WO2019051828A1 - 一种埃及伊蚊及其生产方法 - Google Patents

一种埃及伊蚊及其生产方法 Download PDF

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WO2019051828A1
WO2019051828A1 PCT/CN2017/102060 CN2017102060W WO2019051828A1 WO 2019051828 A1 WO2019051828 A1 WO 2019051828A1 CN 2017102060 W CN2017102060 W CN 2017102060W WO 2019051828 A1 WO2019051828 A1 WO 2019051828A1
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aedes aegypti
eggs
wpip
microinjection
culex pipiens
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PCT/CN2017/102060
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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/CN2017/102060 priority Critical patent/WO2019051828A1/zh
Publication of WO2019051828A1 publication Critical patent/WO2019051828A1/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
    • A01K67/30—Rearing or breeding invertebrates

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  • the present invention belongs to the field of artificial infection, and in particular, the present invention relates to an Aedes aegypti and a method for producing the same.
  • Aedes aegypti 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 aegypti 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.
  • 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 great harm.
  • the purpose of the present invention is to overcome the prior art that no specific drugs and vaccines can be Effectively preventing the defects of mosquito-borne diseases such as dengue fever, providing an artificially infected Aedes aegypti, which can effectively inhibit the population of Aedes aegypti in nature and reduce the occurrence and spread of mosquito-borne diseases such as dengue from the source, and An artificial production method for artificially infected Aedes aegypti is provided.
  • the present invention provides an Aedes aegypti, which is artificially infected with a wpip that harasses Culex.
  • the artificial infection is achieved by injecting the cytoplasm of the Culex pipiens eggs into the Aedes aegypti eggs by microinjection.
  • the amount of microinjection is from 1*10 -5 to 1.5*10 -5 ⁇ L.
  • the Aedes aegypti is male.
  • the Aedes aegypti has a cytoplasmic incompatibility with an Aedes aegypti that has not been artificially infected.
  • the invention provides a method of producing Aedes aegypti, comprising: artificially infecting Aedes aegypti to harassing wpip of Culex pipiens.
  • the step of artificially infecting comprises: injecting the cytoplasm of the Culex pipiens eggs into the Aedes aegypti eggs by microinjection.
  • the amount of microinjection is from 1*10 -5 to 1.5*10 -5 ⁇ L.
  • the method further comprises: large-scale breeding of Aedes aegypti, which is artificially infected with wpip that harassed Culex pipiens.
  • an Aedes aegypti (especially male) artificially infected with wpip that infects Culex pipiens, having a very high CI intensity (for example, 95-100%), or the Aedes aegypti of the present invention or through the present invention
  • the male Aedes aegypti obtained by the method is placed in nature and can significantly reduce the Aedes aegypti.
  • the rate of spawning thus effectively controlling the generation and population of offspring of Aedes aegypti, can effectively reduce 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 aegypti after artificial infection.
  • the invention provides an Aedes aegypti, which is artificially infected with a wpip that harasses Culex pipiens.
  • wpip is a subtype of Wolbachia, a kind of maternal inheritance of Gram-negative bacteria.
  • the Aedes aegypti which is artificially infected with wpip that is artificially infected with Culex pipiens, has an extremely high CI (for example, 95-100%), and is placed in nature to substantially reduce the rate of oviposition of Aedes aegypti in nature. Thereby effectively controlling the generation and population of offspring of Aedes aegypti, which can effectively reduce the occurrence and spread of mosquito-borne diseases such as dengue fever.
  • Aedes aegypti 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 aegypti.
  • the artificial infection is achieved by injecting the cytoplasm of the Culex pipiens eggs into the Aedes aegypti eggs by microinjection.
  • the amount of the microinjection is not particularly limited as long as the Aedes aegypti to be artificially infected can be successfully infected with wpip which harasses Culex.
  • the amount of microinjection is from 1*10 -5 to 1.5*10 -5 ⁇ L.
