Method for obtaining amphoteric strain by removing endosymbiotic bacteria Ricktsia of small belly bees

Abstract

The invention provides a method for obtaining an amphoteric strain by removing a symbiotic bacterium Ricktsia in Pingbelly, and belongs to the technical field of arthropod reproductive mode regulation, and the method comprises the following steps: continuously feeding Pingbelly with rifampicin for multiple generations so as to remove Ricktsia carried by Pingbelly and obtain a fertile amphoteric strain. According to the method, the antibiotic rifampicin can be used for stably removing the symbiotic bacteria Ricktsia in the parthenogenetic female Pingetsia to obtain a fertile amphoteric strain, so that a foundation is laid for further regulating and controlling an arthropod reproduction mode by using the Ricktsia and exploring the interaction of the symbiotic bacteria and a host; the method provides a practical path for studying changes of biology, behavioristics, physiology and the like of hosts before and after bacterium removal, and has good application value.

Description

Technical Field

[0001] The invention belongs to the technical field of arthropod reproductive mode regulation, and particularly relates to a method for removing symbiotic bacteria Rickettsia in flat-bellied wasps to obtain hermaphroditic strains. Background Art

[0002] Rickettsia is a Gram-negative bacterium belonging to the α-subgroup Rickettsiaceae of the Proteobacteria class. It is a morphologically diverse eukaryotic secondary endosymbiont. Rickettsia was first discovered in hemophilic arthropods and is widely known as a vertebrate pathogen. However, Rickettsia also exists in non-hemophilic arthropods, such as Bemisiata baci, Acyrthosi phonpisum, and Pnigalio soemius, inducing parthenogenesis and male killing in the host. In addition, Rickettsia can also serve as a nutritional symbiont, enhance host resistance, and improve the host's ability to resist natural enemies, high temperatures, or other lethal factors.

[0003] Previous studies have found that Anastatus gansuensis mainly produces females indoors, and the offspring parasitized on eggs of Caligula japonica at different ages and Antheraea pernyi at different states are all female. 100% of Anastatus gansuensis collected in the wild are infected with Rickettsia, and the inventors speculate that the reproduction pattern of Anastatus gansuensis is affected by Rickettsia.

[0004] In actual production applications, it is mainly female bees that exert biological control effects. Indoor breeding of parthenogenetic strains can save more production costs than hermaphroditic strains. Moreover, when released in the field, parasitic bees with parthenogenetic reproductive ability are more conducive to establishing field populations and maximizing the biological control effect. Therefore, it is very important to explore how the symbiotic bacteria Rickettsia mediates the transformation of the reproductive mode of parasitic bees and obtain hermaphroditic strains without Rickettsia as a control. This will help to further improve the breeding efficiency of other parasitic bees through transfection and other methods in the future, and promote the better practice and development of the use of natural enemy insects for pest biological control. Summary of the invention

[0005] In order to obtain a hermaphroditic strain of a flat-bellied wasp, the present invention provides a method for removing the endosymbiotic bacteria Rickettsia of the flat-bellied wasp to obtain a hermaphroditic strain. The method uses the antibiotic rifampicin to stably remove the endosymbiotic bacteria Rickettsia of the parthenogenetic flat-bellied wasp to obtain a fertile hermaphroditic strain, laying a foundation for further using Rickettsia to regulate the reproductive mode of arthropods and explore the interaction between symbiotic bacteria and hosts, and provides a practical path for studying the changes in the biology, behavior, physiology, etc. of the host before and after the removal of the bacteria, and has good application value.

[0006] The present invention is achieved through the following technical solutions:

[0007] The invention provides a method for removing symbiotic bacteria Rickettsia in flat-abdomen wasps to obtain hermaphroditic strains. The method comprises: feeding rifampicin to flat-abdomen wasps for multiple generations to remove Rickettsia carried by the flat-abdomen wasps and obtain fertile hermaphroditic strains.

[0008] Furthermore, the rifampicin concentration is 0.01-10.00 mg / mL, and the feeding time for each generation is 24±1 h.

[0009] Furthermore, the method specifically includes: feeding the flat-bellied wasps with a fructose solution containing rifampicin for 7 consecutive generations to remove the Rickettsia carried by the flat-bellied wasps and obtain a fertile hermaphroditic strain.

