WO2019041207A1 - 一种致倦库蚊及其生产方法 - Google Patents

一种致倦库蚊及其生产方法 Download PDF

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WO2019041207A1
WO2019041207A1 PCT/CN2017/099802 CN2017099802W WO2019041207A1 WO 2019041207 A1 WO2019041207 A1 WO 2019041207A1 CN 2017099802 W CN2017099802 W CN 2017099802W WO 2019041207 A1 WO2019041207 A1 WO 2019041207A1
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culex pipiens
pipiens pallens
walbb
pallens
aedes albopictus
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PCT/CN2017/099802
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English (en)
French (fr)
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梁永康
侯汲虹
刘菊莲
汤其仙
唐周兰
杨翠
巫羲琏
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广州威佰昆生物科技有限公司
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Priority to PCT/CN2017/099802 priority Critical patent/WO2019041207A1/zh
Publication of WO2019041207A1 publication Critical patent/WO2019041207A1/zh

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    • 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

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  • 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.
  • 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).
  • CI cytoplasmic incompatibility
  • 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.
  • simple horizontal transfection cannot be performed. Therefore, there are still many difficulties and challenges in this study.
  • 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.
  • the present invention provides a Culex pipiens pallens which is artificially infected with wAlbB of Aedes albopictus.
  • the artificial infection is by microinjection of the cytoplasm of Aedes albopictus eggs into Cucumber Cucumber eggs, and screening for a depletion library infected with the wAlbB Mosquitoes are realized.
  • the amount of microinjection is from 1*10 -5 to 1.5*10 -5 ⁇ L.
  • the Culex pipiens pallens is not naturally infected with Wolbachia prior to being artificially infected with wAlbB of Aedes albopictus.
  • the Culex pipiens pallens is treated with antibiotics prior to being artificially infected with wAlbB of Aedes albopictus.
  • the antibiotic treatment can be carried out by soaking the antibiotic with the feed and feeding the treated feed to the Culex pipiens pallens.
  • the antibiotic is at least one of tetracycline, penicillin, and fosfomycin.
  • the concentration of the antibiotic is from 0.5 to 2% by weight.
  • the Culex pipiens pallens has cytoplasmic incompatibility with Culex pipiens pallens that has not been artificially infected.
  • the present invention provides a method of producing Culex pipiens, comprising: artificially infecting Culex sinensis to wAlbB of Aedes albopictus.
  • 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 wAlbB Culex pipiens.
  • the amount of microinjection is from 1*10 -5 to 1.5*10 -5 ⁇ L.
  • the method further comprises: treating the Culex pipiens pallens with an antibiotic prior to artificially infecting wAlbB of Aedes albopictus.
  • 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 antibiotic is at least one of tetracycline, penicillin, and fosfomycin.
  • the concentration of the antibiotic is from 0.5 to 2% by weight.
  • the method further comprises: performing large-scale breeding of Culex pipiens, which is artificially infected with wAlbB of Aedes albopictus.
  • Culex pipiens pallens artificially infected with WAlbB of Aedes albopictus with high CI intensity (for example, 80-90%) can be obtained, or 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 can be 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.
  • Fig. 1 is a graph showing representative results of PCR detection of Culex pipiens pallens after artificial infection.
  • the invention provides a Culex pipiens, which is artificially infected with wAlbB of Aedes albopictus.
  • wAlbB is a subtype of Wolbachia, which is a kind of maternal-inherited Gram-negative bacteria.
  • the CIA of the artificially infected WAlbB of Aedes albopictus has a high CI intensity (for example, 80-90%), 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.
  • the Culex pipiens pallens is only artificially infected with wAlbB of Aedes albopictus, but not wAlbA (also a subtype of Wolbachia).
  • various artificial infections well known to those skilled in the art can be used to artificially infect Culex pipiens, as long as wAlbB carried by Aedes albopictus can be introduced into Culex pipiens.
  • the artificial infection is achieved by injecting the cytoplasm of Aedes albopictus eggs into the Culex pipiens pallens by microinjection and screening for Culex pipiens pallens infected with the wAlbB.
  • Aedes albopictus in nature usually carries wAlbA and/or wAlbB, and even if the A.
  • screening can be carried out after microinjection and cultivation into adults to screen Culex pipiens pallens carrying wAlbA without carrying wAlbB.
