germ-free wasp embryo collection and rearing with nasonia-rearing medium (nrm)

饲养无菌 Nasonia 黄蜂

Germ-free animals are animals that have no detectable living microorganisms and parasites1. Germ-free embryos can be obtained by dissecting the mother under aseptic conditions and subsequently raised in barrier systems2. Such animals can be used to study the effects of microorganisms on animals, such as on the intestinal microbiota, immune system, and metabolism1. With certain technical means, many insects and even mammals can be rendered sterile3,4. Germ-free animals have a unique role and have been widely used in various aspects of microbiology research5. For example, the use of germ-free Nasonia wasps has revealed that microorganisms can help hosts adapt to new environments under long-term exogenous environmental stress6,7.
Nasonia parasitoids are small parasitic wasps that inject their eggs into the pupae of flies4. There are four known species of Nasonia, including Nasonia vitripennis, Nasonia longicornis, Nasonia giraulti, and Nasonia oneida8. N. vitripennis can be found worldwide, while the other three species have limited ranges in North America4. Nasonia parasitoid wasps are regarded as ideal model insects because of their characteristics, such as easy cultivation, short reproduction cycle, sequenced genome, and long term diapause8,9. They can be used to study various aspects of insect evolution, genetics, development, behavior, and symbiosis10. Moreover, Nasonia parasitoid wasps can also help control harmful flies in agriculture and disease11. The successful establishment of a sterile insect system involves two major steps: (1) sterilization of the embryos and (2) provision of sterile food to the larvae in vitro. In order to obtain sterile food, Brucker and Bordenstein12 developed Nasonia rearing medium (NRMv1) in 2012 by using chemicals such as antibiotics, bleach, and fetal bovine serum to kill bacteria12. However, the chemical sterilization method resulted in low survival and eclosion rates of N. vitripennis13. Then, in 2016, Shropshire et al. developed NRMv2 by using a filter sterilization method instead of a chemical sterilization method to eliminate the hazards of antibiotics and other substances, and optimized the breeding process13. Unfortunately, this method still has some disadvantages, such as the challenges associated with preparing and using the medium, as well as the risks of drowning, underfeeding, or dehydration for the embryos, larvae, and closed pupae14. Wang and Brucker14 recently improved the Nasonia rearing media version 3 (NRMv3) and the germ-free rearing version 2 (GFRv2) protocols. These improvements reduced the cost and media consumption. However, the NRMv3 has a very short storage time and is highly susceptible to contamination.
Building on NRMv3, the NRM preparation tool storage method and nutrient ratio were optimized in this study. This methodological refinement facilitates using N. vitripennis as a model for microbiome studies. Compared with the NRMv3 developed by Wang et al.14, the improved tool for squeezing Sarcophaga bullata pupa, one of the NRM raw materials, greatly enhances the production efficiency of S. bullata pupa tissue fluid compared with the 60 mL syringe with a bottom hole used by Wang et al.14. We adjusted the nutrient ratio of NRM, which led to a certain increase in the survival rate of germ-free Nasonia wasps without affecting their development time. In addition, the NRM was packed into small-capacity centrifuge tubes (1.5 mL) and frozen in a -20 &#176;C refrigerator to extend the storage time. It is worth noting that while we used the housefly Lucilia sericata as the host and source for NRM preparation, this protocol can likely be adapted for other Nasonia hosts that are available in the laboratory.

作者聚焦：优化无菌黄蜂的饲养程序  

优化无菌黄蜂的饲养程序

Our research focuses on the study of insect-microbiota interactions, specifically investigating the impact of in those symbiotic bacteria and exploring the dynamics of host-microbe relationships. Germ-free Nasonia is an essential tool for conducting this research. Current research utilize technologies such as high-throughput sequencing, germ-free insect rearing, transcriptoptomics, proteomics, and advanced microscope.This tool enable comprehensive analysis and virtualization of insect-microbe host interaction for advancing our understanding of this field. The optimized the procedure for preparing the Nasonia Rearing Medium provides economic advantages and saves time and effort. Additionally, it effectively reduces the risk of NRM contamination during the feeding process, greatly improving the efficiency of producing aseptic Nasonia

Watch this Scientific Journal Video about Germ-Free Wasp Embryo Collection and Rearing with Nasonia-Rearing Medium NRM at JoVE.com

Germ-Free Wasp Embryo Collection and Rearing with Nasonia-Rearing Medium (NRM)  

使用 Nasonia饲养基（NRM）进行无菌黄蜂胚胎收集和饲养

首先，将大约40只露西莉亚蛹寄生到新出现的成年雄性和雌性Nasonia蜂上，交配1.5天。在寄生后 12 至 24 小时内，使用无菌解剖针小心地打开蛹壳的一端。握住壳的另一端并找到黄蜂胚胎。然后将胚胎转移到用PBS润湿的无菌细胞过滤器中。移植20至30个胚胎后，用1， 000微升10%次氯酸钠溶液洗涤它们，然后用无菌PBS和70%乙醇洗涤它们。最后，用无菌PBS清洗三次。接下来，将用PBS预润湿的聚丙烯网片放入24孔板中。使用无菌的小刷子，轻轻地将黄蜂胚胎从细胞过滤器转移到聚丙烯网片上。对于黄蜂饲养，用一张纸向每个孔中加入 50 微升准备好的 NRM。然后在每口井之间加入一毫升无菌水，以保持潮湿的生长环境。第二天，使用酒精消毒镊子将含有幼虫的聚丙烯网从一个孔转移到另一个孔中，并加入50微升平衡的NRM。进食9至11天后，超过80%的幼虫应发育成白色或黄色蛹。然后将网格转移到干净的孔板中。本研究显示，与NRM版本3饲养无菌黄蜂相比，无菌黄蜂从幼虫到蛹的存活率显著提高。此外，无菌黄蜂和常规饲养的黄蜂在生成周期上没有差异。

germ-free-wasp-embryo-collection-rearing-with-nasonia-rearing-medium

Research

JoVE Journal

Biology

Germ-Free Wasp Embryo Collection and Rearing with Nasonia-Rearing Medium (NRM)

