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)

Criação de vespas Nasonia livres de germes

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.

Autor em destaque: Otimizando o procedimento de criação de vespas livres de germes  

Otimizando o procedimento de criação de vespas livres de germes

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)  

Coleta e Criação de Embriões de Vespa Livre de Germes com Meio de Criação de Nasonia (NRM)

Para começar, parasitar cerca de 40 pupas de Lucilia sericata em vespas Nasonia adultas adultas emergentes acasaladas por 1,5 dias. Dentro de 12 a 24 horas após a parasitização, abra cuidadosamente uma extremidade da concha pupal usando uma agulha dissecante estéril. Segure a outra extremidade da casca e localize o embrião da vespa.Em seguida, transfira o embrião para um filtro de células estéril umedecido com PBS. Uma vez transferidos de 20 a 30 embriões, lave-os com 1.000 microlitros de solução de hipoclorito de sódio a 10%, seguido de PBS estéril e etanol a 70%. Por fim, lave-os três vezes com PBS estéril.Em seguida, coloque uma folha de malha de polipropileno pré-molhada com PBS em uma placa de 24 poços. Usando uma pequena escova esterilizada, transfira suavemente os embriões de vespa do filtro de células para a folha de malha de polipropileno. Para a criação da vespa, adicione 50 microlitros de NRM preparado a cada poço com uma folha.Em seguida, adicione um mililitro de água estéril entre cada poço para manter um ambiente úmido para o crescimento. No dia seguinte, transfira a malha de polipropileno contendo a larva de um poço para outro poço usando pinças desinfetadas com álcool e adicione 50 microlitros de NRM equilibrado. Após nove a 11 dias de alimentação, mais de 80% das larvas devem evoluir para pupas brancas ou amarelas.em seguida, transfira a malha para uma placa de poço limpo. No presente estudo, a taxa de sobrevivência das vespas livres de germes de larva para pupa foi significativamente melhorada em comparação com a criação de vespas livres de germes com NRM versão três. Além disso, não houve diferença no período de geração entre as vespas livres de germes e criadas convencionalmente.

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 ...