Funding: this work was supported by the National Science Foundation of China (32270538), the National Key R&#38;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.

1. Preparation of germ-free&#160;Nasonia rearing medium
<ol>
	<li>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). 
	NOTE: After conducting many experiments, it was found that the medium made from the dark old pupae easily turned dark and stopped the development of germ-free wasps. This phenomenon did not occur when using the young brownish-red pupae to produce medium. The reason for this is still unclear and requires further investigation to verify it.</li>
	<li>Add enough deionized water to the beaker to cover the entire surface of the pupae. Wrap the mouth of the beaker with tinfoil or gauze, shake the beaker to clean the impurities on the surface of the pupae, and then pour out the water. Repeat three to five times.</li>
	<li>Place the cleaned L. sericata pupae in the filter tank of a garlic press, squeeze hard, and collect the tissue fluid of the L. sericata pupae in a 50 mL sterile centrifuge tube. Then, pour out the pupa dregs and put new pupae in the filter tank of the garlic press until all are squeezed. 
	NOTE: Compared to the needle extrusion device used by Wang et al.14, the garlic press is more convenient and can separate the interstitial fluid more thoroughly. This advancement lays the foundation for large-scale production and long-term storage of NRM.</li>
	<li>Centrifuge the mixture at 4 &#176;C (25,000 x g) for 10 min. After centrifugation, the mixture will be separated into three layers from bottom to top: sediment layer, protein layer, and fat layer (Figure 1).</li>
	<li>To prevent clogging during filtration, aspirate the protein layer using a 18&#160;G sterile needle and transfer it to a new 50 mL conical sterile polypropylene centrifuge tube.</li>
	<li>Add commercial liquid Drosophila medium (see Table of Materials) to the protein extract at a 1:1 ratio. 
	NOTE: More convenient commercialized L. sericata was used as the host and NRM raw material, compared with NRMv3. Therefore, the nutritional ratio was adjusted from 1:2 to 1:1 based on NRMv3, which was more suitable for the growth and development of sterile Nasonia (Figure 2).</li>
	<li>Filter the mixed medium (Drosophila medium and protein extract of L. sericata pupa) using a vacuum filtration system with different pore sizes (8, 1.2, 0.8, and 0.45 &#181;m; see Table of Materials) to remove particles of different sizes. To prevent clogging, change the filter paper when the flow slows down. 
	NOTE: The use of a new 50 mL sterile centrifuge tube is required for each filtration.</li>
	<li>Centrifuge the liquid again at 4 &#176;C (15,000 x g) for 10 min and sterilize the supernatant medium through a 0.22 &#181;m syringe filter. Repeat the above steps once.</li>
	<li>Store the culture medium at -20 &#176;C after aliquoting (Figure 3A) for long-term storage. 
	&#8203;NOTE: To prevent contamination and repeated freezing and thawing, it is recommended to open each tube of NRM only once when using the culture medium after packaging (Figure 3A).</li>
</ol>
2. Germ-free egg collection
<ol>
	<li>To ensure a stable male-to-female ratio in offspring, place the pupae in a Drosophila vial during the pupal stage at a ratio of 50 females to 15 males because of their haploid genetic characteristics (males develop from haploid cells, while females develop from diploid cells that result from fertilized eggs)4,15.
	<ol>
		<li>After emerging to adults, allow the males and females to mate for 1.5 days, and then put about 40 L. sericata pupae into the vial. Within 12-24 h after parasitization, use a sterile dissecting needle to carefully open one end of the pupal shell under a stereomicroscope (Figure 4).</li>
		<li>Hold the other end by hand and find the wasp embryos. Use a dissecting needle to transfer embryos from the surface of the L. sericata pupa tissue to a sterile cell strainer with phosphate-buffered saline (PBS)14. 
		NOTE: Older L. sericata pupae should be used for parasitism because the dry L. sericata pupa tissue is easier to transfer embryos. In order to ensure the smooth transfer of eggs from the L. sericata pupa tissue surface, try to be careful not to puncture the tissue when the dissecting needle opens the pupal case so that the interstitial fluid does not flow out.</li>
	</ol>
	</li>
	<li>Place 20-30 embryos on a cell strainer and wash them evenly with 1,000 &#181;L of 10% commercial sodium hypochlorite solution (see Table of Materials), followed by another wash with 1,000 &#181;L of 1x sterile PBS. Then, wash once with 1,000 &#181;L of 70% ethanol solution and wash three times with 1,000 &#181;L of 1x sterile PBS.</li>
	<li>First, place a 5 mm diameter polypropylene mesh sheet (see Table of Materials) that has been pre-wetted with 1x PBS into a 24-well plate. Then, using a sterilized small brush, gently brush the wasp embryos on the cell strainer onto the polypropylene mesh sheet. This can be done for all four wells in a vertical column of the 24-well plate.</li>
</ol>
3. Germ-free wasp rearing
<ol>
	<li>Add 50 &#181;L of NRM to each well in a laminar flow hood. To maintain a humid environment for growth, add 1 mL of sterile water between each well in the 24-well plate. A small beaker containing 30 mL of sterile water can also be placed in the 5 L sterile plastic box with the 24-well plate.
	<ol>
		<li>Throughout the entire experiment, keep the sterile plastic box and the 24-well plate in a climate chamber at a constant temperature of 25 &#177; 2 &#176;C and under constant light.</li>
	</ol>
	</li>
	<li>Before transferring and adding NRM every day, thaw the frozen NRM on a laminar flow bench. In a sterile environment, use alcohol-disinfected tweezers to transfer the polypropylene mesh with larvae on it from one well to another. Lastly, add 50 &#181;L of NRM that has been equilibrated to room temperature (Figure 3B). Repeat the above operations every day until pupae are observed. 
	NOTE: With the development of wasps, the amount of NRM should be increased appropriately to ensure that the germ-free wasps have sufficient nutrition. For example, the fourth instar larvae were supplied with 60 to 70 &#181;L of NRM. Since different developmental stages may coexist in the same well, use a sterilized brush to transfer out the pupae. Add a small amount of NRM to the remaining larvae. Use aseptic techniques to avoid microbial invasion and pollution.</li>
	<li>After feeding for 9 to 11 days, more than 80% of the larvae develop into white or yellow pupae. At this time, move the filter to a clean well plate and stop adding culture medium to wait for eclosion.</li>
</ol>

