WO2019028585A1 - 一种白纹伊蚊及其生产方法 - Google Patents

一种白纹伊蚊及其生产方法 Download PDF

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WO2019028585A1
WO2019028585A1 PCT/CN2017/096169 CN2017096169W WO2019028585A1 WO 2019028585 A1 WO2019028585 A1 WO 2019028585A1 CN 2017096169 W CN2017096169 W CN 2017096169W WO 2019028585 A1 WO2019028585 A1 WO 2019028585A1
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aedes albopictus
antibiotic
culex pipiens
wpip
eggs
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PCT/CN2017/096169
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English (en)
French (fr)
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侯汲虹
梁永康
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/CN2017/096169 priority Critical patent/WO2019028585A1/zh
Publication of WO2019028585A1 publication Critical patent/WO2019028585A1/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 an Aedes albopictus and a production method thereof.
  • Aedes albopictus is an important vector for various diseases such as dengue fever, yellow fever, chikungunya fever and West Nile fever.
  • various diseases such as dengue fever, yellow fever, chikungunya fever and West Nile fever.
  • 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.
  • the prevention and control of the vector Aedes albopictus is the main means to prevent and control the dengue epidemic.
  • 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, so there are still many in this study. Difficulties and challenges.
  • the purpose of the present invention is to overcome the prior art that no specific drugs and vaccines can be Effectively prevent the defects of mosquito-borne diseases such as dengue fever, and provide an artificially infected Aedes albopictus, which can effectively inhibit the population of Aedes albopictus in nature and reduce the occurrence and spread of mosquito-borne diseases such as dengue fever from the source. And provide a method for cultivating and producing the artificially infected Aedes albopictus.
  • the present invention provides an Aedes albopictus which is artificially infected with wpip of Culex pipiens.
  • the artificial infection is achieved by injecting the cytoplasm of Culex pipiens pipiens eggs into Aedes albopictus eggs by microinjection.
  • the amount of microinjection is from 1*10 -5 to 1.5*10 -5 ⁇ L.
  • the Aedes albopictus is not naturally infected with Wolbachia prior to being artificially infected with wpip of Culex pipiens pallens.
  • the Aedes albopictus is treated with antibiotics prior to being artificially infected with wpip of Culex pipiens pallens.
  • the antibiotic treatment can be carried out by soaking the antibiotic with the feed and feeding the treated feed to the Aedes albopictus.
  • 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 Aedes albopictus has cytoplasmic incompatibility with Aedes albopictus which is not artificially infected.
  • the present invention provides a method of producing Aedes albopictus comprising: artificially infecting Aedes albopictus to cause wpip of Culex pipiens pallens.
  • the step of artificially infecting comprises: injecting the cytoplasm of Culex pipiens pipiens eggs into Aedes albopictus eggs by microinjection.
  • the amount of microinjection is from 1*10 -5 to 1.5*10 -5 ⁇ L.
  • the method further comprises: treating the Aedes albopictus with an antibiotic prior to artificially infecting wpip of Culex pipiens pallens.
  • the step of treating the Aedes albopictus with an antibiotic comprises: soaking the feed with an antibiotic and feeding the treated feed to the Aedes albopictus.
  • 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: large-scale breeding of Aedes albopictus infected with wpip artificially infected with Culex pipiens pallens.
  • an Aedes albopictus artificially infected with Wpip of Culex pipiens pallens with extremely high CI intensity (for example, 90-99.9%), which is obtained by the Aedes albopictus of the present invention or by the method of the present invention.
  • Aedes albopictus is placed in nature, which can significantly reduce the rate of oviposition of Aedes albopictus, thus effectively controlling the generation and population of Aedes albopictus, thus effectively reducing the occurrence and spread of mosquito-borne diseases such as dengue fever. .
  • Fig. 1 is a graph showing representative results of PCR detection of Aedes albopictus after artificial infection.
  • the invention provides an Aedes albopictus, which is artificially infected with wpip of Culex pipiens.
  • wpip is a subtype of Wolbachia, a kind of maternal inheritance of Gram-negative bacteria.
  • the Ips of Aedes albopictus which is artificially infected with Culex pipiens pallens, has a very high CI intensity (for example, 90-99.9%), and is placed in nature to substantially reduce the spawning of Aedes albopictus in nature.