[0010] Alternatively, the present invention provides a method for removing the endosymbiotic bacteria Rickettsia of the flat-bellied wasp to obtain a hermaphroditic strain, the method comprising:

[0011] Collect female flat-bellied wasps infected with Rickettsia and use them as parents for breeding;

[0012] The parents and offspring were fed with rifampicin, and the infection and content of Rickettsia endosymbiotic bacteria in each generation of adult bees were determined;

[0013] If the identification results show the presence of Rickettsia infection, continue to feed the next generation with rifampicin until all the identification results are negative, then replace rifampicin with fructose solution for feeding, continue for multiple generations, and identify the infection status and content of Rickettsia symbiotic bacteria in each generation of adult bees;

[0014] If there is no Rickettsia symbiosis in adult bees for three consecutive generations, the fourth generation without Rickettsia symbiosis is the strain of flat-bellied wasps that is not infected with Rickettsia;

[0015] From the strain of flat-bellied wasps not infected with Rickettsia, male and female wasps without mating experience are selected for mating, and the obtained offspring are the hermaphroditic strain of flat-bellied wasps not infected with Rickettsia.

[0016] Furthermore, the feeding of rifampicin to the adult worms of the parent and offspring specifically comprises:

[0017] The parents and the adults of the offspring are fed with a rifampicin-fructose solution for 24±1 hours per generation, wherein the concentration of rifampicin in the rifampicin-fructose solution is 0.01-10.00 mg / mL.

[0018] Furthermore, the identification of the infection status and content of Rickettsia endosymbiotic bacteria in each generation of adult bees specifically includes:

[0019] Conventional PCR and quantitative PCR were used to identify the infection and content of Rickettsia endosymbiotic bacteria in each generation of adult bees.

[0020] Among them, the primers used in ordinary PCR are as follows:

[0021] gltA-F:5'-GCTCTTCTCATCCTATGGCTATTAT-3';

[0022] gltA-R:5'-CAGGGTCTTCGTGCATTTCTT-3';

[0023] The primers used for quantitative PCR are as follows:

[0024] glt375-F:5'-TGGTATTGCATCGCTTTGGG-3';

[0025] glt574-R:5'-TTTCTTTAAGCACTGCAGCACG-3'.

[0026] Furthermore, the identification of the infection status and content of Rickettsia endosymbiotic bacteria in each generation of adult bees by conventional PCR and quantitative PCR respectively specifically includes:

[0027] Conventional PCR and quantitative PCR were used to identify the infection status and content of the symbiotic bacteria Rickettsia in each generation of adult bees. The products amplified by conventional PCR were subjected to agarose gel electrophoresis. If the Rickettsia test was negative, it indicated that the wasp was not infected with Rickettsia. The quantitative PCR titer analysis was performed. If the titer was 0 copies / mic, it further proved that the wasp was not infected with Rickettsia.

[0028] Furthermore, if the identification results show the presence of Rickettsia infection, continue to feed the next generation with rifampicin until all the identification results are negative, then replace rifampicin with fructose solution for feeding, and continue for multiple generations. At the same time, identify the infection status and content of Rickettsia symbiotic bacteria in each generation of adult bees, including:

[0029] If the identification results show the presence of Rickettsia infection, continue to feed the next generation with rifampicin-fructose solution until all identification results are negative. Then replace the rifampicin-fructose solution with fructose solution for feeding the next generation. Repeat for multiple generations and use conventional PCR and quantitative PCR to identify the infection status and content of the endosymbiotic bacteria Rickettsia in each generation of adult bees.

[0030] Based on the same inventive concept, the present invention provides a hermaphroditic strain of Ornithogalum that does not contain the endosymbiotic bacterium Rickettsia. The hermaphroditic strain of Ornithogalum is constructed by the above-mentioned method of obtaining a hermaphroditic strain by removing the endosymbiotic bacterium Rickettsia of Ornithogalum.

[0031] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0032] 1. The present invention discloses a method for removing the symbiotic bacteria Rickettsia in flat-bellied wasps to obtain hermaphroditic strains. The method finds for the first time that continuous feeding of 0.01 mg / mL of rifampicin for multiple generations can completely remove the Rickettsia carried by the flat-bellied wasps, and feeding 0.01 mg / mL of rifampicin has no adverse effect on the reproduction of subsequent populations, and obtains hermaphroditic strains that are fertile for normal mating, which lays a foundation for further using Rickettsia to regulate the reproductive mode of arthropods and explore the interaction between symbiotic bacteria and hosts, provides a practical path for studying the changes in the biology, behavior, physiology, etc. of the host before and after the removal of the bacteria, promotes better practice and development of the use of natural enemy insects for biological pest control, and has good application value.