Rearing Germ-Free Nasonia Wasps

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nasonia-microbiome+associations:+a+model+for+evolutionary+hologenomics+research.">Nasonia-microbiome associations: a model for evolutionary hologenomics research.</a> Trends in Parasitology. 39 (2), 101-112 (2022).</li><li>Li, J., Wei, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+an+efficient+germ-free+animal+system+to+support+functional+microbiome+research.">Establishment of an efficient germ-free animal system to support functional microbiome research.</a> Science China Life Sciences. 62 (10), 1400-1403 (2019).</li><li>Wang, G. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+microbiome+confer+multigenerational+host+resistance+after+sub-toxic+pesticide+exposure.">Changes in microbiome confer multigenerational host resistance after sub-toxic pesticide exposure.</a> Cell Host Microbe. 27 (2), 213-224 (2020).</li><li>Wang, G. H., Dittmer, J., Douglas, B., Huang, L., Brucker, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coadaptation+between+host+genome+and+microbiome+under+long-term+xenobiotic-induced+selection.">Coadaptation between host genome and microbiome under long-term xenobiotic-induced selection.</a> Science Advances. 7 (19), (2021).</li><li>Dittmer, J., Brucker, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=When+your+host+shuts+down:+larval+diapause+impacts+host-microbiome+interactions+in+Nasonia+vitripennis.">When your host shuts down: larval diapause impacts host-microbiome interactions in Nasonia vitripennis.</a> Microbiome. 9 (1), 85 (2021).</li><li>Dittmer, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disentangling+a+holobiont-recent+advances+and+perspectives+in+Nasonia+wasps.">Disentangling a holobiont-recent advances and perspectives in Nasonia wasps.</a> Frontiers in Microbiology. 7, 1478 (2016).</li><li>Brooks, A. W., Kohl, K. D., Brucker, R. M., van Opstal, E. J., Bordenstein, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylosymbiosis:+relationships+and+functional+effects+of+microbial+communities+across+host+evolutionary+history.">Phylosymbiosis: relationships and functional effects of microbial communities across host evolutionary history.</a> PLoS Biology. 14 (11), e2000225 (2016).</li><li>Heavner, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partial+venom+gland+transcriptome+of+a+Drosophila+parasitoid+wasp,+Leptopilina+heterotoma,+reveals+novel+and+shared+bioactive+profiles+with+stinging+Hymenoptera.">Partial venom gland transcriptome of a Drosophila parasitoid wasp, Leptopilina heterotoma, reveals novel and shared bioactive profiles with stinging Hymenoptera.</a> Gene. 526 (2), 195-204 (2013).</li><li>Brucker, R. M., Bordenstein, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+cultivation+of+the+hymenoptera+genetic+model,+Nasonia.">In vitro cultivation of the hymenoptera genetic model, Nasonia.</a> PLoS One. 7 (12), e51269 (2012).</li><li>Shropshire, J. D., van Opstal, E. J., Bordenstein, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+approach+to+germ-free+rearing+in+the+jewel+wasp+Nasonia.">An optimized approach to germ-free rearing in the jewel wasp Nasonia.</a> PeerJ. 4, e2316 (2016).</li><li>Wang, G. H., Brucker, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+method+for+Nasonia+germ-free+rearing.">An optimized method for Nasonia germ-free rearing.</a> Scientific Reports. 12 (1), 219 (2022).</li><li>Brucker, R. M., Bordenstein, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hologenomic+basis+of+speciation:+gut+bacteria+cause+hybrid+lethality+in+the+genus+Nasonia.">The hologenomic basis of speciation: gut bacteria cause hybrid lethality in the genus Nasonia.</a> Science. 341 (6146), 667-669 (2013).</li><li>Fontaine, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+free+of+germs+is+germ-free?+Detection+of+bacterial+contamination+in+a+germ+free+mouse+unit.">How free of germs is germ-free? Detection of bacterial contamination in a germ free mouse unit.</a> Gut Microbes. 6 (4), 225-233 (2015).</li><li>Mazmanian, S. K., Liu, C. H., Tzianabos, A. O., Kasper, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+immunomodulatory+molecule+of+symbiotic+bacteria+directs+maturation+of+the+host+immune+system.">An immunomodulatory molecule of symbiotic bacteria directs maturation of the host immune system.</a> Cell. 122 (1), 107-118 (2005).</li><li>Weersma, R. K., Zhernakova, A., Fu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+between+drugs+and+the+gut+microbiome.">Interaction between drugs and the gut microbiome.</a> Gut. 69 (8), 1510-1519 (2020).</li></ol>