  • the rate thus effectively controlling the generation and population of offspring of Aedes albopictus, can effectively reduce the occurrence and spread of mosquito-borne diseases such as dengue fever.
  • Aedes albopictus can be artificially infected by various artificial infection methods well known to those skilled in the art, as long as the wpip carried by Culex pipiens can be introduced into Aedes albopictus.
  • the artificial infection is achieved by injecting the cytoplasm of Culex pipiens pipiens eggs into Aedes albopictus eggs by microinjection.
  • the amount of the microinjection is not particularly limited as long as the Aedes albopictus to be artificially infected can be successfully infected with the wpip of Culex pipiens.
  • the amount of microinjection is from 1*10 -5 to 1.5*10 -5 ⁇ L.
  • the CI phenomenon may be certain.
  • the effect for example, if the wpip of Culex pipiens pallens is directly artificially infected with A. cerevisiae which is naturally infected with Wolbachia, the CI intensity of the artificially infected Aedes albopictus obtained in the present invention may be lowered.
  • Aedes albopictus which is not naturally infected with Wolbachia, can be screened prior to artificial infection.
  • the Aedes albopictus is not naturally infected with Wolbachia prior to being artificially infected with wpip of Culex pipiens pallens.
  • the artificially infected Aedes albopictus population can be selected or treated by various methods well known to those skilled in the art prior to artificial infection to remove Aedes albopictus to be artificially infected.
  • Naturally infected Wolbachia resulting in uninfected Wolbachia Aedes albopictus.
  • the Aedes albopictus is treated with antibiotics prior to being artificially infected with wpip of Culex pipiens.
  • the step of antibiotic treatment can be carried out in various ways as long as the antibiotic can be applied to Wolbachia which is naturally infected in Aedes albopictus to be removed.
  • the antibiotic treatment can be carried out by soaking the antibiotic with the feed and feeding the treated feed to the Aedes albopictus. Antibiotics were thus introduced into these Aedes albopictus and Wolbachia, which is naturally infected in the Aedes albopictus, was removed.
  • 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 artificially infected Aedes albopictus with Wolbachia has a high CI intensity, and there is often a cytoplasmic incompatibility at the time of mating, so that the population of Aedes albopictus can be effectively controlled.
  • the Aedes albopictus has cytoplasmic incompatibility with Aedes albopictus which is not artificially infected.
  • the present invention provides a method of producing Aedes albopictus comprising: artificially infecting Aedes albopictus to cause wpip of Culex pipiens pallens.
  • Aedes albopictus can be artificially infected by various artificial infection methods well known to those skilled in the art, as long as the wpip carried by Culex pipiens can be introduced into Aedes albopictus.
  • the step of artificially infecting comprises: injecting the cytoplasm of Culex pipiens pipiens eggs into Aedes albopictus eggs by microinjection.
  • the amount of the microinjection is not particularly limited as long as the Aedes albopictus to be artificially infected can be successfully infected with the wpip of Culex pipiens.
  • the amount of microinjection is from 1*10 -5 to 1.5*10 -5 ⁇ L.
  • the CI phenomenon may be certain. Effects, for example, if the wpip of Culex pipiens pallens is directly infected with A. cerevisiae, which is naturally infected with Wolbachia, may result in artificially infected whites obtained in the present invention.
  • the A. sinensis has a reduced CI intensity.
  • the artificially infected Aedes albopictus population can be selected or treated by various methods well known to those skilled in the art prior to artificial infection to remove Aedes albopictus to be artificially infected.
  • Wolbachia which is naturally infected, gives Aedes albopictus uninfected with Wolbachia.
  • the method for producing Aedes albopictus further comprises: treating the Aedes albopictus with an antibiotic prior to artificially infecting wpip of Culex pipiens pallens.
  • the step of antibiotic treatment can be carried out in various ways as long as the antibiotic can be applied to Wolbachia which is naturally infected in Aedes albopictus to be removed.
  • the step of treating the Aedes albopictus with an antibiotic comprises: soaking the feed with an antibiotic and feeding the treated feed to the Aedes albopictus. Antibiotics were thus introduced into these Aedes albopictus and Wolbachia, which is naturally infected in the Aedes albopictus, was removed.
