WO2019041208A1 - Culex quinquefasciatus and method for obtaining same - Google Patents

Abstract

A method for producing Culex pipiens, comprising: artificially infecting Culex pipiens pv. By this method, a CuA-induced Culex pipiens pallens artificially infected with A. albopictus with high CI intensity (for example, 95-100%) can be obtained, and the Culex pipiens pallens obtained by the method can be reduced in nature. The rate of oviposition of Culex pipiens pallens effectively controls the generation and population of Culex pipiens, which can effectively reduce the occurrence and spread of epidemic encephalitis.

Description

Culex pipiens and its production method Technical field

The invention belongs to the field of artificial infection, and in particular, the invention relates to a Culex pipiens pallens and a production method thereof.

Background technique

In recent years, there has been a marked increase in the prevalence of mosquito-borne diseases in the world. Many of them are diseases with strong transmission, wide popularity, high incidence and high risk, and have become a worldwide public health problem. Culex pipiens pallens is an important vector for diseases such as epidemic encephalitis. Therefore, the prevention of Culex pipiens pallens is the main means to prevent and control epidemic encephalitis.

Wolbachia belongs to the genus Wolbachia belonging to Proteobacteria, Alphaproteobacteria, Rickettsiales, Rickettsiaceae. Is a type of maternally inherited Gram-negative bacterium widely found in arthropods, subtypes including wAlbA, wAlbB and wpip. Because Wolbachia can induce cytoplasmic incompatibility (CI, which refers to bacterial-induced cytoplasmic incompatibility between sperm and egg cells, which leads to no or less progeny), induces cytoplasmic incompatibility (CI). Mechanisms such as parthenogenesis-inducing (PI), feminizing, and male-killing alter and influence the reproduction of their hosts, and are therefore used for biological control. The method has the characteristics of sustainable, high-efficiency, environmental protection and no bio-safety hazard, especially in the control of mosquitoes and mosquito-borne diseases, which has attracted extensive attention from researchers at home and abroad. Different types of Wolbachia in mosquito infection have different effects on their fertility and virus resistance. Differentiation of Wolbachia with different genotypes is the premise and key to its application in mosquito control. However, due to the variety of species and the differences in genetic background between different species, simple horizontal transfection cannot be performed. Therefore, there are still many difficulties and challenges in this study.

Summary of the invention

Since mosquito-borne diseases have a significant increasing trend in the global epidemic, many of them are diseases with strong spread, wide popularity, high incidence, and high risk. The purpose of the present invention is to overcome the difficulty in simply and effectively preventing epidemics in the prior art. A deficiency of mosquito-borne diseases such as Japanese encephalitis, providing an artificially infected Culex pipiens pallens, which can effectively inhibit the population of Culex pipiens in nature, and reduce mosquito vectors such as Japanese encephalitis from the source. The occurrence and spread of the disease, and provide a method for cultivating and producing the artificially infected Culex pipiens.

In order to achieve the above object, in one aspect, the present invention provides a Culex pipiens pallens which is artificially infected with wAlbA of Aedes albopictus.

In a preferred embodiment, the artificial infection is by injecting the cytoplasm of Aedes albopictus eggs into the Culex pipiens pallens by microinjection, and screening the deficient library infected with the wAlbA. Mosquitoes are realized.

In a preferred embodiment, the amount of microinjection is from 1*10 ^{-5 to} 1.5*10 ^-5 μL.

In a preferred embodiment, the Culex pipiens pallens is not naturally infected with Wolbachia prior to being artificially infected with wAlbA of Aedes albopictus.

In a preferred embodiment, the Culex pipiens pallens is treated with antibiotics prior to being artificially infected with wAlbA of Aedes albopictus.

In a preferred embodiment, the antibiotic treatment can be carried out by soaking the antibiotic with the feed and feeding the treated feed to the Culex pipiens pallens.

In a preferred embodiment, the antibiotic is at least one of tetracycline, penicillin, and fosfomycin.

In a preferred embodiment, the concentration of the antibiotic is from 0.5 to 2% by weight.

In a preferred embodiment, the Culex pipiens pallens has cytoplasmic incompatibility with Culex pipiens pallens that has not been artificially infected.

In another aspect, the present invention provides a method of producing Culex pipiens, comprising: artificially infecting Culex sinensis to wAlbA of Aedes albopictus.

In a preferred embodiment, the step of artificially infecting comprises: injecting the cytoplasm of Aedes albopictus eggs into the Culex pipiens pallens by microinjection, and screening for infection with the wAlbA Culex pipiens.

