Name: Preparation of Germ-Free Nasonia Rearing Medium (NRM)
Uploaded: 2023-07-21T14:40:00
Description: Watch this Scientific Journal Video about Preparation of Germ-Free Nasonia Rearing Medium NRM at JoVE.com

preparation of germ-free nasonia rearing medium (nrm)

Rearing Germ-Free Nasonia Wasps

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.

Author Spotlight: Optimizing the Rearing Procedure of Germ-Free Wasps  

Optimizing the Rearing Procedure of Germ-Free Wasps

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 Preparation of Germ-Free Nasonia Rearing Medium NRM at JoVE.com

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.

Research

JoVE Journal

Biology

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