[0033] 2. The present invention provides a method for removing the endosymbiotic bacteria Rickettsia of flat-abdomen wasps to obtain a hermaphroditic strain. The method uses low concentrations of rifampicin to continuously feed flat-abdomen wasps infected with Rickettsia for 7 generations, screens out female and male bees that are not infected with Rickettsia, mates the flat-abdomen wasps that are not infected with Rickettsia, and uses conventional PCR and quantitative PCR to detect the presence of hermaphroditic bacteria in F 0 、F 1 、F 4 、F 6 、F 7 、F 11 、F 15 and F 17The infection of Rickettsia in three generations of D. pygma was detected to confirm the stability of Rickettsia and D. pygma, thus establishing a Rickettsia-free hermaphroditic strain of D. pygma.

[0034] 3. The present invention discloses a method for removing symbiotic bacteria Rickettsia in flat-bellied wasps to obtain hermaphroditic strains. The inventors first discovered that the use of the antibiotic rifampicin to feed flat-bellied wasps can effectively remove Rickettsia and reverse the reproductive mode of flat-bellied wasps, that is, change from parthenogenetic reproduction to parthenogenetic androgenesis. However, feeding high concentrations of antibiotics is extremely harmful to the wasps, resulting in a reduced emergence rate, increased mortality, etc. The present invention feeds flat-bellied wasps with 0.01 mg / mL of rifampicin for multiple generations to successfully remove the symbiotic bacteria Rickettsia in the flat-bellied wasps. Combined with molecular technology and biological verification, the flat-bellied wasps with a reproductive mode of parthenogenetic reproduction can be converted into parthenogenetic androgenesis. Since the antibiotic concentration of each generation of adult bees is low and the continuous generation time is long, the toxic effects of antibiotic feeding on the host can be effectively avoided. The present invention can establish a stable and long-term hermaphroditic strain of flat-bellied wasps that are not infected with Rickettsia, and achieve stable reproduction and generation of flat-bellied wasps that are not infected with Rickettsia.

[0035] 4. The present invention discloses a method for removing the symbiotic bacteria Rickettsia in the flat-bellied wasp to obtain a hermaphroditic strain. The method is simple to operate and has strong controllability. The prepared rifampicin solution can be directly fed, and the feeding time of each generation is short and controllable. The low concentration of antibiotics can greatly reduce the damage to the parasitic wasp. The acquisition of the hermaphroditic strain not infected with Rickettsia is of great significance for studying the biological functions of the flat-bellied wasp, the bee-bacteria interaction, and the development of new technologies for pest control of other parthenogenetic parasitic wasps using the symbiotic bacteria Rickettsia transfection. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0037] Figure 1 This is a survival analysis diagram of Gansu flat-bellied wasp after feeding different concentrations of rifampicin solution;

[0038] Figure 2 This is an analysis chart of the male emergence rate of Gansu flat-bellied wasp after feeding with different concentrations of rifampicin solution;

[0039] Figure 3This is an analysis chart of the emergence rate of Gansu flat-bellied wasp after feeding with different concentrations of rifampicin solution;

[0040] Figure 4 This is an analysis of the infection of Rickettsia in Gansu Province after continuous treatment of multiple generations with rifampicin;

[0041] Figure 5 This is a graph showing the titer changes of Rickettsia gansuensis after continuous treatment of multiple generations of rifampicin. DETAILED DESCRIPTION

[0042] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0043] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0044] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0045] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0046] The following is a detailed description of a method for removing the endosymbiotic bacteria Rickettsia of the flat-bellied wasp to obtain a hermaphroditic strain according to the present invention in combination with examples and experimental data.

[0047] Test insects

[0048] The Gansu flat-bellied wasp used in the experiment was collected from a walnut orchard in Gansu Province, China. The parasitic wasp was identified based on morphological characteristics and further confirmed by COI sequence (GenBank: MK373759). Gansu flat-bellied wasp was raised with tussah silkworm eggs, and the parasitized tussah silkworm eggs were placed in a culture room for eclosion. Through 16S third-generation full-length sequencing, the bee was severely infected with and only with the existing Rickettsia that induces parthenogenesis.

[0049] Example 1

[0050] This embodiment provides a method for removing the endosymbiotic bacteria Rickettsia of the flat-bellied wasp to obtain a hermaphroditic strain.