  • 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 the Aedes albopictus of the present invention after the artificial infection of the Wpip Aedes aegypti with the Culex pipiens pallens of the present invention by the method of the present invention, it may preferably further comprise: Aedes albopictus, which is artificially infected with wpip of Culex pipiens pallens, was raised on a large scale. By carrying out large-scale rearing under suitable feeding conditions, the population of Aedes albopictus of the present invention can be increased, so that it can be placed in nature to effectively control the generation and population of Aedes albopictus in nature. .
  • the method of producing Aedes albopictus according to the present invention may include the following steps during actual operation:
  • Aedes albopictus and Culex pipiens pallens are collected directly from nature. After collecting Aedes albopictus and Culex pipiens pallens, they are raised under laboratory conditions to prepare for subsequent spawning; they will be treated with antibiotics.
  • the feed (for example, syrup containing 1% by weight of tetracycline) is fed to Aedes albopictus to remove Wolbachia from Aedes albopictus, so as to prevent Wolbachia, which is naturally infected by Aedes albopictus, from affecting infection and test results; Afterwards, several Aedes albopictus and Culex pipiens pallens 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 Aedes albopictus and Culex pipiens, Aedes albopictus Arrange with the eggs of Culex pipiens, such as sorting two rows of Aedes albopictus and Culex pipiens pallens or pointing the tails of all eggs to the same, etc., 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 the microinjection, the cytoplasm of the tip of the donor egg (the Culex pipiens) can be aspirated under a microscope with a microinjector, and then Injection into the tip of the recipient egg (Aedes albopictus), and the eggs
  • Somatic eggs to obtain microscopic injection of A. albopictus eggs with the cytoplasm of Culex pipiens pipiens eggs, wherein the amount of microinjection is 1*10 -5 -1.5*10 -5 ⁇ L;
  • the Aedes mosquitoes were transferred to a culture condition of about 27 ° C and 80% RH to incubate to obtain the WPip Aedes aegypti artificially infected with Culex pipiens pallens.
  • Aedes albopictus and Culex pipiens pallens were collected from nature and kept in the laboratory. In the breeding, Aedes albopictus was fed with syrup containing 1% by weight of tetracycline. 10 adults of Aedes albopictus and Culex pipiens sinensis, which were selected for 5 days, were placed in spawning for 60 min. Among them, the spawning cup containing the female Culex pipiens was placed at night. Environmental conditions in the incubator to promote spawning.
  • the eggs of Aedes albopictus and Culex pipiens pallens were collected and transferred to moist filter paper, and the Aedes albopictus eggs and Culex pipiens pallens 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 the eyepiece 10 ⁇ and the 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 Aedes albopictus eggs is gently torn off from the slide and stored at 27 ° C, 80%. RH in the glass tube.
  • the mosquito 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 old, they are kept in single tube, and G0 white lines are obtained after adult. Aedes.
  • G0 generation Aedes albopictus 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 Aedes albopictus 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 generation Aedes albopictus was obtained in the same manner as in Example 1, except that Aedes albopictus was treated without using an antibiotic.
  • Genotype Aedes albopictus obtained in Examples 1-4 were selected, and G0 male Aedes albopictus was directly tested by PCR.
  • G0 female Aedes albopictus was mated with unnaturally infected male Aedes albopictus.
  • PCR was performed after spawning to obtain the infection rate of G0 generation Aedes albopictus. See Table 1 for the specific results. Then, the G0 generation female Aedes albopictus and the unnaturally infected male Aedes albopictus were reared and reared at the age of 5, and the G1 generation Aedes albopictus was obtained after the adult. Similarly, 100 were selected respectively.
  • G1 generation Aedes albopictus obtained in Examples 1-4 (all positive, that is, G1 generation Aedes albopictus), G1 generation Aedes albopictus was directly tested by PCR, G1 generation Aedes albopictus PCR was performed after mating with unnaturally infected male Aedes albopictus, and the results are also shown in Table 1. The above steps were repeated to obtain G2 and G3 Aedes albopictus and their infection rates were examined. See Table 1 for specific results. In addition, representative results of PCR detection of Aedes albopictus after artificial infection are shown in Fig. 1, as can be seen from Fig. 1: injection Strain 1-4 was positive at wpip, indicating successful carrying of wpip with Culex pipiens.