459 次观看.  Chinese Academy of Sciences. 首先，将大约40只露西莉亚蛹寄生到新出现的成年雄性和雌性Nasonia蜂上，交配1.5天。在寄生后 12 至 24 小时内，使用无菌解剖针小心地打开蛹壳的一端。握住壳的另一端并找到黄蜂胚胎。然后将胚胎转移到用PBS润湿的无菌细胞过滤器中。移植20至30个胚胎后，用1， 000微升10%次氯酸钠溶液洗涤它们，然后用无菌PBS和70%乙醇洗涤它们。最后，用无菌PBS清洗三次。接下来?...

视频: Germ-Free Wasp Embryo Collection and Rearing with Nasonia-Rearing Medium (NRM)  

Optimizing the Rearing Procedure of Germ-Free Wasps

Aseptic rearing technology is a method of culturing insects under sterile or almost sterile conditions, which can effectively eliminate the influence of external microorganisms on insect microbiota and thus promote the rapid development of insect microbiota research. Nasonia (wasp genus) is a parasitic wasp insect that has many advantages, such as a short lifespan, high genetic variation, easy operation, etc., and is widely used as an insect model system. Unlike antibiotic treatment, which can only reduce the number of microorganisms in animals, aseptic rearing techniques can control both the composition and quantity of microorganisms in animals, further facilitating the study of host-microbe interactions. However, previous versions of Nasonia rearing medium (NRM) have some defects and problems, such as a complex and time-consuming preparation process, easy contamination by bacteria or fungi, and short storage time. Therefore, this study solves these problems by optimizing the tools used in the NRM preparation process, storage conditions, and component ratios. The optimized medium could allow storage at -20 &#176;C for at least 3 months and eliminate the possibility of NRM contamination during feeding sterile wasps. This further improves the survival rate and health level of aseptic Nasonia, which is important for using Nasonia as a model for microbial research.

Nasonia wasp embryos were dissected from Lucillia sericata pupae after parasitization for 12-24 h and washed with alcohol and 10% sodium hypochlorite solution to obtain germ-free embryos. After rearing the germ-free embryos&#160;and supplying them with Nasonia rearing medium to grow and develop in vitro, germ-free Nasonia adults were obtained.

Aseptic rearing technology is a method of culturing insects under sterile or almost sterile conditions, which can effectively eliminate the influence of ...

科研

生物学

Preparation of Germ-Free Nasonia Rearing Medium (NRM)

Preparation of Germ-Free Nasonia Rearing Medium (NRM)  

To begin, place commercially available Lucillia sericata pupae on a sheet of paper. Discard any underdeveloped larvae, dark old pupae, empty pupil shells, sawdust, or other impurities. Transfer young brownish red colored pupae to a beaker and immerse the pupae in deionized water.Wrap the beaker with tinfoil or gauze and shake it to clean any surface impurities off the pupae. Then discard the water. Place the cleaned pupae in the filter tank of a garlic press, and squeeze hard.Then collect the pupae tissue fluid in a 50-millimeter sterile centrifuge tube, and centrifuge it at 25, 000 x g for 10 minutes at four degrees Celsius. Using a 22-gauge sterile needle, collect the protein layer into a new 50-millimeter polypropylene centrifuge tube, and add commercial liquid drosophila medium at an equal ratio to the protein extract. To remove the particles of different sizes from the mixture, filter it using a vacuum filtration system with filter paper of different pore sizes.To prevent clogging change the filter paper when the flow slows down. Once the filtrate is centrifuged, sterilize the supernatant through a 0.22 micrometer syringe filter, and store the prepared nasonia rearing medium, or NRM aliquots, at 20 degrees Celsius until further use.

无菌 纳索尼亚 饲养培养基（NRM）的制备

To begin, parasitize about 40 Lucilia sericata pupae onto emerging adult male and female Nasonia wasps mated for 1.5 days. Within 12 to 24 hours after parasitization, carefully open one end of the pupal shell using a sterile dissecting needle. Hold the other end of the shell and locate the wasp embryo.Then transfer the embryo to a sterile cell strainer moistened with PBS. Once 20 to 30 embryos have been transferred, wash them with 1, 000 microliters of 10%sodium hypochlorite solution followed by sterile PBS and 70%ethanol. Finally, wash them three times with sterile PBS.Next, place a polypropylene mesh sheet pre-wetted with PBS into a 24 well plate. Using a sterilized small brush, gently transfer the wasp embryos from the cell strainer to the polypropylene mesh sheet. For the wasp rearing, add 50 microliters of prepared NRM to each well with a sheet.Then add one milliliter of sterile water between each well to maintain a humid environment for growth. The next day, transfer the polypropylene mesh containing the larva from one well to another well using alcohol disinfected tweezers, and add 50 microliters of equilibrated NRM. After nine to 11 days of feeding, over 80%of the larva should develop into white or yellow pupae.then transfer the mesh to a clean well plate. In the present study, the survival rate of the germ-free wasps from larva to pupae was significantly improved compared to rearing germ-free wasps with NRM version three. Additionally, there was no difference in the generation period between the germ-free and conventionally reared wasps.