  • 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 wAlbB and negative at wAlbA, indicating that the sample carries wAlbB instead of Carry wAlbA.
  • the amount of the microinjection is not particularly limited as long as it can successfully infect the WAlbB of Aedes albopictus to be artificially infected.
  • the amount of microinjection is from 1*10 -5 to 1.5*10 -5 ⁇ L.
  • 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 wAlbB may cause a decrease in the CI intensity of the artificially infected Culex pipiens pallens obtained in the present invention.
  • Culex pipiens pallens which is not naturally infected with Wolbachia, can be screened prior to artificial infection.
  • the Culex pipiens pallens is not naturally infected with Wolbachia prior to being artificially infected with wAlbB of Aedes albopictus.
  • 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.
  • the Culex pipiens pallens is treated with antibiotics prior to being artificially infected with wAlbB of Aedes albopictus.
  • 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.
  • 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.
  • the method of the above-described method is used to treat Culex pipiens pallens with an antibiotic
  • the antibiotic is at least one of tetracycline, penicillin, and fosfomycin.
  • 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.
  • the Culex pipiens pallens has cytoplasmic incompatibility with Culex pipiens pallens that has not been artificially infected.
  • the present invention also provides a method of producing Culex pipiens, comprising: causing said Culex pipiens pallens artificially infected wAlbB of Aedes albopictus.
  • 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.
  • Aedes albopictus in nature usually carries wAlbA and/or wAlbB, and even if the A.
  • the amount of the microinjection is not particularly limited as long as the Ca. sinensis to be artificially infected can be successfully infected with wAlbB of Aedes albopictus.
  • the amount of microinjection is from 1*10 -5 to 1.5*10 -5 ⁇ L.
  • 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 wAlbB may cause a decrease in the CI intensity of the artificially infected Culex pipiens pallens obtained in the present invention.
  • 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.
  • the method for producing Culex pipiens quinquefolia further comprises: treating the Culex pipiens pallens with an antibiotic prior to artificially infecting wAlbB of Aedes albopictus.
  • 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.
  • 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.
  • the method of the above-described method is used to treat Culex pipiens pallens with an antibiotic
  • the antibiotic is at least one of tetracycline, penicillin, and fosfomycin.
  • the concentration of the antibiotic is from 0.5 to 2% by weight.
  • the present invention in order to further enlarge the population of Culex pipiens pallens of the present invention after obtaining the artificially infected WAlbB of the Aedes albopictus of the present invention by the method of the present invention, it may preferably further comprise : Culex pipiens pallens artificially infected with wAlbB of Aedes albopictus was cultured on a large scale.
  • 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. .
  • the method of producing Culex pipiens pers. 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.
  • 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 ⁇ L;
  • 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 WAlbB of Aedes albopictus.
  • 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.
  • 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 ⁇ L.
  • 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.
  • 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.
  • 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 ⁇ L.
  • 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 ⁇ L.
  • 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.
  • 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 wAlbB infected without wAlbA in the total). See Table 1 for the specific results.
  • 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.
  • Example 1 Example 2 Example 3 Example 4 G0 30% twenty four% 26% 27% G1 50% 48% 48% 46% G2 100% 100% 100% 100% G3 100% 100% 100% 100% 100%
  • Example 1 Example 2
  • Example 3 Example 4
  • Number of eggs hatched 85 90 84
  • Total number of eggs 757 728 740 770
  • Hatching rate 11.23% 12.36% 11.54% 11.95%
  • CI strength 88.77% 87.64%
  • Example 1 Example 2
  • Example 3 Example 4
  • Number of eggs hatched 3 4 5 600
  • 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%, 48%, 48% and 46% (increased compared with the G0 generation), the G2 generation of Culex pipiens pallens showed 100% infection rate, that is, all the artificial infections of the G2 generation Culex pipiens 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.
  • the Culex pipiens pallens of the present invention exhibits bidirectional CI characteristics, and the CI intensity of females is higher than that of males.
  • the Wolbacia carried by Culex pipiens pallens has little effect on the CI results of the male mosquitoes, but the female mosquitoes CI
  • a large effect is exerted, so that antibiotic treatment of Culex pipiens pallens before artificial infection can be carried out under preferred conditions.
  • Culex pipiens pallens artificially infected with Wolbachia can be easily obtained, and the obtained Culex pipiens pallens has a high CI intensity, 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.