  • the inventors of the present invention have found that the Aedes aegypti, which is artificially infected with Wolbachia, has a very high CI intensity, especially when the Aedes aegypti is male, the CI intensity can reach 100%, and there is often a cytoplasm in the mating. Compatible, so it can effectively control the population of Aedes aegypti.
  • the Aedes aegypti is male. More preferably, the Aedes aegypti has cytoplasmic incompatibility with Aedes aegypti that has not been artificially infected.
  • the present invention provides a method of producing Aedes aegypti, comprising: artificially infecting Aedes aegypti to harass wpip of Culex pipiens.
  • Aedes aegypti 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 aegypti.
  • the step of artificially infecting comprises: injecting the cytoplasm of the Culex pipiens eggs into the Aedes aegypti eggs by microinjection.
  • the amount of the microinjection is not particularly limited as long as the Aedes aegypti to be artificially infected can be successfully infected with wpip which harasses Culex.
  • the amount of microinjection is from 1*10 -5 to 1.5*10 -5 ⁇ L.
  • the method further comprises: Large-scale breeding of Aedes aegypti with wpip that harassed Culex pipiens.
  • Large-scale breeding of Aedes aegypti with wpip that harassed Culex pipiens By carrying out large-scale rearing under suitable feeding conditions, the population of Aedes aegypti in the present invention can be increased, so that it can be placed in nature to effectively control the generation and population of Aedes aegypti in nature.
  • the method of producing Aedes aegypti according to the present invention may include the following steps during actual operation:
  • Mosquitoes provide the nighttime environment needed for spawning to promote spawning; after collecting Aedes aegypti and harassing Culex pipiens eggs, they can also arrange eggs of Aedes aegypti and Culex pipiens, such as Aedes aegypti and harassment Culex pipiens are sorted in two columns or the tails of all eggs are oriented the same, which makes subsequent microinjection more convenient; in addition, the surface of the egg can be covered with water-saturated oil before microinjection to prevent excessive drying of the eggs; During the microinjection, the cytoplasm of the tail tip of the donor egg (harassing Cucurbita eggs) can be aspirated under a microscope with a microinjector, and then injected into the tail tip of the recipient egg (Aedes aegypti egg).
  • the eggs arranged as described above are separately aspirated and injected; after the injection is completed, the donor eggs and the individual recipient eggs which are not injected can be picked up to obtain the cytoplasm of the microinjection of the Culex mosquito eggs.
  • Aedes aegypti eggs wherein the amount of microinjection is 1*10 -5 -1.5*10 -5 ⁇ L; finally, the eggs of Aedes aegypti are transferred to about 27 ° C, 80% RH for incubation.
  • Aedes aegypti and harassing Culex pipiens from nature, and rearing them in the laboratory.
  • 10 Aedes aegypti and 5 females of Culex pipiens were randomly placed in the eggs to lay eggs for 60 minutes.
  • Spawning cups containing female Culex pipiens are placed in an incubator equipped with nighttime environmental conditions to promote spawning.
  • Eggs of Aedes aegypti and Culex pipiens were collected and transferred to wet filter paper, and the Aedes aegypti eggs and Culex cunea eggs were sorted in 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 ⁇ L.
  • the donor eggs and the individual recipient eggs that were not injected were picked, and the double-sided tape with the Aedes aegypti eggs was gently torn off from the slide and stored at 27 ° C, 80% RH.
  • 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 after being adult, G0 is obtained. mosquito.
  • G0 generation Aedes aegypti was obtained in the same manner as in Example 1, except that the amount of microinjection was 1*10 -5 ⁇ L.
  • G0 generation Aedes aegypti was obtained in the same manner as in Example 1, except that the amount of microinjection was 1.2*10 -5 ⁇ L.
  • the G0 generation female Aedes aegypti and the wild-type male Aedes aegypti were reared and spawned at the age of 5, and the G1 generation Aedes aegypti was obtained after the adult. Similarly, 100 examples 1 were selected respectively.