  • Example 1 Example 2
  • Example 3 Example 4
  • Number of eggs hatched 11 15 18 17 Total number of eggs 714 725 695 709 Hatching rate 1.54% 2.1% 2.6% 2.4% CI strength 98.46% 97.9% 97.4% 97.6%
  • Example 1 Example 2
  • Example 3 Example 4
  • Number of eggs hatched 8 15 17 648 Total number of eggs 687 700 680 675 Hatching rate 1.16% 2.1% 2.5% 96% CI strength 98.84% 97.9% 97.5% 4%
  • G2 generation Aedes albopictus showed 100% infection rate, that is, G2 generation Aedes albopictus was all artificially infected with Wolbachia, G3 generation Aedes albopictus was the same as G2 generation Aedes albopictus, so artificial according to the method of the present invention Infected with Aedes albopictus, it is only necessary to breed two generations after artificial infection to obtain a population of Aedes albopictus with an infection rate of 100%.
  • Aedes albopictus has cytoplasmic incompatibility; in summary, the Aedes albopictus of the present invention exhibits bidirectional CI characteristics.
  • Wolbacia carried by Aedes albopictus has almost no effect on the results of male CI, but it will be CI As a result, a large effect is exerted, so that the Aedes albopictus before artificial infection can be treated with antibiotics in a preferred case.
  • Aedes albopictus artificially infected with Wolbachia can be easily obtained, and the obtained Aedes albopictus has extremely high CI intensity, so if the Aedes albopictus of the present invention is placed in nature In this way, the oviposition rate of Aedes albopictus can be greatly reduced, thereby effectively controlling the generation and population of Aedes albopictus, thereby effectively reducing the occurrence and spread of mosquito-borne diseases such as dengue fever.

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Abstract

一种白纹伊蚊及其生产方法，包括：使白纹伊蚊人工感染致倦库蚊的wpip。通该方法，可以得到CI强度极高(例如90-99.9％)的人工感染有致倦库蚊的wpip的白纹伊蚊，将该白纹伊蚊或通过该方法得到的白纹伊蚊投放于自然界中，可以大幅降低白纹伊蚊的产卵率，从而有效地控制白纹伊蚊的后代产生和种群数量，从而可以有效地减少登革热等蚊媒病的发生及传播。

Description

一种白纹伊蚊及其生产方法 技术领域
本发明属于人工感染领域，具体地，本发明涉及一种白纹伊蚊及其生产方法。
背景技术
白纹伊蚊(Aedes albopictus)是登革热、黄热病、基孔肯雅热和西尼罗热等多种疾病的重要传播媒介。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。当前，由于登革热及登革出血热尚无特效药物和疫苗，对传播媒介白纹伊蚊的防制是预防和控制登革热疫情的主要手段。
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尚无特效药物和疫苗能够有效地预防登革热等蚊媒病的缺陷，提供一种人工感染的白纹伊蚊，其能够有效地抑制自然界中白纹伊蚊的种群数量，从源头上减少登革热等蚊媒病的发生及传播，并提供了该种人工感染的白纹伊蚊的培育生产方法。
为了实现上述目的，在一方面，本发明提供了一种白纹伊蚊，所述白纹伊蚊被人工感染有致倦库蚊的wpip。
在一个优选的实施方式中，所述人工感染是通过将致倦库蚊卵的胞浆以显微注射的方式注射到白纹伊蚊卵中来实现的。
