After the Trichogramma pine caterpillars emerged, PCR was used to detect whether the Trichogramma pine caterpillars were infected with Wolbachia. The artificial egg solution was prepared according to the following proportions: 3 ml of silkworm pupa hemolymph, 2.5 ml of egg yolk, 1 ml of 10% milk powder solution, 1 ml of Nathanheimer's salt solution, 0.1 g of trehalose, 0.1 g of sterile water, and 180 μl of penicillin-streptomycin solution. The Nathanheimer's salt solution was prepared using 7.5 g of NaCl, 0.1 g of KCl, 20.2 g of CaCl2, 0.2 g of NaHCO3, and 1000 ml of distilled water. The preparation steps of the artificial host egg card are as follows: Take a polyethylene film with a thickness of 20 µm, and use a glass grinding rod to press out a hemispherical depression on it with a diameter of 2-3 mm and a height of 2-3 mm; use a soft brush to evenly coat the outer surface of the protrusion of the film with a 10% polyvinyl alcohol aqueous solution, and place it in a clean bench to dry; then, add 4 µl of the artificial egg fluid mixture to the depression of the polyethylene film; take another flat polyethylene film, open it at all four sides and heat seal it to make an artificial host egg card.

2. The method for non-invasive infection of Wolbachia to Trichogramma by using artificial host according to claim 1, characterized in that, The culture conditions in the constant temperature and humidity incubator were: 27±1℃, RH 80±5%, and photoperiod 14 L:10 D.

Images