[0051] 1. Experimental Methods

[0052] 1. Collection of Rickettsia strains infected by Gansu flat-bellied wasp:

[0053] The parasitic wasps collected in the wild were first identified as Gansu flat-bellied wasps species by morphology and molecular biology, and then sequenced with Rickettsia universal primers to determine the infection status.

[0054] Use a fine brush to collect all the adult bees into a plastic jar (size 12.5*6.5cm), cover the jar mouth with a white cloth cut to a suitable size, and fix it with a rubber band. There are 20 female bees in each jar, and use a small brush to brush 30% honey water onto the jar wall. Be careful not to stick the parasitic bees to death. Then place the jar in a cultivation greenhouse under constant conditions of temperature (24±1)℃, humidity (70±5)%, and light-dark ratio of 14:10. Wait for about 5 days and give 300 tussah eggs for parasitism to breed the next generation (F 0 ).

[0055] The adults that were 100% infected with Rickettsia were used as parent adults (F 0 ).

[0056] 2. Feeding with different concentrations of rifampicin solution:

[0057] Rifampicin powder was mixed with 1 mol / L fructose water to prepare 0.01, 0.05, 0.10, 1.00, and 10.00 mg / mL rifampicin solutions, respectively, and stored in centrifuge tubes for later use.

[0058] 3. Screening for appropriate rifampicin solution concentration:

[0059] Select small bees and form a jar (12.5*6.5cm). Use a small brush to carefully brush antibiotic fructose water on the wall of the jar to form small mist-like droplets on the wall of the jar. Feed them once every 24 hours. Feed them with each concentration of antibiotic fructose water for 1, 3, 5, 7, 10, 15, and 20 days. During this period, the number of deaths of small bees is recorded daily to facilitate survival analysis.

[0060] Take out a single bee that has been fed for a certain number of days and place it in a glass tube (1.5*10cm), feed it with enough antibiotic fructose water of corresponding concentration and give it fresh tussah egg cards for parasitism of the bee. After 24 hours of parasitism, remove the bee, and record the male birth rate and eclosion rate of the offspring when feeding with the corresponding concentration of antibiotic fructose water solution, so as to eliminate the antibiotic concentration that has a greater impact on the eclosion of the bee.

[0061] The control group was fed only fructose water without antibiotics, and all other conditions were the same as those of the experimental group.

[0062] The survival rate, male emergence rate and eclosion rate of each group of bees were counted. The experimental results are as follows Figure 1 ,2 3. The results showed that low concentration of rifampicin solution could achieve 100% male offspring, and there was no significant difference in mortality compared with the control.

[0063] 4. Obtaining the infection-free Rickettsia strain:

[0064] Select newly emerged Rickettsia gansuensis (F 0 ), placed them separately in glass tubes (1.5*10cm), and brushed 0.01mg / mL rifampicin-fructose aqueous solution onto the tube wall with a small brush. After feeding for 24h, they were parasitized with tussah silkworm eggs. After 24h of parasitism, the female bees were removed.

[0065] If all the offspring are female, continue to breed the next generation; if the offspring are both female and male, pair the male and female.

[0066] Each generation is tested for Rickettsia infection. If the females and males in the offspring can mate and no Rickettsia is detected in the female bees, the antibiotic feeding is stopped immediately and the next generation is fed with a fructose aqueous solution instead. If no Rickettsia is detected in the offspring for three consecutive generations, it is proved that a hermaphroditic strain of the Gansu flat-bellied wasp that is not infected with Rickettsia has been obtained.

[0067] Or you can also use the following method:

[0068] Each generation is tested for Rickettsia infection. If the identification results show the presence of Rickettsia infection, the next generation will continue to be fed with rifampicin-fructose solution until all identification results are negative. Then the rifampicin-fructose solution will be replaced with fructose solution for feeding the next generation. This will be repeated for multiple generations. At the same time, ordinary PCR and quantitative PCR are used to identify the infection status and content of the symbiotic bacteria Rickettsia in each generation of adult bees. When there is no Rickettsia symbiosis in adult bees for three consecutive generations, the fourth generation without Rickettsia symbiosis is a strain of flat-bellied wasps that is not infected with Rickettsia.

[0069] From a strain of flat-bellied wasps that has not been infected with Rickettsia, unmated female and male bees are selected for mating, and the resulting offspring are the hermaphroditic strain of flat-bellied wasps that has not been infected with Rickettsia.