In a preferred embodiment, the amount of microinjection is from 1*10 ^{-5 to} 1.5*10 ^-5 μL.

In a preferred embodiment, the method further comprises: treating the Culex pipiens pallens with an antibiotic prior to artificially infecting wAlbA of Aedes albopictus.

In a preferred embodiment, the step of treating the Culex pipiens pallens with an antibiotic comprises: soaking the feed with an antibiotic and feeding the treated feed to the Culex pipiens pallens.

In a preferred embodiment, the antibiotic is at least one of tetracycline, penicillin, and fosfomycin.

In a preferred embodiment, the concentration of the antibiotic is from 0.5 to 2% by weight.

In a preferred embodiment, the method further comprises: carrying out large-scale breeding of Culex pipiens, which is artificially infected with WAlbA of Aedes albopictus.

By the method of the present invention, Culex pipiens pallens artificially infected with WAlbA of Aedes albopictus can be obtained with extremely high CI intensity (for example, 95-100%), and the Culex pipiens pallens of the present invention can be obtained by the method of the present invention. The Culex pipiens pallens is placed in nature and can significantly reduce the rate of oviposition of Culex pipiens, thus effectively controlling the generation and population of Culex pipiens, which can effectively reduce mosquitoes such as Japanese encephalitis. The occurrence and spread of vector diseases.

DRAWINGS

Fig. 1 is a graph showing representative results of PCR detection of Culex pipiens pallens after artificial infection.

Detailed ways

Specific embodiments of the present invention will be described in detail below. It is to be understood that the specific embodiments described herein are merely illustrative and not restrictive.

The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value. Ranges or values should be understood to include values that are close to those ranges or values. For numerical ranges, the endpoint values of the various ranges, the endpoint values of the various ranges and the individual point values, and the individual point values can be combined with one another to yield one or more new ranges of values. The scope should be considered as specifically disclosed herein.

In one aspect, the invention provides a Culex pipiens pallens, which is artificially infected with wAlbA of Aedes albopictus.

Among them, wAlbA is a subtype of Wolbachia, which is a kind of maternal-inherited Gram-negative bacteria. According to the present invention, the CuA of A. cerevisiae, which is artificially infected with A. cerevisiae, is extremely high in CI (for example, 95-100%), and is placed in nature to substantially reduce the production of Culex pipiens in nature. The egg rate can effectively control the generation and population of the Culex pipiens, which can effectively reduce the occurrence and spread of mosquito-borne diseases such as Japanese encephalitis. Furthermore, in a preferred embodiment, the Culex pipiens pallens is only artificially infected with wAlbA of Aedes albopictus, but not wAlbB (also a subtype of Wolbachia).

According to the present invention, various artificial infections well known to those skilled in the art can be used to artificially infect Culex pipiens, as long as wAlbA carried by Aedes albopictus can be introduced into Culex pipiens. Preferably, the artificial infection is achieved by injecting the cytoplasm of Aedes albopictus eggs into Culex pipiens pallens by microinjection and screening for Culex pipiens pallens infected with the wAlbA. Among them, since Aedes albopictus in nature usually carries wAlbA and/or wAlbB, and even if the A. sinensis of the injection strain carries both wAlbA and wAlbB, the artificially infected offspring does not necessarily carry both at the same time, and It is more inclined to carry only one of them, for example, only wAlbA or wAlbB is carried. Therefore, in order to separately study the biological effect of Culex pipiens carrying wAlbA, a screening step can be performed after microinjection and cultivation into adults to screen Culex pipiens pallens carrying wAlbA without carrying wAlbB. Among them, screening can be carried out by a conventional screening method (for example, polymerase chain reaction, PCR) in the art. A representative result plot of PCR can be as shown in Figure 1, which indicates that the test sample (i.e., the artificially infected Culex pipiens pallens) is positive at wAlbA and negative at wAlbB, indicating that the sample carries wAlbA without Carry wAlbB.

According to the present invention, the amount of the microinjection is not particularly limited as long as it can successfully infect the WAlbA of Aedes albopictus to be artificially infected. Preferably, the amount of microinjection is from 1*10 ^{-5 to} 1.5*10 ^-5 μL.

In a preferred embodiment, it is considered that Wolbachia, which may be naturally infected by Culex pipiens, may also have an effect on CI phenomenon, for example, if the natural infection with Wolbachia is directly infected with Aedes albopictus wAlbA may cause a decrease in the CI intensity of the artificially infected Culex pipiens pallens obtained in the present invention. In order to rule out the above effects, Culex pipiens pallens, which is not naturally infected with Wolbachia, can be screened prior to artificial infection. Preferably, the Culex pipiens pallens is not naturally infected with Wolbachia prior to being artificially infected with wAlbA of Aedes albopictus.