The authors have nothing to disclose.

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 composition and function of the microbial population in the host is very difficult because the composition of microorganisms in each natural individual is not exactly the same16. Therefore, an ideal model is needed to explore the interaction and related mechanisms between microorganisms and hosts. Nasonia wasps can be used as an ideal model in the microbiome field because of its many biological advantages and the characteristics of germ-free wasps6. Compared to antibiotic treatment, using sterile animals allows for control over the composition and quantity of microorganisms, which is crucial for studying microbial function. Additionally, antibiotic treatment may lead to several side effects, including antibiotic resistance, intestinal dysfunction, and intestinal barrier damage18. Therefore, developing a more efficient method to obtain germ-free wasps is of great significance for studying host-microbiome interactions.
The acquisition of germ-free Nasonia mainly includes two steps - obtaining sterile embryos and providing sterile food. The embryos were obtained by dissecting L. sericata pupae 12-24 h after N. vitripennis parasitization under a stereoscope13. This step required enough skill to move the embryos faster and without damaging them to the cell strainer. Based on experience, it is more convenient to dissect the old L. sericata pupae that are black. However, young red L. sericata pupae are preferred for preparing NRM.
Moreover, the normal development of sterile embryos depended on the production of sterile food and the operation process of changing the medium every day12. We found that the main source of contamination for rearing germ-free wasps by Wang et al.&#39;s14 GFRv2 was NRM. Therefore, the culture was packed and stored in the refrigerator at -20 &#176;C in the present study, ensuring that each medium is only opened once. This not only had no effect on the growth and development of Nasonia wasps, but also greatly prolonged the culture medium&#39;s storage time and eliminated the chance of NRM contamination. In total, this method further increased the survival rate of aseptic wasps.
However, the present method still has some drawbacks. For example, the daily transfer of sterile embryos in a 24-well plate may increase the risk of pollution. In addition, the process of squeezing a mass of fly pupae is also time-consuming and labor-intensive. If the specific nutrients that play a role in the fly pupae can be identified, they could be purchased and added directly to the medium, which would greatly improve the efficiency of NRM preparation. It is expected that this method will be further optimized in future 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 various aspects of microbiology research5. For example, the use of germ-free Nasonia wasps has revealed that microorganisms can help hosts adapt to new environments under long-term exogenous environmental stress6,7.
Nasonia parasitoids are small parasitic wasps that inject their eggs into the pupae of flies4. There are four known species of Nasonia, including Nasonia vitripennis, Nasonia longicornis, Nasonia giraulti, and Nasonia oneida8. N. vitripennis can be found worldwide, while the other three species have limited ranges in North America4. Nasonia parasitoid wasps are regarded as ideal model insects because of their characteristics, such as easy cultivation, short reproduction cycle, sequenced genome, and long term diapause8,9. They can be used to study various aspects of insect evolution, genetics, development, behavior, and symbiosis10. Moreover, Nasonia parasitoid wasps can also help control harmful flies in agriculture and