An optimized method for Nasonia germ-free rearing

Guan-Hong Wang,Robert M Brucker

doi:10.1038/s41598-021-04363-9

Guan-Hong Wang, Robert M Brucker

Open Access

https://doi.org/10.1038/s41598-021-04363-9

Copy DOI

Abstract

A germ-free rearing system is a crucial method for host–microbiota interactions using Nasonia as a model system. The previous rearing media in 2012 introduced toxic factors like bleach and antibiotics, required significant effort and volume of media preparation, and the rearing protocols in 2012 and 2016 often resulted in embryos, larvae, and enclosing pupae drowning, underfed, or desiccating. In this work, we optimize the germ-free rearing media that excludes the toxic factors and provide a substrate for the developing animals to have constant access to media without the risk of drowning or desiccation. The new process resulted in an increase in full maturation of larvae to adults from 33 to 65%, with no effect on the rate of growth or final adult size. This significantly improves the applicability of germ-free rearing of Nasonia and potentially other parasitoids.

Highlights

A germ-free rearing system is a crucial method for host–microbiota interactions using Nasonia as a model system
The previous Germ-free rearing version 1 (GFRv1) protocol continued feeding the wasps with Nasonia Rearing Media (NRM) for 11 days to yellow pupae so that some pupae died in the wet well environment
We compared the 1-day-old female body weights between conventional rearing (Wasps were parasitized into S. bullata pupae and developed in normal incubator) and optimized Nasonia Rearing Media version 3 (NRMv3), and with rearing protocol germ-free rearing version 2 (GFRv2), there are no significant differences (Fig. 3A)

Summary

Materials and methods

All wasps were reared in fly vials with Sarcophaga bullata pupae (fly pupae) from Carolina Biological Supply (Burlington, NC) in a 25 °C incubator with constant light. Each fly vial contained 50 female and 15 male wasps with 25 fresh fly pupae. After they were incubated together for [12–24] h, we opened the fly pupae carefully, removing the puparium, and collected the wasp embryos. For the conventional rearing (CV) and NRMv3 with GFRv2 rearing wasp, we collected the 1-day-old female adult and compared their body weight. We counted the number of dead larvae and pupae remaining on d 20 and calculated the survival rate from larvae to adulthood for each well following the approach from Shropshire et al.[3]: Survival rate from d 3 to adult

Results and discussion

Add a 2:1 ratio of Schneider’s Drosophila medium to the protein extract

Progressive filter sterilization