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Abstract

一种致倦库蚊及其生产方法，该生产致倦库蚊的方法包括：使致倦库蚊人工感染白纹伊蚊的wAlbB。通过该方法，可以得到CI强度较高(例如80-90％)的人工感染有白纹伊蚊的wAlbB的致倦库蚊，将通过该方法得到的致倦库蚊投放于自然界中，可以大幅降低致倦库蚊的产卵率，从而有效地控制致倦库蚊的后代产生和种群数量，从而可以有效地减少流行性乙型脑炎等蚊媒病的发生及传播。

Description

一种致倦库蚊及其生产方法 技术领域
本发明属于人工感染领域，具体地，本发明涉及一种致倦库蚊及其生产方法。
背景技术
近些年来，蚊媒病在全球流行呈现明显增加趋势，其中许多是传播力强、流行面广、发病率高、危害性大的疾病，已经成为世界性的公共卫生问题。致倦库蚊是流行性乙型脑炎等疾病的重要传播媒介。因此，对传播媒介致倦库蚊的防制是预防和控制流行性乙型脑炎的主要手段。
沃尔巴克氏体(Wolbachia)隶属于变形菌门(Proteobacteria)、α亚纲(Alphaproteobacteria)、立克次体目(Rickettsiales)、立克次体科(Rickettsiaceae)的沃尔巴克氏体属(Wolbachia)，是一类母性遗传的革兰阴性细菌，在节肢动物体内广泛存在，其亚型包括wAlbA、wAlbB和wpip。由于Wolbachia可以通过诱导宿主间杂交的胞质不相容(cytoplasmic incompatibility，CI，是指细菌诱导的精子和卵细胞之间的细胞质不亲和，其会导致不产生或产生较少的后代)、诱导单性生殖(parthenogenesis-inducing，PI)、雌性化(feminizing)和杀雄作用(male-killing)等机制改变和影响其宿主的繁殖，因而被用于生物防治。该方法具有可持续、高效能、绿色环保以及无生物安全隐患等特点，尤其在蚊虫及蚊媒病控制方面引起国内外研究者的广泛关注。蚊虫感染不同类型的Wolbachia对其繁殖力和病毒的抵抗力有不同的影响，区分不同基因型的Wolbachia是将其应用于蚊媒防制的前提和关键。但是由于物种繁多，且不同的物种之间的遗传背景上都存在差异，并不能进行简单的水平转染，因此在该研究中仍存在着许多困难与挑战。
发明内容
由于蚊媒病在全球流行呈现明显增加趋势，其中许多是传播力强、流行面广、发病率高、危害性大的疾病，本发明的目的在于克服现有技术中难以简单有效地预防流行性乙型脑炎等蚊媒病的缺陷，提供一种人工感染的致倦库蚊，其能够有效地抑制自然界中致倦库蚊的种群数量，从源头上减少流行性乙型脑炎等蚊媒病的发生及传播，并提供了该种人工感染的致倦库蚊的培育生产方法。
为了实现上述目的，在一方面，本发明提供了一种致倦库蚊，所述致倦库蚊被人工感染有白纹伊蚊的wAlbB。
在一个优选的实施方式中，所述人工感染是通过将白纹伊蚊卵的胞浆以显微注射的方式注射到致倦库蚊卵中，并筛选出感染有所述wAlbB的致倦库蚊来实现的。
在一个优选的实施方式中，所述显微注射的量为1*10-5-1.5*10-5μL。
在一个优选的实施方式中，在被人工感染有白纹伊蚊的wAlbB之前，所述致倦库蚊未自然感染有Wolbachia。
在一个优选的实施方式中，在被人工感染有白纹伊蚊的wAlbB之前，所述致倦库蚊经过抗生素处理。
在一个优选的实施方式中，所述抗生素处理可以通过将抗生素浸泡饲料，并将处理过的饲料喂食给所述致倦库蚊来进行。
在一个优选的实施方式中，所述抗生素为四环素、青霉素和磷霉素中的至少一种。
在一个优选的实施方式中，所述抗生素的浓度为0.5-2重量％。
在一个优选的实施方式中，所述致倦库蚊与未被人工感染的致倦库蚊存在胞质不相容性。