[0070] (1) Common PCR detection of Rickettsia infection in Gansu flat-bellied wasps:

[0071] For the conventional PCR detection of the symbiotic bacteria Rickettsia, a kit (TIANamp Genomic DNA kit) was used to extract the DNA of the Gansu flat-bellied wasp. For specific operation steps, please refer to the instructions for use.

[0072] PCR was used to detect the infection of Gansu flat-bellied wasps, using a pair of primers targeting the gltA gene sequence of Rickettsia: the sequences of gltA-F and gltA-R are:

[0073] gltA-F: 5'-GCTCTTCTCATCCTATGGCTATTAT-3'

[0074] gltA-R:5'-CAGGGTCTTCGTGCATTTCTT-3'

[0075] Reaction system:

[0076]

[0077]

[0078] Total volume 25 μL

[0079] Amplification conditions:

[0080] Pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 53.7°C for 30 s, extension at 72°C for 30 s, 35 cycles; extension at 72°C for another 10 min, and cycle at 4°C.

[0081] The PCR product was detected by 1.5% agarose gel electrophoresis. A 2kb marker was used as a control. 5 μL of the PCR product and the marker were added to a 1.5% agarose gel and immediately placed in 1×TAE buffer for electrophoresis. The electrophoresis conditions were 120 V for 20 min.

[0082] Use a gel imaging system to take a picture of it and save the picture to get the result. The band diagram is as follows Figure 4 Show.

[0083] (2) Determination of Rickettsia titer:

[0084] To determine the abundance of Rickettsia in D. gansuensis, we used Ex Taq TM (Takara) and Bio-Rad CFX96 TM Real-Time System (BioRad) was used for determination;

[0085] The gel of gltA gene obtained by the above method was used to perform the following steps: gel recovery and purification of DNA fragments, connection of target gene with vector, heat shock transformation of Escherichia coli competent cells, positive clone screening and sequencing, and plasmid extraction. All the steps were performed according to the instructions of the kit.

[0086] Quantitative PCR detection of Rickettsia titer

[0087] The standard curve was drawn by the standard plasmid samples extracted above, and the concentrations were 10 4 , 10 5 , 10 6 , 10 7 and 10 8 Gene copy number / mic.

[0088] Primer sequence: glt375-F: 5′-TGGTATTGCATCGCTTTGGG-3′

[0089] glt574-R 5′-TTTCTTTAAGCACTGCAGCACG-3′.

[0090] The bee samples of each generation obtained by the above 0.01 mg / mL rifampicin-fructose aqueous solution were disinfected (75% alcohol for 1 min, 2% NaClO for 30 s, ddH 2 O 3 times, 3 min each time, all the above operations were carried out in a clean bench). After DNA extraction, the abundance of Rickettsia in the bees under different treatments was determined by absolute quantification.

[0091] Reaction system

[0092]

[0093]

[0094] The primer sequences are:

[0095] glt375-F:5′-TGGTATTGCATCGCTTTGGG-3′

[0096] glt574-R 5′-TTTCTTTAAGCACTGCAGCACG-3′.

[0097] Total volume 20 μL

[0098] Amplification conditions:

[0099] Pre-denaturation at 95℃ for 30s; denaturation at 95℃ for 5s, extension at 60℃ for 30s, 40 cycles; denaturation at 95℃ for 10s; melting curve from 65℃ to 95℃. The temperature was increased every 5s, and the temperature was increased by 0.5℃ each time.

[0100] (3) Biological verification: A strain of Gansu flat-bellied wasp that could not be detected with Rickettsia for three consecutive generations was verified. If unmated female bees only produced male offspring, while mated female bees produced both male and female offspring, it means that a hermaphroditic strain of Gansu flat-bellied wasp that was not infected with Rickettsia has been obtained.

[0101] 2. Experimental Results

[0102] 1. Effects of different concentrations of rifampicin on bee mortality

[0103] Figure 1 (L: represents rifampicin, ***p<0.001) is the survival analysis of Gansu flat-bellied bees fed with different rifampicin concentrations. It can be seen from the figure that compared with the control, high concentrations of rifampicin solution (1.00, 10.00 mg / mL) have obvious toxic effects on the bees, and the three low concentrations of rifampicin solutions (0.01, 0.05, 0.10 mg / mL) have no significant effect on the mortality rate of the bees.