  • G1 generation Aedes aegypti obtained in 3 (all positive, that is, G1 generation Aedes aegypti)
  • G1 generation male Aedes aegypti was directly tested by PCR
  • G1 generation female Aedes aegypti and wild type male Aedes aegypti were mated PCR detection was performed after the eggs, and the specific results are also shown in Table 1.
  • the above steps were repeated to obtain G2 and G3 generations of Aedes aegypti and their infection rates were examined. See Table 1 for specific results.
  • representative results of PCR detection of Aedes aegypti after artificial infection are shown in Fig. 1. It can be seen from Fig. 1 that the injected strains were positive at wpip, indicating that all of the injected strains successfully carried wpip.
  • Example 1 Example 2
  • Example 3 Number of eggs hatched 0 0 0
  • Example 1 Example 2 Example 3 Number of eggs hatched 685 693 670 Total number of eggs 718 720 700 Hatching rate 95.4% 96.3% 95.7% CI strength 4.6% 3.7% 4.3%
  • G2 generation Aedes aegypti showed 100% infection rate, that is, G2 generation Aedes aegypti were all artificially infected with Wolbachia, G3 generation Aedes aegypti results with G2 Aedes aegypti, thus artificially infecting Aedes aegypti according to the method of the present invention, only need to breed two generations after artificial infection to obtain an Aedes aegypti population with an infection rate of 100%.
  • Aedes aegypti artificially infected with Wolbachia can be easily obtained, and the obtained Aedes aegypti (especially male) has extremely high CI intensity, so if the male Aedes aegypti of the present invention is to be used Putting it into nature can greatly reduce the egg production rate of Aedes aegypti, thus effectively controlling the generation and population of Aedes aegypti, thus effectively reducing the occurrence and spread of mosquito-borne diseases such as dengue fever.

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Abstract

一种埃及伊蚊及其生产方法，所述埃及伊蚊被人工感染有骚扰库蚊的wpip。所述生产埃及伊蚊的方法包括：使埃及伊蚊人工感染骚扰库蚊的wpip。

Description

一种埃及伊蚊及其生产方法 技术领域
本发明属于人工感染领域，具体地，本发明涉及一种埃及伊蚊及其生产方法。
背景技术
埃及伊蚊(Aedes aegypti)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，对传播媒介埃及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埃及伊蚊，其能够有效地抑制自然界中埃及伊蚊的种群数量，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埃及伊蚊的培育生产方法。
为了实现上述目的，在一方面，本发明提供了一种埃及伊蚊，所述埃及伊蚊被人工感染有骚扰库蚊的wpip。
