Introduction

The hymenopteran parasitoid, genus Nasonia, is a well-established genetic model for research in evolution, behavior, development, and endosymbiosis1. The germ-free rearing (GFR) approach2,3 makes Nasonia a versatile animal model for host-microbiota interaction4,5,6 as well. However, the previous Nasonia Rearing Media (NRM) techniques introduced potential toxic factors like bleach, antibiotics, and/or Drosophila medium to the media. What is more, both embryos and pupating larvae are susceptible to drowning at the bottom of the rearing plate, leading to a 35% survival rate from larvae to adults2,3. Here we developed an efficient Nasonia Rearing Media version 3 (NRMv3) and an optimized germ-free rearing version 2 (GFRv2) protocol.

Materials and methods

Nasonia rearing and embryo collection

The N. vitripennis strain AsymCX(u) (Wolbachia free)7 was used. 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.

Comparative analysis of development

For the conventional rearing (CV) and NRMv3 with GFRv2 rearing wasp, we collected the 1-day-old female adult and compared their body weight. As the wasp is very small (~ 1.2 mm × 0.4 mm), we observed the mass of the empty 1.5 ml tube and then calculated the difference after putting one female wasp into the tube to calculate the female wasp body weight.

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:

$$Survival \, rate\,from\,d\,3\,to\,adult= \frac{Number\, of\, d \,3\, larvae-Number\, of\, dead\, larvae\, and\, pupae\, on \,d \,20 }{Number\, of\, d \,3 \,larvae}\times 100$$

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 (PBS) by volume instead of Schneider's Drosophila medium to the protein extract to exclude the potential unknown effect for wasp development.

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

Schematic of the step for Nasonia rearing media (NRMv3).

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Optimized germ-free rearing (GFRv2)

We improved the GFRv2 (Table 1) by switching the 3 mm pore transwell polyester membrane $2.55 per each (Thincert for 24-well plate, 8.0 um pore size, translucent, $122.33 for 48/case from USA Scientific, Part Number 5666-2638Q) to 100 um polypropylene filter $0.046 per each (Polypropylene Mesh Sheet, Opaque White, 12″ Width, 24″ Length, 105 microns Mesh Size, 25% Open Area, $25.17 per case which can make 550, 1.4-cm diameter filter from Amazon, Part Number CMP-0105-D) (Fig. 2), which can sharply decrease the cost. In this way, each well only needs 50 μl fresh media instead of 250 μl (GFRv1) 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.

Figure 2
figure 2

Photos of different stages Nasonia vitripennis in the rearing chamber.

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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 NRMv12 and NRMv23 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.

Figure 3
figure 3

Survival and size of Nasonia germ-free adult females. (A) One-day-old and germ-free females reared on NRMv3 with GFRv2, and females reared conventionally. Body-weight was not significantly different between germ-free and conventional rearing (Mann–Whitney U test, p > 0.05). Vertical bars with caps represent the standard deviation from the mean. (B) The proportion of larval to adult survival in the chamber.

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