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，在被人工感染有致倦库蚊的wpip之前，所述白纹伊蚊未自然感染有Wolbachia。
在一个优选的实施方式中，在被人工感染有致倦库蚊的wpip之前，所述白纹伊蚊经过抗生素处理。
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白纹伊蚊来进行。
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，所述白纹伊蚊与未被人工感染的白纹伊蚊存在胞质不相容性。
在另一方面，本发明提供了一种生产白纹伊蚊的方法，包括：使所述白纹伊蚊人工感染致倦库蚊的wpip。
在一个优选的实施方式中，所述人工感染的步骤包括：将致倦库蚊卵的胞浆以显微注射的方式注射到白纹伊蚊卵中。
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：在人工感染致倦库蚊的wpip之前，用抗生素处理所述白纹伊蚊。
在一个优选的实施方式中，用抗生素处理所述白纹伊蚊的步骤包括：用抗生素浸泡饲料，并将处理过的饲料喂食给所述白纹伊蚊。
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：将人工感染有致倦库蚊的wpip的白纹伊蚊进行大规模饲养。
通过本发明的方法，可以得到CI强度极高(例如90-99.9％)的人工感染有致倦库蚊的wpip的白纹伊蚊，将本发明的白纹伊蚊或通过本发明的方法得到的白纹伊蚊投放于自然界中，可以大幅降低白纹伊蚊的产卵率，从而有效地控制白纹伊蚊的后代产生和种群数量，从而可以有效地减少登革热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白纹伊蚊的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，本发明提供了一种白纹伊蚊，所述白纹伊蚊被人工感染有致倦库蚊的wpip。
其中，wpip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致倦库蚊的wpip的白纹伊蚊的CI强度极高(例如90-99.9％)，将其投放于自然界中，可以大幅降低自然界中白纹伊蚊的产卵率，从而有效地控制白纹伊蚊的后代产生和种群数量，从而可以有效地减少登革热等蚊媒病的发生及传播。
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白纹伊蚊，只要能够将致倦库蚊携带的wpip引入白纹伊蚊即可。优选地，所述人工感染是通过将致倦库蚊卵的胞浆以显微注射的方式注射到白纹伊蚊卵中来实现的。此外，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白纹伊蚊成功感染致倦库蚊的wpip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，考虑到白纹伊蚊可能自然感染的Wolbachia(可能包括wAlbA和wAlbB中的至少一种，其根据种群以及地理位置等都会有所差异)对CI现象会产生一定的影响，例如，如果对自然感染有Wolbachia的白纹伊蚊直接人工感染致倦库蚊的wpip，可能会导致本发明中得到的人工感染的白纹伊蚊的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的白纹伊蚊。优选地，在被人工感染有致倦库蚊的wpip之前，所述白纹伊蚊未自然感染有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白纹伊蚊群体进行一定的选择或处理，以除去待人工感染的白纹伊蚊中自然感染的Wolbachia，从而得到未感染有Wolbachia的 白纹伊蚊。优选地，在被人工感染有致倦库蚊的wpip之前，所述白纹伊蚊经过抗生素处理。
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白纹伊蚊体内自然感染的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白纹伊蚊来进行。从而将抗生素引入这些白纹伊蚊体内，并除去这些白纹伊蚊体内自然感染的Wolbachia。
此外，在使用如上所述的方法来用抗生素处理白纹伊蚊时，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的白纹伊蚊具有较高的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白纹伊蚊的种群数量。
优选地，所述白纹伊蚊与未被人工感染的白纹伊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白纹伊蚊的方法，包括：使所述白纹伊蚊人工感染致倦库蚊的wpip。
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白纹伊蚊，只要能够将致倦库蚊携带的wpip引入白纹伊蚊即可。优选地，所述人工感染的步骤包括：将致倦库蚊卵的胞浆以显微注射的方式注射到白纹伊蚊卵中。此外，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白纹伊蚊成功感染致倦库蚊的wpip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，考虑到白纹伊蚊可能自然感染的Wolbachia(可能包括wAlbA和wAlbB中的至少一种，其根据种群以及地理位置等都会有所差异)对CI现象会产生一定的影响，例如，如果对自然感染有Wolbachia的白纹伊蚊直接人工感染致倦库蚊的wpip，可能会导致本发明中得到的人工感染的白 纹伊蚊的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的白纹伊蚊的步骤。
