We thank members of the Department of Biological Sciences at George Washington University (GWU) for critical reading of the manuscript. GT was supported through a Harlan summer fellowship from GWU. All graphical figures were made using BioRender.

1. Fly rearing
NOTE: The D. melanogaster life cycle is divided into four stages: embryo, larva, pupa, and adult. The generation time with optimal rearing conditions in the laboratory (~25 &#176;C, 60% humidity, and sufficient food) is approximately 10 days from fertilized egg to eclosed adult.Females lay ~100 embryos per day, and embryogenesis lasts about 24 h22. The larvae undergo three developmental stages (instars; L1-L3) in ~4 days (L1 and L2...

<ol>
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M., O'Halloran, D., Eleftherianos, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterorhabditis+bacteriophora+excreted-secreted+products+enable+infection+by+Photorhabdus+luminescens+through+suppression+of+the+Imd+pathway.">Heterorhabditis bacteriophora excreted-secreted products enable infection by Photorhabdus luminescens through suppression of the Imd pathway.</a> Frontiers in Immunology. 10, 2372 (2019).</li><li>Cherry, S., Silverman, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host-pathogen+interactions+in+Drosophila:+New+tricks+from+an+old+friend.">Host-pathogen interactions in Drosophila: New tricks from an old friend.</a> Nature Immunology. 7 (9), 911-917 (2006).</li><li>Younes, S., Al-Sulaiti, A., Nasser, E., Najjar, H., Kamareddine, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+as+a+model+organism+in+host-pathogen+interaction+studies.">Drosophila as a model organism in host-pathogen interaction studies.</a> Frontiers in Cellular and Infection Microbiology. 10, 214 (2020).</li><li>Kenney, E., Hawdon, J. M., O'Halloran, D. M., Eleftherianos, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secreted+virulence+factors+from+Heterorhabditis+bacteriophora+highlight+its+utility+as+a+model+parasite+among+Clade+V+nematodes.">Secreted virulence factors from Heterorhabditis bacteriophora highlight its utility as a model parasite among Clade V nematodes.</a> International Journal for Parasitology. 51 (5), 321-325 (2021).</li><li>Castillo, J. C., Reynolds, S. E., Eleftherianos, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insect+immune+responses+to+nematode+parasites.">Insect immune responses to nematode parasites.</a> Trends in Parasitology. 27 (12), 537-547 (2011).</li><li>Leit&#227;o, A. B., Bian, X., Day, J. P., Pitton, S., Demir, E., Jiggins, F. 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The authors declare no competing interests.

Drosophila melanogaster is among the most valuable, experimentally manipulated models used for investigations of innate immunity and pathogenesis of various microbial infections. This is due to its simple and fast life cycle, simple upkeep in a laboratory, well-established evolutionary genetics, and diverse genetic toolbox. Previous methods of D. melanogaster larvae injections, such as using a hybrid microfluidic device or a Narishige micromanipulator, require highly specialized equipment and can be cos...