在另一方面，本发明提供了一种生产致倦库蚊的方法，包括：使所述致倦库蚊人工感染白纹伊蚊的wAlbB。
在一个优选的实施方式中，所述人工感染的步骤包括：将白纹伊蚊卵的胞浆以显微注射的方式注射到致倦库蚊卵中，并筛选出感染有所述wAlbB的致倦库蚊。
在一个优选的实施方式中，所述显微注射的量为1*10-5-1.5*10-5μL。
在一个优选的实施方式中，进一步包括：在人工感染白纹伊蚊的wAlbB之前，用抗生素处理所述致倦库蚊。
在一个优选的实施方式中，用抗生素处理所述致倦库蚊的步骤包括：用抗生素浸泡饲料，并将处理过的饲料喂食给所述致倦库蚊。
在一个优选的实施方式中，所述抗生素为四环素、青霉素和磷霉素中的至少一种。
在一个优选的实施方式中，所述抗生素的浓度为0.5-2重量％。
在一个优选的实施方式中，进一步包括：将人工感染有白纹伊蚊的wAlbB的致倦库蚊进行大规模饲养。
通过本发明的方法，可以得到CI强度较高(例如80-90％)的人工感染有白纹伊蚊的wAlbB的致倦库蚊，将本发明的致倦库蚊或通过本发明的方法得到的致倦库蚊投放于自然界中，可以大幅降低致倦库蚊的产卵率，从而有效地控制致倦库蚊的后代产生和种群数量，从而可以有效地减少流行性乙型脑炎等蚊媒病的发生及传播。
附图说明
图1是示出了人工感染后的致倦库蚊的PCR检测的代表性结果的图。
具体实施方式
以下对本发明的具体实施方式进行详细说明。应当理解的是，此处所描述的具体实施方式仅用于说明和解释本发明，并不用于限制本发明。
在本文中所披露的范围的端点和任何值都不限于该精确的范围或值，这些 范围或值应当理解为包含接近这些范围或值的值。对于数值范围来说，各个范围的端点值之间、各个范围的端点值和单独的点值之间，以及单独的点值之间可以彼此组合而得到一个或多个新的数值范围，这些数值范围应被视为在本文中具体公开。
在一方面，本发明提供了一种致倦库蚊，所述致倦库蚊被人工感染有白纹伊蚊的wAlbB。
其中，wAlbB是Wolbachia(沃尔巴克氏体)的一种亚型，是一类母性遗传的革兰阴性细菌。根据本发明的被人工感染有白纹伊蚊的wAlbB的致倦库蚊的CI强度较高(例如80-90％)，将其投放于自然界中，可以大幅降低自然界中致倦库蚊的产卵率，从而有效地控制致倦库蚊的后代产生和种群数量，从而可以有效地减少流行性乙型脑炎等蚊媒病的发生及传播。此外，在一个优选的实施方式中，所述致倦库蚊仅被人工感染有白纹伊蚊的wAlbB，而没有wAlbA(也是Wolbachia的一种亚型)。
根据本发明，可以采用本领域技术人员熟知的各种人工感染方式来人工感染致倦库蚊，只要能够将白纹伊蚊携带的wAlbB引入致倦库蚊即可。优选地，所述人工感染是通过将白纹伊蚊卵的胞浆以显微注射的方式注射到致倦库蚊卵中，并筛选出感染有所述wAlbB的致倦库蚊来实现的。其中，由于自然界中的白纹伊蚊通常携带wAlbA和/或wAlbB，而且即便注射株的白纹伊蚊同时携带wAlbA和wAlbB二者，其人工感染的后代也不一定会同时携带二者，并且更倾向于仅携带其中一种，例如仅携带wAlbA或wAlbB，因此，为了单独研究携带wAlbB的致倦库蚊的生物学效果，可以在显微注射并培育成成虫后进行筛选的步骤，以筛选出携带wAlbA而不携带wAlbB的致倦库蚊。其中，筛选可以通过本领域中常规筛选方法(例如聚合酶链式反应，PCR)来进行。PCR的代表性结果图可以如图1所示，其表示检测样本(即被人工感染的致倦库蚊)在wAlbB处呈阳性，而在wAlbA处呈阴性，即表示该样本携带wAlbB，而不携带wAlbA。
根据本发明，对于所述显微注射的量没有特别的限制，只要能够让待人工 感染的致倦库蚊成功感染白纹伊蚊的wAlbB即可。优选地，所述显微注射的量为1*10-5-1.5*10-5μL。