In another preferred embodiment, the artificially infected Culex pipiens population can be selected or treated by various methods well known to those skilled in the art prior to artificial infection to remove Culex pipiens pallens to be artificially infected. Naturally infected Wolbachia, resulting in Culex pipiens pallens uninfected with Wolbachia. Preferably, the Culex pipiens pallens is treated with antibiotics prior to being artificially infected with wAlbA of Aedes albopictus.

Among them, the step of antibiotic treatment can be carried out in various ways as long as antibiotics can be applied to Wolbachia which is naturally infected in Culex pipiens pallens to remove it. Preferably, the antibiotic treatment can be carried out by soaking the antibiotic with the feed and feeding the treated feed to the Culex pipiens pallens. Antibiotics are thus introduced into these Culex pipiens pallens, and Wolbachia, which is naturally infected in Culex pipiens, is removed.

Further, when the method of the above-described method is used to treat Culex pipiens pallens with an antibiotic, there is no particular limitation on the kind and concentration of the antibiotic as long as it can remove Wolbachia. In a preferred embodiment, the antibiotic is at least one of tetracycline, penicillin, and fosfomycin. In another preferred embodiment, the concentration of the antibiotic is from 0.5 to 2% by weight.

The Culex pipiens pallens artificially infected with Wolbachia according to the present invention has a high CI intensity, and there is often a cytoplasmic incompatibility at the time of mating, so that the population of Culex pipiens pallens can be effectively controlled.

Preferably, the Culex pipiens pallens has cytoplasmic incompatibility with Culex pipiens pallens that has not been artificially infected.

In another aspect, the present invention also provides a method of producing Culex pipiens, comprising: causing said Culex pipiens pallens artificially infected wAlbA of Aedes albopictus.

According to the present invention, various artificial infections well known to those skilled in the art can be used to artificially infect Culex pipiens, as long as wAlbA carried by Aedes albopictus can be introduced into Culex pipiens. Preferably, the step of artificially infecting comprises: injecting the cytoplasm of Aedes albopictus eggs into the Culex pipiens pallens by microinjection, and screening for Culex pipiens pallens infected with the wAlbA. Among them, since Aedes albopictus in nature usually carries wAlbA and/or wAlbB, and even if the A. sinensis of the injection strain carries both wAlbA and wAlbB, the artificially infected offspring does not necessarily carry both at the same time, and It is more inclined to carry only one of them, for example, only wAlbA or wAlbB is carried. Therefore, in order to separately study the biological effect of Culex pipiens carrying wAlbA, a screening step can be performed after microinjection and cultivation into adults to screen Culex pipiens pallens carrying wAlbA without carrying wAlbB.

According to the present invention, the amount of the microinjection is not particularly limited as long as the Ca. sinensis to be artificially infected can be successfully infected with wAlbA of Aedes albopictus. Preferably, the amount of microinjection is from 1*10 ^{-5 to} 1.5*10 ^-5 μL.

In a preferred embodiment, it is considered that Wolbachia, which may be naturally infected by Culex pipiens, may also have an effect on CI phenomenon, for example, if the natural infection with Wolbachia is directly infected with Aedes albopictus wAlbA may cause a decrease in the CI intensity of the artificially infected Culex pipiens pallens obtained in the present invention. In order to rule out the above effects, it is possible to include a step of screening for Culex pipiens pallens that is not naturally infected with Wolbachia prior to artificial infection.

In another preferred embodiment, the artificially infected Culex pipiens population can be selected or treated by various methods well known to those skilled in the art prior to artificial infection to remove Culex pipiens pallens to be artificially infected. Naturally infected Wolbachia, resulting in Culex pipiens pallens uninfected with Wolbachia. Preferably, the method for producing Culex pipiens pallens further comprises: treating the Culex pipiens pallens with an antibiotic prior to artificial infection with wAlbA of Aedes albopictus.

Among them, the step of antibiotic treatment can be carried out in various ways as long as antibiotics can be applied to Wolbachia which is naturally infected in Culex pipiens pallens to remove it. Preferably, the step of treating the Culex pipiens pallens with an antibiotic comprises: soaking the feed with an antibiotic, and feeding the treated feed to the shelter Said Culex pipiens. Antibiotics are thus introduced into these Culex pipiens pallens, and Wolbachia, which is naturally infected in Culex pipiens, is removed.

