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Biology

Optimizing the Rearing Procedure of Germ-Free Wasps

Published: July 21st, 2023

DOI:

10.3791/65292

1State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, 2College of Life Sciences, Hebei University
* These authors contributed equally

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 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 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 °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.

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 va....

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1. Preparation of germ-free Nasonia rearing medium

  1. Place the commercially available L. sericata pupae (see Table of Materials) on a surface that can accommodate all the pupae, such as a tray or a sheet of paper. Discard any underdeveloped larvae, dark old pupae, empty pupal shells, sawdust, or other impurities. Keep only young pupae that are brownish-red in color and transfer them to a beaker (approximately 3,000-4,000 pupae).
    NO.......

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The preparation efficiency of NRM was greatly improved by improving the preparation tools. In addition, the problem of NRM pollution in the feeding process was eliminated by optimizing the strategy and preservation method. At the same time, the adjusted NRM had a more suitable nutritional ratio for the growth and development of germ-free wasps with L. sericata as hosts. The survival rate of germ-free wasps from larvae to pupae was significantly improved compared to germ-free wasps reared with NRMv3 using GFRv2 (.......

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With the application of high-throughput detection technologies such as genomics and metabolomics, researchers have gradually realized that there is huge genetic diversity and metabolic complexity in the gut microbiota16. These symbiotic bacteria are closely related to various physiological or pathological states, such as host nutritional metabolism, tumors, immunity, and aging through complex interactions with the host17. However, the research related to the network of the .......

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Funding: this work was supported by the National Science Foundation of China (32270538), the National Key R&D Program of China (2022YFF0710603), the Natural Science Foundation of Beijing (6222046), and the CAS strategic funding via the CAS-CSIRO funding scheme (152111KYSB20210011) awarded to G.H.W. Author contributions: all authors developed the scope and focus of the review and contributed to the writing of the manuscript.

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Name Company Catalog Number Comments
0.22 Sterile vacuum filter NEST 331011
10% SodiumHypochlorite LIRCON XB-84BS-1
1x PBS solution Solarbio P1020
200 mesh nylon net BIOBYING BY-378Z
24 well-plate NEST 702001
8, 1.2, 0.8, and 0.45 µm filters Shanghai Xingya Purification Material Factory HN-AA-JT-10079
Absolute ethyl alcohol Macklin E809057-500ml
Cell Strainer BIOLOGIX 15-1100
Commercial Drosophila Medium Boer B645446-500ml
Dissecting needle Bioroyee 17-9140
Garlic press Taobao No Catalog numbers Purchase on Taobao
Lucillia sericata pupae Hefei Dayuan Biotechnology Co., Ltd. No Catalog numbers Purchase on Taobao
Small writing brush Cestidur BL0508
Stereoscope SOPTOP RX50
Tweezers SALMART A109001-56

  1. Diviccaro, S., et al. Exploring the impact of the microbiome on neuroactive steroid levels in germ-free animals. International Journal of Molecular Sciences. 22 (22), 12551 (2021).
  2. Pang, X., et al. Inter-species transplantation of gut microbiota from human to pigs. The ISME Journal. 1 (2), 156-162 (2007).
  3. Uzbay, T. Germ-free animal experiments in the gut microbiota studies. Current Opinion in Pharmacology. 49, 6-10 (2019).
  4. Zhu, Z., Liu, Y., Hu, H., Wang, G. -. H. Nasonia-microbiome associations: a model for evolutionary hologenomics research. Trends in Parasitology. 39 (2), 101-112 (2022).
  5. Li, J., Wei, H. Establishment of an efficient germ-free animal system to support functional microbiome research. Science China Life Sciences. 62 (10), 1400-1403 (2019).
  6. Wang, G. H., et al. Changes in microbiome confer multigenerational host resistance after sub-toxic pesticide exposure. Cell Host Microbe. 27 (2), 213-224 (2020).
  7. Wang, G. H., Dittmer, J., Douglas, B., Huang, L., Brucker, R. M. Coadaptation between host genome and microbiome under long-term xenobiotic-induced selection. Science Advances. 7 (19), (2021).
  8. Dittmer, J., Brucker, R. M. When your host shuts down: larval diapause impacts host-microbiome interactions in Nasonia vitripennis. Microbiome. 9 (1), 85 (2021).
  9. Dittmer, J., et al. Disentangling a holobiont-recent advances and perspectives in Nasonia wasps. Frontiers in Microbiology. 7, 1478 (2016).
  10. Brooks, A. W., Kohl, K. D., Brucker, R. M., van Opstal, E. J., Bordenstein, S. R. Phylosymbiosis: relationships and functional effects of microbial communities across host evolutionary history. PLoS Biology. 14 (11), e2000225 (2016).
  11. Heavner, M. E., et al. Partial venom gland transcriptome of a Drosophila parasitoid wasp, Leptopilina heterotoma, reveals novel and shared bioactive profiles with stinging Hymenoptera. Gene. 526 (2), 195-204 (2013).
  12. Brucker, R. M., Bordenstein, S. R. In vitro cultivation of the hymenoptera genetic model, Nasonia. PLoS One. 7 (12), e51269 (2012).
  13. Shropshire, J. D., van Opstal, E. J., Bordenstein, S. R. An optimized approach to germ-free rearing in the jewel wasp Nasonia. PeerJ. 4, e2316 (2016).
  14. Wang, G. H., Brucker, R. M. An optimized method for Nasonia germ-free rearing. Scientific Reports. 12 (1), 219 (2022).
  15. Brucker, R. M., Bordenstein, S. R. The hologenomic basis of speciation: gut bacteria cause hybrid lethality in the genus Nasonia. Science. 341 (6146), 667-669 (2013).
  16. Fontaine, C. A., et al. How free of germs is germ-free? Detection of bacterial contamination in a germ free mouse unit. Gut Microbes. 6 (4), 225-233 (2015).
  17. Mazmanian, S. K., Liu, C. H., Tzianabos, A. O., Kasper, D. L. An immunomodulatory molecule of symbiotic bacteria directs maturation of the host immune system. Cell. 122 (1), 107-118 (2005).
  18. Weersma, R. K., Zhernakova, A., Fu, J. Interaction between drugs and the gut microbiome. Gut. 69 (8), 1510-1519 (2020).

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