在一个优选的实施方式中，考虑到致倦库蚊可能自然感染的Wolbachia对CI现象也会产生一定的影响，例如，如果对自然感染有Wolbachia的致倦库蚊直接人工感染白纹伊蚊的wAlbB，可能会导致本发明中得到的人工感染的致倦库蚊的CI强度降低。为了排除上述影响，可以在人工感染之前筛选未自然感染有Wolbachia的致倦库蚊。优选地，在被人工感染有白纹伊蚊的wAlbB之前，所述致倦库蚊未自然感染有Wolbachia。
在另一个优选的实施方式中，可以在人工感染之前通过本领域技术人员熟知的各种方法对待人工感染的致倦库蚊群体进行一定的选择或处理，以除去待人工感染的致倦库蚊中自然感染的Wolbachia，从而得到未感染有Wolbachia的致倦库蚊。优选地，在被人工感染有白纹伊蚊的wAlbB之前，所述致倦库蚊经过抗生素处理。
其中，经过抗生素处理的步骤可以以各种方法进行，只要能够将抗生素作用于致倦库蚊体内自然感染的Wolbachia以将其除去即可。优选地，所述抗生素处理可以通过将抗生素浸泡饲料，并将处理过的饲料喂食给所述致倦库蚊来进行。从而将抗生素引入这些致倦库蚊体内，并除去这些致倦库蚊体内自然感染的Wolbachia。
此外，在使用如上所述的方法来用抗生素处理致倦库蚊时，对抗生素的种类和浓度也没有特别的限制，只要其能够除去Wolbachia即可。在一个优选的实施方式中，所述抗生素为四环素、青霉素和磷霉素中的至少一种。在另一个优选的实施方式中，所述抗生素的浓度为0.5-2重量％。
根据本发明的人工感染有Wolbachia的致倦库蚊具有较高的CI强度，在交配时往往存在胞质不相容，因此能够有效地控制致倦库蚊的种群数量。
优选地，所述致倦库蚊与未被人工感染的致倦库蚊存在胞质不相容性。
在另一方面，本发明还提供了一种生产致倦库蚊的方法，包括：使所述致 倦库蚊人工感染白纹伊蚊的wAlbB。
根据本发明，可以采用本领域技术人员熟知的各种人工感染方式来人工感染致倦库蚊，只要能够将白纹伊蚊携带的wAlbB引入致倦库蚊即可。优选地，所述人工感染的步骤包括：将白纹伊蚊卵的胞浆以显微注射的方式注射到致倦库蚊卵中，并筛选出感染有所述wAlbA的致倦库蚊。其中，由于自然界中的白纹伊蚊通常携带wAlbA和/或wAlbB，而且即便注射株的白纹伊蚊同时携带wAlbA和wAlbB二者，其人工感染的后代也不一定会同时携带二者，并且更倾向于仅携带其中一种，例如仅携带wAlbA或wAlbB，因此，为了单独研究携带wAlbB的致倦库蚊的生物学效果，可以在显微注射并培育成成虫后进行筛选的步骤，以筛选出携带wAlbB而不携带wAlbA的致倦库蚊。
根据本发明，对于所述显微注射的量没有特别的限制，只要能够让待人工感染的致倦库蚊成功感染白纹伊蚊的wAlbB即可。优选地，所述显微注射的量为1*10-5-1.5*10-5μL。
在一个优选的实施方式中，考虑到致倦库蚊可能自然感染的Wolbachia对CI现象也会产生一定的影响，例如，如果对自然感染有Wolbachia的致倦库蚊直接人工感染白纹伊蚊的wAlbB，可能会导致本发明中得到的人工感染的致倦库蚊的CI强度降低。为了排除上述影响，可以在人工感染之前包括筛选未自然感染有Wolbachia的致倦库蚊的步骤。
在另一个优选的实施方式中，可以在人工感染之前通过本领域技术人员熟知的各种方法对待人工感染的致倦库蚊群体进行一定的选择或处理，以除去待人工感染的致倦库蚊中自然感染的Wolbachia，从而得到未感染有Wolbachia的致倦库蚊。优选地，生产致倦库蚊的方法还包括：在人工感染白纹伊蚊的wAlbB之前，用抗生素处理所述致倦库蚊。