disease11. The successful establishment of a sterile insect system involves two major steps: (1) sterilization of the embryos and (2) provision of sterile food to the larvae in vitro. In order to obtain sterile food, Brucker and Bordenstein12 developed Nasonia rearing medium (NRMv1) in 2012 by using chemicals such as antibiotics, bleach, and fetal bovine serum to kill bacteria12. However, the chemical sterilization method resulted in low survival and eclosion rates of N. vitripennis13. Then, in 2016, Shropshire et al. developed NRMv2 by using a filter sterilization method instead of a chemical sterilization method to eliminate the hazards of antibiotics and other substances, and optimized the breeding process13. Unfortunately, this method still has some disadvantages, such as the challenges associated with preparing and using the medium, as well as the risks of drowning, underfeeding, or dehydration for the embryos, larvae, and closed pupae14. Wang and Brucker14 recently improved the Nasonia rearing media version 3 (NRMv3) and the germ-free rearing version 2 (GFRv2) protocols. These improvements reduced the cost and media consumption. However, the NRMv3 has a very short storage time and is highly susceptible to contamination.
Building on NRMv3, the NRM preparation tool storage method and nutrient ratio were optimized in this study. This methodological refinement facilitates using N. vitripennis as a model for microbiome studies. Compared with the NRMv3 developed by Wang et al.14, the improved tool for squeezing Sarcophaga bullata pupa, one of the NRM raw materials, greatly enhances the production efficiency of S. bullata pupa tissue fluid compared with the 60 mL syringe with a bottom hole used by Wang et al.14. We adjusted the nutrient ratio of NRM, which led to a certain increase in the survival rate of germ-free Nasonia wasps without affecting their development time. In addition, the NRM was packed into small-capacity centrifuge tubes (1.5 mL) and frozen in a -20 &#176;C refrigerator to extend the storage time. It is worth noting that while we used the housefly Lucilia sericata as the host and source for NRM preparation, this protocol can likely be adapted for other Nasonia hosts that are available in the laboratory.

<table><tbody><tr><td>0.22 Sterile vacuum filter</td><td>NEST</td><td>331011</td><td></td></tr><tr><td>10% SodiumHypochlorite</td><td>LIRCON</td><td>XB-84BS-1</td><td></td></tr><tr><td>1x PBS solution</td><td>Solarbio</td><td>P1020</td><td></td></tr><tr><td>200 mesh nylon net</td><td>BIOBYING</td><td>BY-378Z</td><td></td></tr><tr><td>24 well-plate</td><td>NEST</td><td>702001</td><td></td></tr><tr><td>8, 1.2, 0.8, and 0.45 &amp;#181;m filters</td><td>Shanghai Xingya Purification Material Factory</td><td>HN-AA-JT-10079</td><td></td></tr><tr><td>Absolute ethyl alcohol</td><td>Macklin</td><td>E809057-500ml</td><td></td></tr><tr><td>Cell Strainer</td><td>BIOLOGIX</td><td>15-1100</td><td></td></tr><tr><td>Commercial Drosophila Medium</td><td>Boer</td><td>B645446-500ml</td><td></td></tr><tr><td>Dissecting needle</td><td>Bioroyee</td><td>17-9140</td><td></td></tr><tr><td>Garlic press</td><td>Taobao</td><td>No Catalog numbers</td><td>Purchase on Taobao</td></tr><tr><td>Lucillia sericata pupae</td><td>Hefei Dayuan Biotechnology Co., Ltd.</td><td>No Catalog numbers</td><td>Purchase on Taobao</td></tr><tr><td>Small writing brush</td><td>Cestidur</td><td>BL0508</td><td></td></tr><tr><td>Stereoscope</td><td>SOPTOP</td><td>RX50</td><td></td></tr><tr><td>Tweezers</td><td>SALMART</td><td>A109001-56</td><td></td></tr></tbody></table>

optimizing the rearing procedure of germ-free wasps

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 &#176;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.

