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

無菌ナ ソニア スズメバチの飼育

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.

著者スポットライト:無菌スズメバチの飼育手順の最適化  

無菌スズメバチの飼育手順の最適化

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)  

無菌スズメバチ胚の採取と ナソニア飼育培地(NRM)による飼育

まず、約40匹のルシリア・セリカータの蛹を、1.5日間交尾した新興の成体のオスとメスのナソニアバチに寄生させます。寄生後12〜24時間以内に、滅菌解剖針を使用して蛹殻の一端を慎重に開きます。殻のもう一方の端を持って、スズメバチの胚を見つけます。次に、胚をPBSで湿らせた滅菌セルストレーナーに移します。20〜30個の胚を移植したら、1, 000マイクロリットルの10%次亜塩素酸ナトリウム溶液とそれに続く滅菌PBSと70%エタノールで洗浄します。最後に、滅菌PBSで3回洗浄します。次に、PBSで予め湿らせたポリプロピレンメッシュシートを24ウェルプレートに入れる。滅菌した小さなブラシを使用して、スズメバチの胚を細胞ストレーナーからポリプロピレンメッシュシートにそっと移します。スズメバチの飼育のために、50マイクロリットルの調製されたNRMをシートと共に各ウェルに加える。次に、各ウェルの間に1ミリリットルの滅菌水を追加して、成長のための湿った環境を維持します。翌日、アルコール消毒ピンセットを使用して幼虫を含むポリプロピレンメッシュを1つのウェルから別のウェルに移し、50マイクロリットルの平衡NRMを追加します。9〜11日の給餌後、幼虫の80%以上が白または黄色の蛹に成長するはずです。次に、メッシュをきれいなウェルプレートに移します。本研究では、幼虫から蛹までの無菌バチの生存率は、NRMバージョン3で無菌バチを飼育した場合と比較して有意に改善されました。また、無菌ハチと従来飼育バチでは発生期間に差はなかった。

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 ビュー.  Chinese Academy of Sciences. まず、約40匹のルシリア・セリカータの蛹を、1.5日間交尾した新興の成体のオスとメスのナソニアバチに寄生させます。寄生後12〜24時間以内に、滅菌解剖針を使用して蛹殻の一端を慎重に開きます。殻のもう一方の端を持って、スズメバチの胚を見つけます。次に、胚をPBSで湿らせた滅菌セルストレーナーに移します。20〜30個の胚を移植したら、1, 000マイクロリッ...

ビデオ: 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 ...

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.

無菌 ナソニア 飼育培地(NRM)の調製

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.