Further, when the method of the above-described method is used to treat Culex pipiens pallens with an antibiotic, there is no particular limitation on the kind and concentration of the antibiotic as long as it can remove Wolbachia. In a preferred embodiment, the antibiotic is at least one of tetracycline, penicillin, and fosfomycin. In another preferred embodiment, the concentration of the antibiotic is from 0.5 to 2% by weight.

According to the present invention, in order to further enlarge the population of Culex pipiens pallens of the present invention after obtaining the WAlbA-inducing Culex pipiens pallens of the present invention by the method of the present invention, it may preferably further include : Culex pipiens pallens artificially infected with WAlbA of Aedes albopictus was cultured on a large scale. By carrying out large-scale rearing under suitable feeding conditions, the population of Culex pipiens pallens of the present invention can be increased, so that it can be placed in nature to effectively control the generation and population of Culex pipiens pallens in nature. .

Specifically, the method of producing Culex pipiens pers. according to the present invention may include the following steps during actual operation:

Culex pipiens and Aedes albopictus are collected directly from nature. After collecting Culex pipiens and Aedes albopictus, they are raised under laboratory conditions to prepare for subsequent spawning; antibiotics will be treated The feed (for example, syrup containing 1% by weight of tetracycline) is fed to Culex pipiens, thereby removing Wolbachia from Culex pipiens, so as to prevent Wolbachia, which is naturally infected with Culex pipiens, from affecting infection and test results; Afterwards, several Culex pipiens and Aedes albopictus were selected and placed in the spawning cups for 45-60 minutes. Among them, Culex pipiens pallens usually lay eggs at night, and the eggs used for injection need to be Provided in a short period of time, it is possible to use the incubator to provide the nighttime environment required for spawning of Culex pipiens to promote spawning; after collecting the eggs of Culex pipiens and Aedes albopictus, the library can also be used. The mosquitoes and Aedes albopictus eggs are arranged, for example, the Culex pipiens and Aedes albopictus are sorted in two columns or the tails of all eggs are oriented the same, which makes subsequent microinjection more convenient; Before microinjection Cover the surface of the egg with water-saturated oil to prevent excessive drying of the egg; during microinjection, the cytoplasm of the tail tip of the donor egg (Aedes albopictus) can be aspirated under a microscope with a microinjector, and then Inject into the tail tip of the recipient egg (the Cucumis sinensis egg), and suck and inject the eggs as described above separately; after the injection, the donor egg can be picked and the individual is not injected. Somatic eggs, to obtain microscopic injection of Culex pipiens var. cerevisiae eggs, wherein the amount of microinjection is 1*10 ^-5 -1.5*10 ^-5 μL; The Culex pipiens pallens eggs were transferred to a culture condition of about 27 ° C and 80% RH for incubation to obtain Culex pipiens pallens which was artificially infected with WAlbA of Aedes albopictus.

The invention will be described in detail below by way of examples.

Example 1

Culex pipiens and Aedes albopictus were collected from nature and kept in the laboratory. In the breeding, Culex pipiens pallens was fed with syrup containing 1% by weight of tetracycline. 10 females of Culex pipiens sinensis and Aedes albopictus, which were selected for 5 days of adult worms, were placed in spawning for 60 min. Among them, the spawning cups containing female Culex pipiens were placed at night. Environmental conditions in the incubator to promote spawning. Eggs of Culex pipiens and Aedes albopictus were collected and transferred to moist filter paper, and the Culex pipiens pipiens and Aedes albopictus eggs were sorted into two columns, and the tails of all eggs were oriented the same. The thick filter paper with the arranged mosquito eggs is reversely attached to the glass slide with double-sided tape, and gently pressed to make the mosquito eggs adhere to the double-sided tape to transfer the eggs. The mosquito eggs were dried at room temperature for about 1 min. Cover the surface of the egg with water-saturated oil to prevent further drying. The slide with the eggs was placed under a microscope of an eyepiece 10× and an objective lens 20×, and the tail tip cytoplasm of the donor egg was aspirated by a microinjector, and then injected into the tip of the recipient egg. The aligned eggs are sequentially aspirated and injected. Among them, the amount of microinjection is 1.5*10 ^-5 μL. After the injection is completed, the donor eggs and individual recipient eggs that have not been injected are picked, and the double-sided tape sticking with Cucumis sinensis eggs is gently peeled off from the slide and stored at 27 ° C, 80% RH. In the glass tube. After 7 days of storage, the eggs are placed in clear water containing hatching solution and hatched. After being hatched, they are transferred to clear water containing food for breeding. After 5 years of age, they are kept in single tube, and they will get G0 generation after being adult. Culex.