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白纹伊蚊群体进行一定的选择或处理，以除去待人工感染的白纹伊蚊中自然感染的Wolbachia，从而得到未感染有Wolbachia的白纹伊蚊。优选地，生产白纹伊蚊的方法还包括：在人工感染致倦库蚊的wpip之前，用抗生素处理所述白纹伊蚊。
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白纹伊蚊体内自然感染的Wolbachia以将其除去即可。优选地，用抗生素处理所述白纹伊蚊的步骤包括：用抗生素浸泡饲料，并将处理过的饲料喂食给所述白纹伊蚊。从而将抗生素引入这些白纹伊蚊体内，并除去这些白纹伊蚊体内自然感染的Wolbachia。
此外，在使用如上所述的方法来用抗生素处理白纹伊蚊时，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致倦库蚊的wpip的白纹伊蚊后，为了进一步扩大本发明的白纹伊蚊的种群数量，优选地，可以进一步包括：将人工感染有致倦库蚊的wpip的白纹伊蚊进行大规模饲养。通过在合适的饲养条件下进行大规模饲养，可以增大本发明的白纹伊蚊的种群数量，从而可以将其投放于自然界中以有效地控制自然界中白纹伊蚊的后代产生和种群数量。
具体地，在实际的操作过程中，根据本发明的生产白纹伊蚊的方法可以包括以下步骤：
直接从自然界中收集白纹伊蚊和致倦库蚊，在收集白纹伊蚊和致倦库蚊后，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)喂食给白纹伊蚊，从而清除白纹伊蚊体内的Wolbachia，以免白纹伊蚊自然感染的Wolbachia对感染以及测试结果产生影响；经过预处理后，选取若干白纹伊蚊和致倦库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；在收集完白纹伊蚊和致倦库蚊的卵之后，还可以对白纹伊蚊和致倦库蚊的卵进行排列，例如将白纹伊蚊和致倦库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(致倦库蚊卵)的尾部尖头的胞浆，然后注射到受体卵(白纹伊蚊卵)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，以得到显微注射有致倦库蚊卵的胞浆的白纹伊蚊卵，其中，所述显微注射的量为1*10-5-1.5*10-5μL；最后将白纹伊蚊卵转移到约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致倦库蚊的wpip的白纹伊蚊。
以下将通过实施例对本发明进行详细描述。
实施例1
从自然界采集白纹伊蚊和致倦库蚊，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白纹伊蚊。选取成虫5天的白纹伊蚊和致倦库蚊雌虫各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。收集白纹伊蚊和致倦库蚊的卵并转移到湿润的滤纸上，将白纹伊蚊卵和致倦库蚊卵分两列排序，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，将粘有白纹伊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代白纹伊蚊。
实施例2
按照与实施例1相同的方式得到G0代白纹伊蚊，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代白纹伊蚊，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代白纹伊蚊，不同的是，不使用抗生素处理白纹伊蚊。
感染率的测试方法：
分别选取100只实施例1-4中得到的G0代白纹伊蚊，将G0代雄性白纹伊蚊直接进行PCR检测，G0代雌性白纹伊蚊与未自然感染的雄性白纹伊蚊交配产卵后再进行PCR检测，以得到G0代白纹伊蚊的感染率，具体结果参见表1。接着，将G0代雌性白纹伊蚊与未自然感染的雄性白纹伊蚊的交配产卵饲养5龄时分单管饲养，待成虫后即得到G1代白纹伊蚊，同样地，分别选取100只实施例1-4中得到的G1代白纹伊蚊(均为阳性即感染成功的G1代白纹伊蚊)，G1代雄性白纹伊蚊直接进行PCR检测，G1代雌性白纹伊蚊与未自然感染的雄性白纹伊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代白纹伊蚊并检测其感染率，具体结果也参见表1。此外，人工感染后的白纹伊蚊的PCR检测的代表性结果示于图1，从图1中可以看出：注射 株1-4在wpip处呈现阳性，表明成功携带有致倦库蚊的wpip。
表1
感染率 实施例1 实施例2 实施例3 实施例4
G0 18％ 16％ 16％ 15％
G1 83％ 60％ 75％ 67％
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代雄性白纹伊蚊与10只野生型雌性白纹伊蚊进行交配产卵，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 11 15 18 17
总卵数 714 725 695 709