在一个优选的实施方式中，所述人工感染是通过将骚扰库蚊卵的胞浆以显微注射的方式注射到埃及伊蚊卵中来实现的。
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，所述埃及伊蚊为雄性。
在一个优选的实施方式中，所述埃及伊蚊与未被人工感染的埃及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埃及伊蚊的方法，包括：使所述埃及伊蚊人工感染骚扰库蚊的wpip。
在一个优选的实施方式中，所述人工感染的步骤包括：将骚扰库蚊卵的胞浆以显微注射的方式注射到埃及伊蚊卵中。
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：将人工感染有骚扰库蚊的wpip的埃及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95-100％)的人工感染有骚扰库蚊的wpip的埃及伊蚊(特别是雄性)，将本发明的雄性埃及伊蚊或通过本发明的方法得到的雄性埃及伊蚊投放于自然界中，可以大幅降低埃及伊蚊的 产卵率，从而有效地控制埃及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埃及伊蚊的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，本发明提供了一种埃及伊蚊，所述埃及伊蚊被人工感染有骚扰库蚊的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骚扰库蚊的wpip的埃及伊蚊的CI强度极高(例如95-100％)，将其投放于自然界中，可以大幅降低自然界中埃及伊蚊的产卵率，从而有效地控制埃及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埃及伊蚊，只要能够将骚扰库蚊携带的wpip引入埃及伊蚊即可。优选地，所述人工感染是通过将骚扰库蚊卵的胞浆以显微注射的方式注射到埃及伊蚊卵中来实现的。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埃及伊蚊成功感染骚扰库蚊的wpip即可。优选地，所述显微注射的量为 1*10-5-1.5*10-5μL。
在人工感染过程中，由于自然界中的埃及伊蚊本身不会携带任何Wolbachia，因此不会对人工感染的结果造成影响，从而也不需要进行任何除去Wolbachia的相关处理。
本发明的发明人发现，本发明的人工感染有Wolbachia的埃及伊蚊具有极高的CI强度，特别是当埃及伊蚊为雄性，CI强度甚至能达到100％，在交配时往往存在胞质不相容，因此能够有效地控制埃及伊蚊的种群数量。
优选地，所述埃及伊蚊为雄性。更优选地，所述埃及伊蚊与未被人工感染的埃及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埃及伊蚊的方法，包括：使所述埃及伊蚊人工感染骚扰库蚊的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埃及伊蚊，只要能够将骚扰库蚊携带的wpip引入埃及伊蚊即可。优选地，所述人工感染的步骤包括：将骚扰库蚊卵的胞浆以显微注射的方式注射到埃及伊蚊卵中。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埃及伊蚊成功感染骚扰库蚊的wpip即可。优选地，所述显微注射的量为1*10-5-1.5*10-5μL。
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骚扰库蚊的wpip的埃及伊蚊后，为了进一步扩大本发明的埃及伊蚊的种群数量，优选地，可以进一步包括：将人工感染有骚扰库蚊的wpip的埃及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，可以增大本发明的埃及伊蚊的种群数量，从而可以将其投放于自然界中以有效地控制自然界中埃及伊蚊的后代产生和种群数量。
具体地，在实际的操作过程中，根据本发明的生产埃及伊蚊的方法可以包括以下步骤：
直接从自然界中收集埃及伊蚊和骚扰库蚊，在收集埃及伊蚊和骚扰库蚊后， 将它们在实验室条件下进行饲养，以为后续的产卵做准备；选取若干埃及伊蚊和骚扰库蚊分别放入产卵杯中，让其产卵45-60min；其中，由于骚扰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骚扰库蚊提供产卵所需的夜间环境以促进产卵；在收集完埃及伊蚊和骚扰库蚊的卵之后，还可以对埃及伊蚊和骚扰库蚊的卵进行排列，例如将埃及伊蚊和骚扰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(骚扰库蚊卵)的尾部尖头的胞浆，然后注射到受体卵(埃及伊蚊卵)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，以得到显微注射有骚扰库蚊卵的胞浆的埃及伊蚊卵，其中，所述显微注射的量为1*10-5-1.5*10-5μL；最后将埃及伊蚊卵转移到约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骚扰库蚊的wpip的埃及伊蚊。
以下将通过实施例对本发明进行详细描述。
实施例1
