An optimized method for germ-free breeding of Nasonia

Nasonia breeding support version 3 (NRMv3)

We improved NRMv3 (Table 1) by modifying a large volume syringe with multiple holes to crush fly pupae, increasing the amount of fly pupae extract by about 10% by volume, and decreasing the time spent to be filtered (Fig. 1). This system works more like an apple squeezer instead of mixing and squeezing the pupae. We also switched to adding 50% Phosphate Buffered Saline (PBS) by volume instead of Schneider’s solution. drosophila medium to the protein extract to rule out the unknown potential effect for wasp development.

Table 1 Comparison between NRMv2 and NRMv3, GFRv1 and GFRv2.
Figure 1

Step diagram for Nasonia rearing medium (NRMv3).

Optimized germ-free breeding (GFRv2)

We improved the GFRv2 (Table 1) by changing the membrane to 3mm pore transwell polyester at $ 2.55 per piece (Thincert for 24 well plate, 8.0 µm pore size, translucent, 122.33 $ for 48 / case from USA Scientific, part number 5666-2638Q) at 100 µm polypropylene filter $ 0.046 per piece (polypropylene mesh sheet, opaque white, 12 “wide, 24” long, mesh size 105 microns, 25% open area, $ 25.17 per case which can be 550, Amazon 1.4cm diameter filter, Part number CMP-0105-D) (Fig. 2), which can greatly reduce the cost. This way, each well only needs 50 L of fresh medium instead of 250 L (GFRv1) each day, which allows us to save 80% of the medium. The previous GFRv1 protocol continued to feed the wasps with NRM for 11 days to yellow pupae, so some pupae died in the wet well environment. We continue to feed for only 8 days to avoid introducing NRMv3 when they are in the white pupae stage and are no longer feeding, thus reducing the proportion of dead white pupae.

Figure 2
Figure 2

Photos of the different stages Nasonia vitripennis in the breeding chamber.

Increased survival from larvae to adults

We compared the body weights of day-old females between conventional breeding (wasps were parasitized in S. bullata pupae and developed in a normal incubator) and optimized NRMv3, and with the GFRv2 breeding protocol, there are no significant differences (Fig. 3A). Survival rates from larvae to adults using NRMv12 and NRMv23 using GFRv1 were very low – around 25% and 35%, respectively. NRMv3 optimized with the GFRv2 breeding protocol significantly increased the survival rate by approximately 65% ​​(Fig. 3B). Here, NRMv3 support with the new GFRv2 breeding protocol is a more efficient and cost effective way to Nasonia breeding without germ.

figure 3
figure 3

Survival and size of Nasonia adult females free from germs. (A) Day-old and germ-free females bred on NRMv3 with GFRv2, and conventionally bred females. Body weight was not significantly different between germ-free and conventional breeding (Mann-Whitney U test, p> 0.05). Vertical bars with capital letters represent the standard deviation from the mean. (B) The proportion of larvae to adult survival in the chamber.

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