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 (Figure 2). Further, the generation period was not different between the aseptic wasps and the conventionally reared wasps (about 14 days a generation)&#160;(Figure 5). These improvements are highly significant for developing the Nasonia model organism and studying the host-microorganism interaction mechanism.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65292/65292fig01.jpg" /> 
Figure 1: Mixture separated by centrifugation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65292/65292fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65292/65292fig02.jpg" /> 
Figure 2: The survival rate of larvae to pupae in each transwell. The survival rate improved significantly compared to germ-free females reared on NRMv3 with GFRv2 (two independent-sample T-tests, *p &#60; 0.05). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65292/65292fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65292/65292fig03.jpg" /> 
Figure 3: Schematic diagram of NRM dispensing and germ-free wasp rearing. (A) The filtered NRM from the 50 mL centrifuge tube was aliquoted into sterile 1.5 mL centrifuge tubes and frozen. Each tube of NRM was only opened once. (B) There was a polypropylene mesh sheet underneath the embryos of the Nasonia wasps. The centrifuge tube contained the packaged NRM. &#34;Day 14-ish&#34; means adult emergence occurs on about the 14th day. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65292/65292fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65292/65292fig04.jpg" /> 
Figure 4: Images of transferred wasp eggs under the stereomicroscope. The eggs laid by the Nasonia wasp are within the red dashed box. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65292/65292fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65292/65292fig05.jpg" /> 
Figure 5: Development time comparison. The development time of germ-free females (about 14 days) was not significantly different from that of the conventionally reared wasps when using the improved NRM of this study (two independent-sample T-tests, ns, not significant,&#160;p &#62; 0.05). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65292/65292fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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Author Spotlight: Optimizing the Rearing Procedure of Germ-Free Wasps  

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&#160;and supplying them with Nasonia rearing medium to grow and develop in vitro, germ-free Nasonia adults were obtained.

Optimizing the Rearing Procedure of Germ-Free Wasps

Aseptic rearing technology is a method of culturing insects under sterile or almost sterile conditions, which can effectively eliminate the influence of ...

optimizing-the-rearing-procedure-of-germ-free-wasps

Nanyang Technological University

Research

JoVE Journal

Biology

2.2K Views.  Chinese Academy of Sciences. 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.

Video: Author Spotlight: Optimizing the Rearing Procedure of Germ-Free Wasps  

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex determination, venom synthesis, and host-symbiont interactions, among others. A major limitation of working with this organism is the lack of effective protocols to perform directed genome modifications. An important part of genome modification is delivery of editing reagents, including CRISPR/Cas9 molecules, into embryos through microinjection. While microinjection is well established in many model organisms, this technique is particularly challenging to perform in N. vitripennis primarily due to its small embryo size, and the fact that embryonic development occurs entirely within a parasitized blowfly pupa. The following procedure overcomes these significant challenges while demonstrating a streamlined, visual procedure for effectively removing wasp embryos from parasitized host pupae, microinjecting them, and carefully transplanting them back into the host for continuation and completion of development. This protocol will strongly enhance the capability of research groups to perform advanced genome modifications in this organism.

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

Microinjection of Nasonia vitripennis embryos is an essential method for generating heritable genome modifications. Described here is a detailed procedure for microinjection and transplantation of Nasonia vitripennis embryos, which will greatly facilitate future genome manipulation in this organism.

The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex ...

Genetics

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts' immune physiology and how coordinated immune reactions are thwarted by this class of parasites.
The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids.
Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts1. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response2. Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited1. Strains of L. boulardi also produce virus-like particles3 although they differ significantly in their ability to succeed on D. melanogaster1. Some of these L. boulardi strains are difficult to grow on D. melanogaster1 as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols.
In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms1,4. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections.
Wasp infection activates immune signaling (Fig. 4)4. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5)5,6. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D).
We present representative data with Toll signal transduction pathway components Dorsal and Sp&auml;tzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