孵化率 1.54％ 2.1％ 2.6％ 2.4％
CI强度 98.46％ 97.9％ 97.4％ 97.6％
同样地，选取10只实施例1-4中得到的G3代雌性白纹伊蚊与10只野生型雄性白纹伊蚊进行交配产卵，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 8 15 17 648
总卵数 687 700 680 675
孵化率 1.16％ 2.1％ 2.5％ 96％
CI强度 98.84％ 97.9％ 97.5％ 4％
从表1的结果可以看出，根据本发明的方法得到的G0代白纹伊蚊分别表现 出18％、16％、16％和15％的感染率，而G1代白纹伊蚊分别表现出83％、60％、75％和67％的感染率(相比于G0代明显增加)，G2代白纹伊蚊均表现出100％的感染率，即G2代白纹伊蚊全部人工感染有Wolbachia，G3代白纹伊蚊结果同G2代白纹伊蚊，因此根据本发明的方法人工感染白纹伊蚊，只需在人工感染后再繁殖两代即可得到感染率为100％的白纹伊蚊种群。
从表2和表3的结果可以看出，当将G3代雄性白纹伊蚊与野生型雌性白纹伊蚊进行交配产卵时，孵化率极低(均在1％-3％)，即本发明的人工感染有Wolbachia的雄性白纹伊蚊与野生型雌性白纹伊蚊存在胞质不相容性，且CI强度极高，能够大幅地减少后代的产生；当将G3代雌性白纹伊蚊与野生型雄性白纹伊蚊进行交配产卵时，孵化率极低(优选地均在1％-3％)，即本发明的人工感染有Wolbachia的雌性白纹伊蚊与野生型雄性白纹伊蚊存在胞质不相容性；综上，本发明的白纹伊蚊表现出双向CI特性。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白纹伊蚊时，白纹伊蚊自身携带的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白纹伊蚊进行抗生素处理。
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的白纹伊蚊，且得到的白纹伊蚊的CI强度极高，因此如果将本发明的白纹伊蚊投放到自然界中，可以大幅降低白纹伊蚊的产卵率，从而有效地控制白纹伊蚊的后代产生和种群数量，从而达到有效减少登革热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. 一种白纹伊蚊，其中，所述白纹伊蚊被人工感染有致倦库蚊的wpip。
  2. 根据权利要求1所述的白纹伊蚊，其中，所述人工感染是通过将致倦库蚊卵的胞浆以显微注射的方式注射到白纹伊蚊卵中来实现的。
  3. 根据权利要求1所述的白纹伊蚊，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所述的白纹伊蚊，其中，在被人工感染有致倦库蚊的wpip之前，所述白纹伊蚊未自然感染有Wolbachia。
  5. 根据权利要求1所述的白纹伊蚊，其中，在被人工感染有致倦库蚊的wpip之前，所述白纹伊蚊经过抗生素处理。
  6. 根据权利要求5所述的白纹伊蚊，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白纹伊蚊来进行。
  7. 根据权利要求5所述的白纹伊蚊，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所述的白纹伊蚊，其中，所述抗生素的浓度为0.5-2重量％。
  9. 根据权利要求1所述的白纹伊蚊，其中，所述白纹伊蚊与未被人工感染的白纹伊蚊存在胞质不相容性。
  10. 一种生产白纹伊蚊的方法，包括：
    使所述白纹伊蚊人工感染致倦库蚊的wpip。
  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：将致倦库蚊卵的胞浆以显微注射的方式注射到白纹伊蚊卵中。
  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：
    在人工感染致倦库蚊的wpip之前，用抗生素处理所述白纹伊蚊。
  14. 根据权利要求13所述的方法，其中，用抗生素处理所述白纹伊蚊的步骤包括：用抗生素浸泡饲料，并将处理过的饲料喂食给所述白纹伊蚊。
  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：将人工感染有致倦库蚊的wpip的白纹伊蚊进行大规模饲养。
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CN101843245A (zh) * 2010-06-12 2010-09-29 成都军区疾病预防控制中心军事医学研究所 库蚊和伊蚊的引诱剂及应用方法
CN102851264A (zh) * 2011-06-30 2013-01-02 复旦大学 一种白纹伊蚊的Apyrase编码基因及其蛋白制备方法和用途
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CN106561577A (zh) * 2016-11-03 2017-04-19 广州威佰昆生物科技有限公司 稳定携带致倦库蚊沃尔巴克氏体的白纹伊蚊的制备方法及其在防治白纹伊蚊中的应用

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CN101843245A (zh) * 2010-06-12 2010-09-29 成都军区疾病预防控制中心军事医学研究所 库蚊和伊蚊的引诱剂及应用方法
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