Name: Germ-Free Wasp Embryo Collection and Rearing with Nasonia-Rearing Medium (NRM)
Uploaded: 2023-07-21T14:40:00
Description: 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)

무균 Nasonia 말벌 사육

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.

저자 스포트라이트: 무균 말벌의 사육 절차 최적화  

무균 말벌의 사육 절차 최적화

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)  

Nasonia-Rearing Medium(NRM)을 사용한 무균 말벌 배아 수집 및 사육

시작하려면 약 40마리의 Lucilia sericata 번데기를 1.5일 동안 짝짓기한 신흥 성인 수컷 및 암컷 Nasonia 말벌에 기생합니다. 기생 후 12-24 시간 이내에 멸균 해부 바늘을 사용하여 번데기 껍질의 한쪽 끝을 조심스럽게 엽니 다. 껍질의 다른 쪽 끝을 잡고 말벌 배아를 찾습니다.그런 다음 배아를 PBS로 적신 멸균 세포 여과기로 옮깁니다. 일단 20 내지 30 배아가 이식되면, 10%의 차아염소산나트륨 용액의 1, 000 마이크로리터로 그들을 세척한 다음 멸균된 PBS와 70% 에탄올로 세척한다. 마지막으로 멸균 PBS로 세 번 세척합니다.다음에, PBS로 미리 습윤된 폴리프로필렌 메쉬 시트를 24웰 플레이트에 놓는다. 멸균 된 작은 브러시를 사용하여 말벌 배아를 세포 여과기에서 폴리 프로필렌 메쉬 시트로 부드럽게 옮깁니다. 말벌 사육을 위해 준비된 NRM 50 마이크로 리터를 시트로 각 우물에 첨가하십시오.그런 다음 각 우물 사이에 1 밀리리터의 멸균 수를 추가하여 성장을위한 습한 환경을 유지하십시오. 다음날, 알코올 소독 핀셋을 사용하여 유충을 함유 한 폴리 프로필렌 메쉬를 한 우물에서 다른 우물로 옮기고 50 마이크로 리터의 평형 NRM을 첨가하십시오. 9-11 일간의 수유 후 유충의 80 % 이상이 흰색 또는 노란색 번데기로 발전해야합니다.그런 다음 메쉬를 깨끗한 우물 판으로 옮깁니다. 본 연구에서는 유충에서 번데기까지 무균 말벌의 생존율이 NRM 버전 3으로 무균 말벌을 사육하는 것에 비해 크게 향상되었습니다. 또한, 무균 말벌과 전통적으로 사육된 말벌 사이의 생성 기간에는 차이가 없었습니다.

Research

JoVE Journal

Biology

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 ...