Immunology and Infection

In Vitro Rearing of Solitary Bees: A Tool for Assessing Larval Risk Factors

Although solitary bees provide crucial pollination services for wild and managed crops, this species-rich group has been largely overlooked in pesticide regulation studies. The risk of exposure to fungicide residues is likely to be especially high if the spray occurs on, or near host plants while the bees are collecting pollen to provision their nests. For species of Osmia that consume pollen from a select group of plants (oligolecty), the inability to use pollen from non-host plants can increase their risk factor for fungicide-related toxicity. This manuscript describes protocols used to successfully rear oligolectic mason bees, Osmia ribifloris sensu lato, from egg to prepupal stage within cell culture plates under standardized laboratory conditions. The in vitro-reared bees are subsequently used to investigate the effects of fungicide exposure and pollen source on bee fitness. Based on a 2 &#215; 2 fully crossed factorial design, the experiment examines the main and interactive effects of fungicide exposure and pollen source on larval fitness, quantified by prepupal biomass, larval developmental time, and survivorship. A major advantage of this technique is that using in vitro-reared bees reduces natural background variability and allows the simultaneous manipulation of multiple experimental parameters. The described protocol presents a versatile tool for hypotheses testing involving the suite of factors affecting bee health. For conservation efforts to be met with significant, lasting success, such insights into the complex interplay of physiological and environmental factors driving bee declines will prove to be critical.

In Vitro Rearing of Solitary Bees: A Tool for Assessing Larval Risk Factors

Fungicide sprays on flowering plants may expose solitary bees to high concentrations of pollen-borne fungicide residues. Using laboratory-based experiments involving in vitro-reared bee larvae, this study investigates the interactive effects of consuming fungicide-treated pollen derived from host and non-host plants.

Although solitary bees provide crucial pollination services for wild and managed crops, this species-rich group has been largely overlooked in pesticide ...

Environment

Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing

The western corn rootworm (WCR) is an important pest of corn and is well known for its ability to rapidly adapt to pest management strategies. Although RNA interference (RNAi) has proved to be a powerful tool for studying WCR biology, it has its limitations. Specifically, RNAi itself is transient (i.e. does not result in long-term Mendelian inheritance of the associated phenotype), and it requires knowing the DNA sequence of the target gene. The latter can be limiting if the phenotype of interest is controlled by poorly conserved, or even novel genes, because identifying useful targets would be challenging, if not impossible. Therefore, the number of tools in WCR&#39;s genomic toolbox should be expanded by the development of methods that could be used to create stable mutant strains and enable sequence-independent surveys of the WCR genome. Herein, we detail the methods used to collect and microinject precellular WCR embryos with nucleic acids. While the protocols described herein are aimed at the creation of transgenic WCR, CRISPR/Cas9-genome editing could also be performed using the same protocols, with the only difference being the composition of the solution injected into the embryos.

Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing

Here we present protocols for collecting and microinjecting precellular western corn rootworm embryos for the purpose of performing functional-genomic assays such as germline transformation and CRISPR/Cas9-genome editing.

The western corn rootworm (WCR) is an important pest of corn and is well known for its ability to rapidly adapt to pest management strategies. Although ...

Bioengineering

Culturing and Genetically Manipulating Entomopathogenic Nematodes

Entomopathogenic nematodes in the genera Heterorhabditis and Steinernema are obligate parasites of insects that live in the soil. The main characteristic of their life cycle is the mutualistic association with the bacteria Photorhabdus and Xenorhabdus, respectively. The nematode parasites are able to locate and enter suitable insect hosts, subvert the insect immune response, and multiply efficiently to produce the next generation that will actively hunt new insect prey to infect. Due to the properties of their life cycle, entomopathogenic nematodes are popular biological control agents, which are used in combination with insecticides to control destructive agricultural insect pests. Simultaneously, these parasitic nematodes represent a research tool to analyze nematode pathogenicity and host anti-nematode responses. This research is aided by the recent development of genetic techniques and transcriptomic approaches for understanding the role of nematode secreted molecules during infection. Here, a detailed protocol on maintaining entomopathogenic nematodes and using a gene knockdown procedure is provided. These methodologies further promote the functional characterization of entomopathogenic nematode infection factors.

Entomopathogenic nematodes live in symbiosis with bacteria and together they successfully infect insects by undermining their innate immune system. To promote research on the genetic basis of nematode infection, methods for maintaining and genetically manipulating entomopathogenic nematodes are described.

Entomopathogenic nematodes in the genera Heterorhabditis and Steinernema are obligate parasites of insects that live in the soil. The main characteristic ...

Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

Aedes aegypti mosquitoes experimentally infected with Wolbachia are being utilized in programs to control the spread of arboviruses such as dengue, chikungunya and Zika. Wolbachia-infected mosquitoes can be released into the field to either reduce population sizes through incompatible matings or to transform populations with mosquitoes that are refractory to virus transmission. For these strategies to succeed, the mosquitoes released into the field from the laboratory must be competitive with native mosquitoes. However, maintaining mosquitoes in the laboratory can result in inbreeding, genetic drift and laboratory adaptation which can reduce their fitness in the field and may confound the results of experiments. To test the suitability of different Wolbachia infections for deployment in the field, it is necessary to maintain mosquitoes in a controlled laboratory environment across multiple generations. We describe a simple protocol for maintaining Ae. aegypti mosquitoes in the laboratory, which is suitable for both Wolbachia-infected and wild-type mosquitoes. The methods minimize laboratory adaptation and implement outcrossing to increase the relevance of experiments to field mosquitoes. Additionally, colonies are maintained under optimal conditions to maximize their fitness for open field releases.

Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

Aedes aegypti mosquitoes infected with Wolbachia are being released into natural populations to suppress the transmission of arboviruses. We describe methods to rear Ae. aegypti with Wolbachia infections in the laboratory for experiments and field release, taking precautions to minimize laboratory adaptation and selection.

Aedes aegypti mosquitoes experimentally infected with Wolbachia are being utilized in programs to control the spread of arboviruses such as dengue, ...

Preparation of Virus-Enriched Inoculum for Oral Infection of Honey Bees (Apis mellifera)

Honey bees are of great ecological and agricultural importance around the world but are also subject to a variety of pressures that negatively affect bee health, including exposure to viral pathogens. Such viruses can cause a wide variety of devastating effects and can often be challenging to study due to multiple factors that make it difficult to separate the effects of experimental treatments from preexisting background infection. Here we present a method to mass produce large quantities of virus particles along with a high throughput bioassay to test viral infection and effects. Necessitated by the current lack of a continuous, virus-free honey bee cell line, viral particles are amplified in vivo using honey bee pupae, which are extracted from the hive in large volumes using minimally stressful methodology. These virus particles can then be used in honey bee cage bioassays to test inocula viability, as well as various other virus infection dynamics, including interactions with nutrition, pesticides, and other pathogens. A major advantage of using such particles is that it greatly reduces the chances of introducing unknown variables in subsequent experimentation when compared to current alternatives, such as infection via infected bee hemolymph or homogenate, though care should still be taken when sourcing the bees, to minimize background virus contamination. The cage assays are not a substitute for large-scale, field-realistic experiments testing virus infection effects at a colony level, but instead function as a method to establish baseline viral responses that, in combination with the semi-pure virus particles, can serve as important tools to examine various dimensions of honey bee-virus physiological interactions.

Preparation of Virus-Enriched Inoculum for Oral Infection of Honey Bees Apis mellifera

Here we describe two protocols: first to propagate, extract, purify, and quantify large quantities of honey bee non-enveloped virus particles, including a method for removing honey bee pupae and second to test the effects of viral infection using a highly repeatable, high-throughput cage bioassay.

Honey bees are of great ecological and agricultural importance around the world but are also subject to a variety of pressures that negatively affect bee ...

Pupal and Adult Injections for RNAi and CRISPR Gene Editing in Nasonia vitripennis

The jewel wasp, Nasonia vitripennis, has become an efficient model system to study epigenetics of haplo-diploid sex determination, B-chromosome biology, host-symbiont interactions, speciation, and venom synthesis. Despite the availability of several molecular tools, including CRISPR/Cas9, functional genetic studies are still limited in this organism. The major limitation of applying CRISPR/Cas9 technology in N. vitripennis stems from the challenges of embryonic microinjections. Injections of embryos are particularly difficult in this organism and in general in many parasitoid wasps, due to small embryo size and the requirement of a host pupa for embryonic development. To address these challenges, Cas9 ribonucleoprotein complex delivery into female&#160;ovaries by adult injection, rather than embryonic microinjection, was optimized, resulting in both somatic and heritable germline edits. The injection procedures were optimized in pupae and female wasps using either ReMOT Control (Receptor-Mediated Ovary Transduction of Cargo) or BAPC (Branched Amphiphilic Peptide Capsules). These methods are shown to be effective alternatives to embryo injection, enabling site-specific and heritable germline mutations.

Pupal and Adult Injections for RNAi and CRISPR Gene Editing in Nasonia vitripennis

Here, we describe methods for efficient pupal and adult injections in Nasonia vitripennis as accessible alternatives to embryo microinjection, enabling functional analysis of genes of interest using either RNA-silencing via RNA interference (RNAi) or gene knockout via CRISPR/Cas9 genome editing.

The jewel wasp, Nasonia vitripennis, has become an efficient model system to study epigenetics of haplo-diploid sex determination, B-chromosome biology, ...