Example 2

G0 subtilis was obtained in the same manner as in Example 1, except that the antibiotic used was 0.5% by weight of penicillin, and the amount of microinjection was 1*10 ^-5 μL.

Example 3

G0 subtilis was obtained in the same manner as in Example 1, except that the antibiotic used was 2% by weight of fosfomycin, and the amount of microinjection was 1.2*10 ^-5 μL.

Example 4

G0-induced Culex pipiens pallens was obtained in the same manner as in Example 1, except that the Culex pipiens pallens was treated without using an antibiotic.

Test method for infection rate:

100 Genotypes of Culex pipiens pallens obtained in Examples 1-4 were selected, and G0 generation male Culex pipiens pallens was directly tested by PCR. G0 generation female Culex pipiens pallens was mated with unnaturally infected male Culex pipiens pallens. PCR was carried out after spawning to obtain the infection rate of G0-induced Culex pipiens (ie, the proportion of wAlbA infected without wAlbB in the total). See Table 1 for the specific results. Then, the G0 generation female Culex pipiens pallens and the unnaturally infected male Culex pipiens pallens were reared and reared in a single tube at the age of 5, and the G1 generation Culex pipiens pallens was obtained after the adult. Similarly, 100 were selected respectively. Only the G1 generation of Culex pipiens pallens obtained in Examples 1-4 (both positive and infection-infected G1-induced Culex pipiens), G1 generation male Culex pipiens pallens were directly tested by PCR, and G1 generation female Culex pipiens PCR was performed after mating with unnaturally infected male Culex pipiens pallens, and the results are also shown in Table 1. The above procedure was repeated to obtain G2 and G3-induced Culex pipiens pallens and the infection rate was examined. The specific results are also shown in Table 1. In addition, representative results of PCR detection of Culex pipiens pallens after artificial infection are shown in Fig. 1. It can be seen from Fig. 1 that samples 1-5 are positive at wAlbA and negative at wAlbB, indicating that these The sample carries wAlbA without carrying wAlbB.

Table 1

Infection rate Example 1 Example 2 Example 3 Example 4 G0 30% twenty four% 26% 27% G1 50% 47% 46% 45% G2 100% 100% 100% 100% G3 100% 100% 100% 100%

CI intensity detection method:

10 G3 male Culex pipiens quinquefolia obtained in Examples 1-4 and 10 wild-type females were selected to cause fatigue Culex pipiens were mated to lay eggs and the hatching rate (%) of the eggs was measured, while CI intensity = 1 - hatching rate. See Table 2 for the specific results.

Table 2

Example 1 Example 2 Example 3 Example 4 Number of eggs hatched 2 3 3 3 Total number of eggs 530 650 579 590 Hatching rate 0.38% 0.46% 0.52% 0.51% CI strength 99.62% 99.54% 99.48% 99.49%

Similarly, 10 G3 female Culex pipiens pallens obtained in Examples 1-4 were mated with 10 wild-type male Culex pipiens pallens, and the hatching rate (%) and CI intensity of the eggs were measured. See Table 3 for the specific results.

table 3

Example 1 Example 2 Example 3 Example 4 Number of eggs hatched 1 2 2 540 Total number of eggs 625 645 590 615 Hatching rate 0.16% 0.31% 0.34% 87.8% CI strength 99.84% 99.69% 99.66% 12.2%

It can be seen from the results of Table 1 that the G0-induced Culex pipiens pallens obtained according to the method of the present invention exhibited infection rates of 30%, 24%, 26%, and 27%, respectively, while the G1-induced Culex pipiens pallens showed The infection rates of 50%, 47%, 46% and 45% (increased compared with the G0 generation), the G2 generation of Culex pipiens pallens showed 100% infection rate, that is, all artificial infections of Cu2 Wolbachia, G3-induced Culex pipiens results, and G2-induced Culex pipiens, therefore artificially infected Culex pipiens pallens according to the method of the present invention, only need to breed two generations after artificial infection to obtain an infection rate of 100% Culex pipiens population.

