An optimized method for Nasonia germ-free rearing

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.


Survival proportion statistics.
We counted the embryo number when we transferred them to the filter (d 1).
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 :

Results and discussion
Nasonia rearing media version 3 (NRMv3). We improved the NRMv3 (Table 1) by modifying a large volume syringe with multiple holes to crush the fly pupae, increasing the fly pupae extract amount by around 10% volume, and decreasing the amount of time spent filtering (Fig. 1). This system works more like an apple press instead of blending and juicing the pupae. We also switched to adding 50% Phosphate Buffered Saline In the beaker, cover pupae with sterile millipore water, allow to sit for 1 min, and strain to remove surface particulates from the puparium surface. Some moisture will remain on the pupae 2. In the beaker, cover pupae with sterile millipore water, allow to sit for 1 min, and strain to remove surface particulates from the puparium surface. Some moisture will remain on the pupae 3. Crush the pupae by hand (covered with powder-free nitrile gloves) and squeeze juices through a 100 mm nylon mesh to remove the S. bullata puparium 3. Crush the pupae by 60 ml syringe with holes bored in the bottom and squeeze extract through a 100 mm polypropylene mesh to remove the S.

20-25 embryos were placed on a circular 100um polypropylene filter, and the filter is rinsed with 200 μl 10% bleach solution once and rinsed with 200 ul 1X PBS once. Then the filter is rinsed with 200 μl 70% ethanol solution once and rinsed with 200 μl 1X PBS three times (d 1)
3. After rinsing, the transwell insert was moved into a 24 well plate with 250 μ of NRM in the well. All plates were stored in a sterile Tupperware box at 25 ± 2 ℃ in constant light conditions for the duration of the experiment 3. After rinsing, the filter was moved into a 24 well plate with 50 μl of NRMv3 in the well. All plates were stored in a sterile Tupperware box at 25 ± 2 ℃ in constant light conditions for the duration of the www.nature.com/scientificreports/ each day so we can save 80% of the media. The previous GFRv1 protocol continued feeding the wasps with NRM for 11 days to yellow pupae so that some pupae died in the wet well environment. We continue feeding for only 8 days to avoid introducing NRMv3 when they are in the white pupae stage and are no longer feeding, thus reducing the dead proportion of white pupae.
Increased survival for larvae to adult. 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 NRMv3, and with rearing protocol GFRv2, there are no significant differences (Fig. 3A). The survival rates from larvae to adult using NRMv1 2 and NRMv2 3 using GFRv1 were very low-around 25% and 35%, respectively. The optimized NRMv3 with rearing protocol GFRv2 significantly increased the survival rate by approximately 65% (Fig. 3B). Herein, the media NRMv3 with new rearing protocol GFRv2 is a more efficient and cost-effective way for Nasonia germ-free rearing.