其中，经过抗生素处理的步骤可以以各种方法进行，只要能够将抗生素作用于致倦库蚊体内自然感染的Wolbachia以将其除去即可。优选地，用抗生素处理所述致倦库蚊的步骤包括：用抗生素浸泡饲料，并将处理过的饲料喂食给所 述致倦库蚊。从而将抗生素引入这些致倦库蚊体内，并除去这些致倦库蚊体内自然感染的Wolbachia。
此外，在使用如上所述的方法来用抗生素处理致倦库蚊时，对抗生素的种类和浓度也没有特别的限制，只要其能够除去Wolbachia即可。在一个优选的实施方式中，所述抗生素为四环素、青霉素和磷霉素中的至少一种。在另一个优选的实施方式中，所述抗生素的浓度为0.5-2重量％。
根据本发明，在通过本发明的方法得到本发明的人工感染有白纹伊蚊的wAlbB的致倦库蚊后，为了进一步扩大本发明的致倦库蚊的种群数量，优选地，可以进一步包括：将人工感染有白纹伊蚊的wAlbB的致倦库蚊进行大规模饲养。通过在合适的饲养条件下进行大规模饲养，可以增大本发明的致倦库蚊的种群数量，从而可以将其投放于自然界中以有效地控制自然界中致倦库蚊的后代产生和种群数量。
具体地，在实际的操作过程中，根据本发明的生产致倦库蚊的方法可以包括以下步骤：
直接从自然界中收集致倦库蚊和白纹伊蚊，在收集致倦库蚊和白纹伊蚊后，将它们在实验室条件下进行饲养，以为后续的产卵做准备；将用抗生素处理过的饲料(例如含1重量％四环素的糖水)喂食给致倦库蚊，从而清除致倦库蚊体内的Wolbachia，以免致倦库蚊自然感染的Wolbachia对感染以及测试结果产生影响；经过预处理后，选取若干致倦库蚊和白纹伊蚊分别放入产卵杯中，让其产卵45-60min；其中，由于致倦库蚊通常在夜间产卵，且用于注射的卵需要在短时间内提供，因此可以使用培养箱为致倦库蚊提供产卵所需的夜间环境以促进产卵；在收集完致倦库蚊和白纹伊蚊的卵之后，还可以对致倦库蚊和白纹伊蚊的卵进行排列，例如将致倦库蚊和白纹伊蚊分两列排序或将所有卵的尾部朝向相同等，这样可以使得后续的显微注射更加方便；此外，在显微注射前可以用水饱和油覆盖卵表面，以防止卵的过度干燥；在显微注射过程中，可以在显微镜下用显微注射仪吸取供体卵(白纹伊蚊卵)的尾部尖头的胞浆，然后注射到 受体卵(致倦库蚊卵)的尾部尖头中，并将如前所述排列的卵分别进行吸取和注射；注射完毕后，可以挑去供体卵以及个别没有进行注射的受体卵，以得到显微注射有白纹伊蚊卵的胞浆的致倦库蚊卵，其中，所述显微注射的量为1*10-5-1.5*10-5μL；最后将致倦库蚊卵转移到约27℃、80％RH的条件下进行培养孵化，以得到本发明的人工感染有白纹伊蚊的wAlbB的致倦库蚊。
以下将通过实施例对本发明进行详细描述。
实施例1
从自然界采集致倦库蚊和白纹伊蚊，并在实验室中饲养，在饲养中，使用含有1重量％四环素的糖水喂食给致倦库蚊。选取成虫5天的致倦库蚊和白纹伊蚊雌虫各10只分别放入产卵中，使其产卵60min，其中，装有致倦库蚊雌虫的产卵杯置于设置有夜间环境条件的培养箱中以促进产卵。收集致倦库蚊和白纹伊蚊的卵并转移到湿润的滤纸上，将致倦库蚊卵和白纹伊蚊卵分两列排序，并且所有卵的尾部朝向相同。将带有排列好蚊卵的厚滤纸反转贴在有双面胶的玻片上，并轻轻按压，使蚊卵粘在双面胶上以转移卵。将蚊卵在室温中干燥1min左右。用水饱和油覆盖卵表面，防止进一步干燥。将带有卵的玻片置于目镜10×和物镜20×的显微镜下，通过显微注射仪吸取供体卵的尾部尖头胞浆，然后注射进入受体卵的尾部尖头。依次将所排列的卵分别进行吸取和注射。其中，显微注射的量为1.5*10-5μL。注射完毕后，挑去供体卵以及个别没有进行注射的受体卵，将粘有致倦库蚊卵的双面胶从玻片上轻轻的撕下来，并将其保存到27℃、80％RH的玻璃管中。保存7天后，将蚊卵置于含有孵化液的清水中进行孵化，待孵化后即转移到含有食物的清水中进行饲养，待5龄后分单管饲养，待成虫后即得到G0代致倦库蚊。