[0104] 2. Effects of different concentrations of rifampicin on the male emergence rate and emergence rate of small bees

[0105] Figure 2 and Figure 3 In order to analyze the male emergence rate and emergence rate of Gansu flat-bellied wasp fed with different concentrations of rifampicin, since 1.00 mg / mL and 10.00 mg / mL have obvious toxic effects on the wasp, only the male emergence rate and emergence rate of Gansu flat-bellied wasp fed with three concentrations of 0.01, 0.05, and 0.10 mg / mL were analyzed.

[0106] In terms of male yield, Figure 2 As shown, after feeding for 10 days with the three antibiotic concentrations, the male virility rate of the offspring increased significantly, reaching about 60%, and there was no significant difference among the three; after feeding for about 15 days, the male virility rate of the offspring reached more than 80%, and there was no significant difference among the three; after feeding for about 20 days, the male virility rate of the offspring could reach 100%, and there was no significant difference among the three.

[0107] In terms of emergence rate, Figure 3 As shown, when the three antibiotic concentrations were fed within 5 days, the offspring emergence rate reached 100%, and there was no significant difference compared with the control; when feeding on the 7th, 10th, 15th, and 20th days, the offspring emergence rate was not significantly different from the control except for the 0.10 mg / mL concentration, the other two concentrations.

[0108] In summary, a rifampicin solution with a concentration of 0.01 mg / mL is preferred for feeding.

[0109] 3. Obtaining a Rickettsia-free strain

[0110] (1) Generation-by-generation verification of infection status of the Gansu flat-bellied wasp infected with Rickettsia treated with antibiotics

[0111] Figure 4 The band diagram of the change in the titer of the endosymbiotic bacteria Rickettsia in the continuous treatment of Gansu flat-bellied wasps during the experiment. The presence of bands indicates that the host is infected with Rickettsia, and the absence of bands indicates that the host is not infected with Rickettsia. As can be seen from the figure, compared with the control (CK), in F 0 、F 1 、F 4 Rickettsia bands were detected in samples of all generations, but only in F 7 In the first generation, no bright bands of Rickettsia could be detected. Then, the animals were continuously fed with fructose aqueous solution without antibiotics, and the male and female offspring were kept to mate and reproduce the next generation. In the following generations, no Rickettsia bands could be detected.

[0112] (2) Titer analysis of Rickettsia infection and antibiotic treatment in Gansu Province

[0113] Figure 5 This is a titer analysis of the endosymbiotic bacteria Rickettsia in Gansu Province treated by the flat-bellied wasp for several generations during the experiment. It can be seen from the figure that compared with the control (CK), the parent (F 0 )100% infected with Rickettsia, the titer of infection with Rickettsia gradually decreased from the first generation, to the sixth generation (F 6 ) It can be clearly seen that the Rickettsia titer decreased significantly until F 7 The titer of the first generation was about 0 copies / mic, and then the offspring were continuously fed with fructose aqueous solution without antibiotics, and the titer of the offspring remained at about 0 copies / mic for many generations.

[0114] (3) Biologically verified hermaphroditic strains

[0115] After feeding with antibiotics, observe the male-female ratio of the offspring. If there are both male and female offspring, pair them up. 4 Mating occurs in the F generation. 7 The infection of Rickettsia was verified by the next generation, at which time Rickettsia had been completely removed. 8The results of biological verification showed that the offspring of mating female individuals (fertilization) were both male and female, while the offspring of unmated female individuals (unfertilized) were only male. 8 Starting from the first generation, antibiotics were stopped and fructose water was fed instead. Figure 4 It can be seen that after stopping the feeding of antibiotics, no Rickettsia bands were detected in the subsequent offspring, and by counting the male status of the offspring after the mating of the parthenogenetic strain and the hermaphroditic strain, the results are shown in Table 6. The offspring of the parthenogenetic strain are only female, the male ratio is 0, and the number of repetitions is 20; while the offspring of the mated hermaphroditic strain are both female and male, and the male ratio is 40.65%, and the number of repetitions is 20. Therefore, a hermaphroditic strain of Gansu flat-bellied wasp that is not infected with Rickettsia and has stable inheritance is obtained.

[0116] Table 6. Male ratio of offspring in parthenogenetic strains and hermaphroditic strains after mating

[0117]

[0118] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0119] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0120] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for removing the endosymbiotic bacteria Rickettsia of flat-bellied wasps to obtain hermaphroditic strains, characterized in that: The method comprises: feeding rifampicin to the flat-bellied wasps for multiple generations continuously to remove Rickettsia carried by the flat-bellied wasps and obtain fertile hermaphroditic strains.