It can be seen from the results of Table 2 and Table 3 that when the G3 male Culex pipiens pallens and the wild-type female Culex pipiens pallens are mated to lay eggs, the hatching rate is extremely low (all below 1%), that is, the present invention The artificial infection with the cytoplasmic incompatibility of the male Culex pipiens pallens with Wolbachia and the wild-type female Culex pipiens pallens, and the high CI intensity, can significantly reduce the generation of offspring; when the G3 generation females cause Culex pipiens With wild-type males When the Culex pipiens palls for mating and laying eggs, the hatching rate is extremely low (preferably less than 1%), that is, the female Culex pipiens pallens infected with Wolbachia of the present invention has no cytoplasmic phase with wild-type male Culex pipiens pallens. Capacitance; In summary, the Culex pipiens pallens of the present invention exhibit bidirectional CI characteristics. In addition, it can be seen from the results of Example 4 that when the Culex pipiens pallens is not treated with antibiotics before artificial infection, the Wolbacia carried by Culex pipiens pallens has little effect on the CI results of the male mosquitoes, but the female mosquitoes CI As a result, a large effect is exerted, so that antibiotic treatment of Culex pipiens pallens before artificial infection can be carried out under preferred conditions.

In summary, according to the method of the present invention, Culex pipiens pallens artificially infected with Wolbachia can be easily obtained, and the obtained CI-induced Culex pipiens pallens is extremely high, so if the Culex pipiens pallens of the present invention is administered to nature In this way, the oviposition rate of Culex pipiens pallens can be greatly reduced, thereby effectively controlling the generation and population of Culex pipiens, thereby effectively reducing the occurrence and spread of mosquito-borne diseases such as Japanese encephalitis.

The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solutions of the present invention within the scope of the technical idea of the present invention. These simple variants All fall within the scope of protection of the present invention.

It should be further noted that the specific technical features described in the above specific embodiments may be combined in any suitable manner without contradiction. To avoid unnecessary repetition, the present invention has various possibilities. The combination method will not be described separately.

In addition, any combination of various embodiments of the invention may be made as long as it does not deviate from the idea of the invention, and it should be regarded as the disclosure of the invention.

Claims (17)

A Culex pipiens pallens, wherein the Culex pipiens pallens is artificially infected with wAlbA of Aedes albopictus. The Culex pipiens pallens according to claim 1, wherein the artificial infection is obtained by microinjecting the cytoplasm of Aedes albopictus eggs into Cucumber Cucumber eggs and screening for infection. The wAlbA-induced Culex pipiens pallens is achieved. The Culex pipiens pallens according to claim 2, wherein the amount of the microinjection is 1*10 ^-5 -1.5*10 ^-5 μL. The Culex pipiens pallens according to claim 1, wherein the Culex pipiens pallens is not naturally infected with Wolbachia prior to being artificially infected with wAlbA of Aedes albopictus. The Culex pipiens pallens according to claim 1, wherein the Culex pipiens pallens is treated with antibiotics prior to being artificially infected with wAlbA of Aedes albopictus. The Culex pipiens pallens according to claim 5, wherein the antibiotic treatment is carried out by soaking the antibiotics with the feed and feeding the treated feed to the Culex pipiens pallens. The Culex pipiens pallens according to claim 5, wherein the antibiotic is at least one of tetracycline, penicillin and fosfomycin. The Culex pipiens pallens according to claim 5, wherein the concentration of the antibiotic is from 0.5 to 2% by weight. The Culex pipiens pallens according to claim 1, wherein the Culex pipiens pallens has a cytoplasmic incompatibility with a Culex pipiens pallens that has not been artificially infected. A method of producing Culex pipiens, comprising: The Culex pipiens pallens was artificially infected with wAlbA of Aedes albopictus. The method according to claim 10, wherein the step of artificially infecting comprises: injecting the cytoplasm of Aedes albopictus eggs into the Culex pipiens pallens by microinjection, and screening for infections as described Culex pipiens pv. The method according to claim 11, wherein the amount of the microinjection is 1*10 ^-5 -1.5*10 ^-5 μL. The method of claim 10 further comprising: The Culex pipiens pallens was treated with antibiotics prior to artificial infection with wAlbA of Aedes albopictus. The method according to claim 13, wherein the step of treating the Culex pipiens pallens with an antibiotic comprises: soaking the feed with an antibiotic, and feeding the treated feed to the Culex pipiens pallens. The method according to claim 13, wherein the antibiotic is at least one of tetracycline, penicillin and fosfomycin. The method according to claim 13, wherein the concentration of the antibiotic is from 0.5 to 2% by weight. The method according to claim 10, further comprising: large-scale rearing of Culex pipiens, which is artificially infected with WAlbA of Aedes albopictus.

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