实施例2
按照与实施例1相同的方式得到G0代致倦库蚊，不同的是，使用的抗生素为0.5重量％的青霉素，显微注射的量为1*10-5μL。
实施例3
按照与实施例1相同的方式得到G0代致倦库蚊，不同的是，使用的抗生素为2重量％的磷霉素，显微注射的量为1.2*10-5μL。
实施例4
按照与实施例1相同的方式得到G0代致倦库蚊，不同的是，不使用抗生素处理致倦库蚊。
感染率的测试方法：
分别选取100只实施例1-4中得到的G0代致倦库蚊，将G0代雄性致倦库蚊直接进行PCR检测，G0代雌性致倦库蚊与未自然感染的雄性致倦库蚊交配产卵后再进行PCR检测，以得到G0代致倦库蚊的感染率(即感染有wAlbB而没有wAlbA的数量占总数的比例)，具体结果参见表1。接着，将G0代雌性致倦库蚊与未自然感染的雄性致倦库蚊的交配产卵饲养5龄时分单管饲养，待成虫后即得到G1代致倦库蚊，同样地，分别选取100只实施例1-4中得到的G1代致倦库蚊(均为阳性即感染成功的G1代致倦库蚊)，G1代雄性致倦库蚊直接进行PCR检测，G1代雌性致倦库蚊与未自然感染的雄性致倦库蚊交配产卵后再进行PCR检测，具体结果也参见表1。重复上述步骤，以得到G2和G3代致倦库蚊并检测其感染率，具体结果也参见表1。此外，人工感染后的致倦库蚊的PCR检测的代表性结果示于图1，从图1中可以看出：样本1-5在wAlbB处呈阳性，而在wAlbA处呈阴性，即表示这些样本携带wAlbB，而不携带wAlbA。
表1
感染率 实施例1 实施例2 实施例3 实施例4
G0 30％ 24％ 26％ 27％
G1 50％ 48％ 48％ 46％
G2 100％ 100％ 100％ 100％
G3 100％ 100％ 100％ 100％
CI强度的检测方法：
选取10只实施例1-4中得到的G3代雄性致倦库蚊与10只野生型雌性致倦 库蚊进行交配产卵，并测量卵的孵化率(％)，而CI强度＝1-孵化率。具体结果参见表2。
表2
  实施例1 实施例2 实施例3 实施例4
孵化的卵数 85 90 84 92
总卵数 757 728 740 770
孵化率 11.23％ 12.36％ 11.54％ 11.95％
CI强度 88.77％ 87.64％ 88.46％ 88.05％
同样地，选取10只实施例1-4中得到的G3代雌性致倦库蚊与10只野生型雄性致倦库蚊进行交配产卵，并测量卵的孵化率(％)和CI强度。具体结果参见表3。
表3
  实施例1 实施例2 实施例3 实施例4
孵化的卵数 3 4 5 600
总卵数 677 680 701 695
孵化率 0.44％ 0.59％ 0.71％ 86.3％
CI强度 99.56％ 99.41％ 99.29％ 13.7％
从表1的结果可以看出，根据本发明的方法得到的G0代致倦库蚊分别表现出30％、24％、26％和27％的感染率，而G1代致倦库蚊分别表现出50％、48％、48％和46％的感染率(相比于G0代明显增加)，G2代致倦库蚊均表现出100％的感染率，即G2代致倦库蚊全部人工感染有Wolbachia，G3代致倦库蚊结果同G2代致倦库蚊，因此根据本发明的方法人工感染致倦库蚊，只需在人工感染后再繁殖两代即可得到感染率为100％的致倦库蚊种群。
从表2和表3的结果可以看出，当将G3代雄性致倦库蚊与野生型雌性致倦库蚊进行交配产卵时，孵化率较低(均在11％-13％)，即本发明的人工感染有Wolbachia的雄性致倦库蚊与野生型雌性致倦库蚊存在胞质不相容性，且CI强度较高，能够大幅地减少后代的产生；当将G3代雌性致倦库蚊与野生型雄性致 倦库蚊进行交配产卵时，孵化率极低(优选地均在1％以下)，即本发明的人工感染有Wolbachia的雌性致倦库蚊与野生型雄性致倦库蚊存在胞质不相容性；综上，本发明的致倦库蚊表现出双向CI特性，且雌性的CI强度比雄性的CI强度更高。此外，从实施例4的结果中可以看出，在人工感染前不使用抗生素处理致倦库蚊时，致倦库蚊自身携带的Wolbacia对雄蚊CI结果几乎无影响，但会对雌蚊CI结果造成很大的影响，因此在优选的情况下可以对人工感染前的致倦库蚊进行抗生素处理。