Automating Aggregate Quantification in Caenorhabditis elegans

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This increased attention has called for the diversification and improvement of animal models capable of reproducing disease phenotypes observed in humans with PCDs. Though murine models have proven invaluable, they are expensive and are associated with laborious, low-throughput methods. Use of the Caenorhabditis elegans nematode model to study PCDs has been justified by its relative ease of maintenance, low cost, and rapid generation time, which allow for high-throughput applications. Additionally, high conservation between the C. elegans and human genomes makes this model an invaluable discovery tool. Nematodes that express fluorescently tagged tissue-specific polyglutamine (polyQ) tracts exhibit age- and polyQ length-dependent aggregation characterized by fluorescent foci. Such reporters are often employed as proxies to monitor changes in proteostasis across tissues. Manual aggregate quantification is time-consuming, limiting experimental throughput. Furthermore, manual foci quantification can introduce bias, as aggregate identification can be highly subjective. Herein, a protocol consisting of worm culturing, image acquisition, and data processing was standardized to support high-throughput aggregate quantification using C. elegans that express intestine-specific polyQ. By implementing a C. elegans-based image processing pipeline using CellProfiler, an image analysis software, this method has been optimized to separate and identify individual worms and enumerate their respective aggregates. Though the concept of automation is not entirely unique, the need to standardize such procedures for reproducibility, elimination of bias from manual counting, and increase throughput is high. It is anticipated that these methods can drastically simplify the screening process of large bacterial, genomic, or drug libraries using the C. elegans model.

Automating Aggregate Quantification in Caenorhabditis elegans

The following protocol describes the development and optimization of a high-throughput workflow for worm culturing, fluorescence imaging, and automated image processing to quantify polyglutamine aggregates as an assessment of changes in proteostasis.

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This ...

Methods for Rearing the Parasitoid Ganaspis brasiliensis, a Promising Biological Control Agent for the Invasive Drosophila suzukii

Native to East Asia, the spotted-wing drosophila, Drosophila suzukii (Matsumura) (Diptera: Drosophilidae), has established widely in the Americas, Europe, and parts of Africa over the last decade, becoming a devastating pest of various soft-skinned fruits in its invaded regions. Biological control, especially by means of self-perpetuating and specialized parasitoids, is expected to be a viable option for sustainable area-wide management of this highly mobile and polyphagous pest. Ganaspis brasiliensis Ihering (Hymenoptera: Figitidae) is a larval parasitoid that is widely distributed in East Asia, and has been found to be one of the most effective parasitoids of D. suzukii.
Following rigorous pre-introduction evaluations of its efficacy and potential non-target risks, one of the more host-specific genetic groups of this species (G1 G. brasiliensis) has been approved recently for introduction and field release in the United States and Italy. Another genetic group (G3 G. brasiliensis), which was also commonly found to attack D. suzukii in East Asia, may be considered for introduction in the near future. There is currently enormous interest in rearing G. brasiliensis for research or in mass-production for field release against D. suzukii. This protocol and associated video article describe effective rearing methods for this parasitoid, both on a small scale for research and a large scale for mass-production and field release. These methods may benefit further long-term research and use of this Asian-native parasitoid as a promising biological control agent for this global invasive pest.

Methods for Rearing the Parasitoid Ganaspis brasiliensis, a Promising Biological Control Agent for the Invasive Drosophila suzukii

Ganaspis brasiliensis-a larval parasitoid of Drosophila suzukii (a global invasive fruit crop pest)-has been approved or is considered for introduction into Europe and the United States for biological control of this pest. This article provides protocols for both small-scale and large-scale rearing of this parasitoid.

Native to East Asia, the spotted-wing drosophila, Drosophila suzukii (Matsumura) (Diptera: Drosophilidae), has established widely in the Americas, Europe, ...

Optimizing the Rearing Procedure of Germ-Free Wasps

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Chapters in this video

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

In Vitro Rearing of Solitary Bees: A Tool for Assessing Larval Risk Factors

Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing

Culturing and Genetically Manipulating Entomopathogenic Nematodes

Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

Preparation of Virus-Enriched Inoculum for Oral Infection of Honey Bees (Apis mellifera)

Pupal and Adult Injections for RNAi and CRISPR Gene Editing in Nasonia vitripennis

Automating Aggregate Quantification in Caenorhabditis elegans

Methods for Rearing the Parasitoid Ganaspis brasiliensis, a Promising Biological Control Agent for the Invasive Drosophila suzukii