从自然界采集埃及伊蚊和骚扰库蚊，并在实验室中饲养，选取成虫5天的埃及伊蚊和骚扰库蚊雌虫各10只分别放入产卵中，使其产卵60min，其中，装有骚扰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。收集埃及伊蚊和骚扰库蚊的卵并转移到湿润的滤纸上，将埃及伊蚊卵和骚扰库蚊卵分两列排序，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，将粘有埃及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代埃及伊蚊。
实施例2
按照与实施例1相同的方式得到G0代埃及伊蚊，不同的是，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代埃及伊蚊，不同的是，显微注射的量为1.2*10-5μL。
感染率的测试方法：
分别选取100只实施例1-3中得到的G0代埃及伊蚊，将G0代雄性埃及伊蚊直接进行PCR检测，G0代雌性埃及伊蚊与野生型雄性埃及伊蚊交配产卵后再进行PCR检测，以得到G0代埃及伊蚊的感染率，具体结果参见表1。接着，将G0代雌性埃及伊蚊与野生型雄性埃及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代埃及伊蚊，同样地，分别选取100只实施例1-3中得到的G1代埃及伊蚊(均为阳性即感染成功的G1代埃及伊蚊)，G1代雄性埃及伊蚊直接进行PCR检测，G1代雌性埃及伊蚊与野生型雄性埃及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代埃及伊蚊并检测其感染率，具体结果也参见表1。此外，人工感染后的埃及伊蚊的PCR检测的代表性结果示于图1，从图1中可以看出：注射株在wpip处均呈现阳性，表明这些注射株全部成功携带有wpip。
表1
感染率 实施例1 实施例2 实施例3
G0 35％ 29％ 33％
G1 55％ 50％ 51％
G2 100％ 100％ 100％
G3 100％ 100％ 100％
CI强度的检测方法：
选取10只实施例1-3中得到的G3代雄性埃及伊蚊与10只野生型雌性埃及伊蚊进行交配产卵，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
孵化的卵数 0 0 0
总卵数 780 756 767
孵化率 0％ 0％ 0％
CI强度 100％ 100％ 100％
同样地，选取10只实施例1-3中得到的G3代雌性埃及伊蚊与10只野生型雄性埃及伊蚊进行交配产卵，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
孵化的卵数 685 693 670
总卵数 718 720 700
孵化率 95.4％ 96.3％ 95.7％
CI强度 4.6％ 3.7％ 4.3％
从表1的结果可以看出，根据本发明的方法得到的G0代埃及伊蚊分别表现出35％、29％和33％的感染率，而G1代埃及伊蚊分别表现出55％、50％和51％的感染率(相比于G0代明显增加)，G2代埃及伊蚊均表现出100％的感染率，即G2代埃及伊蚊全部人工感染有Wolbachia，G3代埃及伊蚊结果同G2代埃及伊蚊，因此根据本发明的方法人工感染埃及伊蚊，只需在人工感染后再繁殖两代即可得到感染率为100％的埃及伊蚊种群。
从表2和表3的结果可以看出，当将G3代雄性埃及伊蚊与野生型雌性埃及伊蚊进行交配产卵时，孵化率极低(均0％)，即本发明的人工感染有Wolbachia的雄性埃及伊蚊与野生型雌性埃及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代雌性埃及伊蚊与野生型雄性埃及伊蚊进行交配产卵时，孵化率较高(均在95％-97％之间)，即本发明的人工感染有Wolbachia的雌性埃及伊蚊与野生型雄性埃及伊蚊不存在胞质不相容性；综上，本发明的埃及伊蚊表现出单向CI特性。
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的埃及伊蚊，且得到的埃及伊蚊(特别是雄性)的CI强度极高，因此如果将本发明的雄性埃及伊蚊投放到自然界中，可以大幅降低埃及伊蚊的产卵率，从而有效地控制埃及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 (9)

  1. 一种埃及伊蚊，其中，所述埃及伊蚊被人工感染有骚扰库蚊的wpip。
  2. 根据权利要求1所述的埃及伊蚊，其中，所述人工感染是通过将骚扰库蚊卵的胞浆以显微注射的方式注射到埃及伊蚊卵中来实现的。
  3. 根据权利要求2所述的埃及伊蚊，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所述的埃及伊蚊，其中，所述埃及伊蚊为雄性。
  5. 根据权利要求1或4所述的埃及伊蚊，其中，所述埃及伊蚊与未被人工感染的埃及伊蚊存在胞质不相容性。
  6. 一种生产埃及伊蚊的方法，包括：
    使所述埃及伊蚊人工感染骚扰库蚊的wpip。
  7. 根据权利要求6所述的方法，其中，所述人工感染的步骤包括：将骚扰库蚊卵的胞浆以显微注射的方式注射到埃及伊蚊卵中。
  8. 根据权利要求7所述的方法，其中，所述显微注射的量为1*10-5-1.5*10-5μL。
  9. 根据权利要求6所述的方法，进一步包括：将人工感染有骚扰库蚊的wpip的埃及伊蚊进行大规模饲养。
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