2. The method for removing the endosymbiotic bacteria Rickettsia of the flat-bellied wasp to obtain a hermaphroditic strain according to claim 1, characterized in that: The rifampicin concentration is 0.01-10.00 mg / mL, and the feeding time for each generation is 24±1 h.

3. The method for removing the endosymbiotic bacteria Rickettsia of the flat-bellied wasp to obtain a hermaphroditic strain according to claim 1, characterized in that: The method specifically comprises: feeding a fructose solution containing rifampicin to the flat-bellied wasps for seven consecutive generations to remove the Rickettsia carried by the flat-bellied wasps and obtain a fertile hermaphroditic strain.

4. The method for removing the endosymbiotic bacteria Rickettsia of the flat-bellied wasp to obtain a hermaphroditic strain according to claim 1, characterized in that: The method comprises: Collect female flat-bellied wasps infected with Rickettsia and use them as parents for breeding; The parents and offspring were fed with rifampicin, and the infection and content of Rickettsia endosymbiotic bacteria in each generation of adult bees were determined; If the identification results show the presence of Rickettsia infection, continue to feed the next generation with rifampicin until all the identification results are negative, then replace rifampicin with fructose solution for feeding, continue for multiple generations, and identify the infection status and content of Rickettsia symbiotic bacteria in each generation of adult bees; When there is no Rickettsia symbiosis in adult bees for three consecutive generations, the fourth generation without Rickettsia symbiosis is the strain of flat-bellied wasps that is not infected with Rickettsia; From the strain of flat-bellied wasps not infected with Rickettsia, male and female wasps without mating experience are selected for mating, and the obtained offspring are the hermaphroditic strain of flat-bellied wasps not infected with Rickettsia.

5. The method for removing the endosymbiotic bacteria Rickettsia of the flat-bellied wasp to obtain a hermaphroditic strain according to claim 4, characterized in that: The feeding of rifampicin to the adults of the parents and offspring specifically comprises: The parents and the adults of the offspring are fed with a rifampicin-fructose solution for 24±1 hours per generation, wherein the concentration of rifampicin in the rifampicin-fructose solution is 0.01-10.00 mg / mL.

6. The method for removing the endosymbiotic bacteria Rickettsia of the flat-bellied wasp to obtain a hermaphroditic strain according to claim 4, characterized in that: The identification of the infection status and content of Rickettsia endosymbiotic bacteria in each generation of adult bees specifically includes: Conventional PCR and quantitative PCR were used to identify the infection and content of Rickettsia endosymbiotic bacteria in each generation of adult bees. Among them, the primers used in ordinary PCR are as follows: gltA-F:5'-GCTCTTCTCATCCTATGGCTATTAT-3'; gltA-R:5'-CAGGGTCTTCGTGCATTTCTT-3'; The primers used for quantitative PCR are as follows: glt375-F:5'-TGGTATTGCATCGCTTTGGG-3'; glt574-R:5'-TTTCTTTAAGCACTGCAGCACG-3'.

7. The method for removing the endosymbiotic bacteria Rickettsia of the flat-bellied wasp to obtain a hermaphroditic strain according to claim 4, characterized in that: If the identification results show that Rickettsia infection exists, rifampicin is continued to be fed to the next generation until all the identification results are negative, then rifampicin is replaced with fructose solution for feeding, and this is continued for multiple generations, and the infection status and content of Rickettsia symbiotic bacteria in each generation of adult bees are identified at the same time, specifically including: If the identification results show the presence of Rickettsia infection, continue to feed the next generation with rifampicin-fructose solution until all identification results are negative. Then replace the rifampicin-fructose solution with fructose solution for feeding the next generation. Repeat for multiple generations and use conventional PCR and quantitative PCR to identify the infection status and content of the symbiotic bacteria Rickettsia in each generation of adult bees.

8. A hermaphroditic strain of a flat-bellied wasp that does not contain the endosymbiotic bacteria Rickettsia, characterized in that: The hermaphroditic strain of the flat-abdomen wasp is constructed by the method for obtaining a hermaphroditic strain by removing the endosymbiotic bacteria Rickettsia of the flat-abdomen wasp according to any one of claims 1 to 7.

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