综上所述，根据本发明的方法能够容易地得到人工感染有Wolbachia的致倦库蚊，且得到的致倦库蚊的CI强度较高，因此如果将本发明的致倦库蚊投放到自然界中，可以大幅降低致倦库蚊的产卵率，从而有效地控制致倦库蚊的后代产生和种群数量，从而达到有效减少流行性乙型脑炎等蚊媒病的发生及传播的目的。
以上详细描述了本发明的优选实施方式，但是，本发明并不限于上述实施方式中的具体细节，在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，这些简单变型均属于本发明的保护范围。
另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本发明对各种可能的组合方式不再另行说明。
此外，本发明的各种不同的实施方式之间也可以进行任意组合，只要其不违背本发明的思想，其同样应当视为本发明所公开的内容。

Claims (17)

  1. 一种致倦库蚊，其中，所述致倦库蚊被人工感染有白纹伊蚊的wAlbB。
  2. 根据权利要求1所述的致倦库蚊，其中，所述人工感染是通过将白纹伊蚊卵的胞浆以显微注射的方式注射到致倦库蚊卵中，并筛选出感染有所述wAlbB的致倦库蚊来实现的。
  3. 根据权利要求2所述的致倦库蚊，其中，所述显微注射的量为1*10-5-1.5*10-5μL。
  4. 根据权利要求1所述的致倦库蚊，其中，在被人工感染有白纹伊蚊的wAlbB之前，所述致倦库蚊未自然感染有Wolbachia。
  5. 根据权利要求1所述的致倦库蚊，其中，在被人工感染有白纹伊蚊的wAlbB之前，所述致倦库蚊经过抗生素处理。
  6. 根据权利要求5所述的致倦库蚊，其中，所述抗生素处理通过将抗生素浸泡饲料，并将处理过的饲料喂食给所述致倦库蚊来进行。
  7. 根据权利要求5所述的致倦库蚊，其中，所述抗生素为四环素、青霉素和磷霉素中的至少一种。
  8. 根据权利要求5所述的致倦库蚊，其中，所述抗生素的浓度为0.5-2重量％。
  9. 根据权利要求1所述的致倦库蚊，其中，所述致倦库蚊与未被人工感染的致倦库蚊存在胞质不相容性。
  10. 一种生产致倦库蚊的方法，包括：
    使所述致倦库蚊人工感染白纹伊蚊的wAlbB。
  11. 根据权利要求10所述的方法，其中，所述人工感染的步骤包括：将白纹伊蚊卵的胞浆以显微注射的方式注射到致倦库蚊卵中，并筛选出感染有所述wAlbB的致倦库蚊。
  12. 根据权利要求11所述的方法，其中，所述显微注射的量为 1*10-5-1.5*10-5μL。
  13. 根据权利要求10所述的方法，进一步包括：
    在人工感染白纹伊蚊的wAlbB之前，用抗生素处理所述致倦库蚊。
  14. 根据权利要求13所述的方法，其中，用抗生素处理所述致倦库蚊的步骤包括：用抗生素浸泡饲料，并将处理过的饲料喂食给所述致倦库蚊。
  15. 根据权利要求13所述的方法，其中，所述抗生素为四环素、青霉素和磷霉素中的至少一种。
  16. 根据权利要求13所述的方法，其中，所述抗生素的浓度为0.5-2重量％。
  17. 根据权利要求10所述的方法，进一步包括：将人工感染有白纹伊蚊的wAlbB的致倦库蚊进行大规模饲养。
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