Drosophila melanogaster has been immensely utilized in biological and biomedical research for several decades, as the sophisticated array of genetic and molecular tools have steadily evolved for analysis of a wide range of studies1,2,3,4. The evolutionarily conserved aspects of development, homeostasis and innate immunity in D. melanogaster have made it a valuable model organism for studying various human and insect diseases5,6. Notably, the fundamental role of the D. melanogaster model for studying immunity has been largely exemplified in adult flies studies. However, D. melanogaster larvae studies have also contributed to the current knowledge and mainly explored cellular immune responses, specifically for wasp and nematode infections that occur through the insect cuticle7,8,9,10. Drosophila melanogaster larvae possess three different types of blood cells, collectively called hemocytes: plasmatocytes, crystal cells, and lamellocytes11,12,13. These cells can mount an array of immune responses when D. melanogaster larvae are infected with pathogens such as bacteria, fungi, viruses, and parasites14,15,16. Cellular immune responses include direct engulfment (phagocytosis) of small molecules or bacteria, melanization, encapsulation of larger pathogens such as parasitoid eggs, and production of reactive oxygen species (ROS) and Nitric oxide synthases (NOS)17,18,19.
In contrast, fewer studies have been published on the use of the D. melanogaster larval model to analyze humoral immune responses. This is mainly due to the application of feeding assays for oral infection of D. melanogaster larvae and several challenges associated with microinjecting larvae including the precise handling of larvae and proper use of the microneedle, especially during penetration20,21. Thus, the limited knowledge of larval infection and technical difficulties (i.e., high mortality) have frequently made the D. melanogaster larval model difficult to use. A larval model will have the potential to identify novel molecular mechanisms that will provide further insights into host-pathogen interactions and the induction of specific host innate immune responses against pathogenic infections.
Here a simple and efficient protocol that can be used to inject D. melanogaster larvae with various pathogens, such as bacteria, is described in detail. In particular, D. melanogaster larvae are used for injections with the human pathogen Photorhabdus asymbiotica and the non-pathogenic bacteria Escherichia coli. This method can be used for the manipulation and analysis of D. melanogaster&#39;s immune responses to various microbial infections.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Fly Food B (Bloomington Recipe)</td>
			<td>LabExpress</td>
			<td>7001-NV</td>
			<td>Food B, in narrow vials, 100 vials/tray</td>
		</tr>
		<tr>
			<td>100 x 15, Mono Petri Dishes Fully Stackable</td>
			<td>VWR</td>
			<td>25384-342</td>
			<td>Diameter 100 x 15 mm</td>
		</tr>
		<tr>
			<td>60 x 15, Mono Petri dishes Fully Stackable</td>
			<td>VWR</td>
			<td>25384-092</td>
			<td>Diameter 60 x 15 mm</td>
		</tr>
		<tr>
			<td>Glass capillaries</td>
			<td>VWR</td>
			<td>53440-186</td>
			<td></td>
		</tr>
		<tr>
			<td>Grade 1 qualitative filter paper standard grade, circle</td>
			<td>VWR</td>
			<td>28450-150</td>
			<td>Diameter 150 mm</td>
		</tr>
		<tr>
			<td>Lab culture Class II Type A2 Biosafety Safety Cabinet</td>
			<td>ESCO</td>
			<td>LA2-4A2-E</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Agar</td>
			<td>Fisher Scientific</td>
			<td>BP1425-500</td>
			<td>LB agar miller powder 500 g</td>
		</tr>
		<tr>
			<td>LB Broth</td>
			<td>Fisher Scientific</td>
			<td>BP1426-500</td>
			<td>LB broth miller powder 500 g</td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Alfa Aesar, Thermo Fisher Scientific</td>
			<td>31911-A1</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop 2000/2000c Spectrophotometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>ND-2000C</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanoject III Programmable Nanoliter Injector</td>
			<td>Drummond</td>
			<td>3-000-207</td>
			<td></td>
		</tr>
		<tr>
			<td>Narrow Drosophila Vials, Polystyrene</td>
			<td>Genesee Scientific</td>
			<td>32-109</td>
			<td></td>
		</tr>
		<tr>
			<td>Needles, hypodermic</td>
			<td>VWR</td>
			<td>89219-316</td>
			<td>22 G, 25 mm</td>
		</tr>
		<tr>
			<td>Next Generation Micropipette Puller</td>
			<td>World Precision Instruments</td>
			<td>SU-P1000</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>VWR</td>
			<td>97062-732</td>
			<td>Buffer PBS tablets biotech grade 200tab</td>
		</tr>
		<tr>
			<td>Prism</td>
			<td>GraphPad</td>
			<td>Version 8</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringes - plastic, disposable</td>
			<td>VWR</td>
			<td>76124-652</td>
			<td>20 mL</td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>Sigma-Aldrich</td>
			<td>T8154</td>
			<td></td>
		</tr>
	</tbody>
</table>

drosophila melanogaster larva injection protocol

The use of unconventional models to study innate immunity and pathogen virulence provides a valuable alternative to mammalian models, which can be costly and raise ethical issues. Unconventional models are notoriously cheap, easy to handle and culture, and do not take much space. They are genetically amenable and possess complete genome sequences, and their use presents no ethical considerations. The fruit fly Drosophila melanogaster, for instance, has provided great insights into a variety of behavior, development, metabolism, and immunity research. More specifically, D. melanogaster adult flies and larvae possess several innate defense reactions that are shared with vertebrate animals. The mechanisms regulating immune responses have been mostly revealed through genetic and molecular studies in the D. melanogaster model. Here a novel larval injection technique is provided, which will further promote investigations of innate immune processes in D. melanogaster larvae and explore the pathogenesis of a wide range of microbial infections.

When performed correctly, injections of D. melanogaster larvae show a bacterium-specific effect. The survival data were collected at several time points following infections of P. asymbiotica (strain ATCC43943), E. coli (strain K12), and PBS (Figure 4). Whereas D. melanogaster larvae are susceptible to P. asymbiotica, which compromises survival rapidly, larvae injected with E. coli or PBS controls exhibit prolonged survivals<sup class="xr...

Watch this Scientific Journal Video about Drosophila melanogaster Larva Injection Protocol at JoVE.com

Drosophila melanogaster Larva Injection Protocol

Drosophila melanogaster adult flies have been extensively utilized as model organisms to investigate the molecular mechanisms underlying host antimicrobial innate immune responses and microbial infection strategies. To promote the D. melanogaster larva stage as an additional or alternative model system, a larval injection technique is described.

Drosophila melanogaster Larva Injection Protocol

The use of unconventional models to study innate immunity and pathogen virulence provides a valuable alternative to mammalian models, which can be costly ...

This protocol addresses the difficulties and challenges with microinjecting larva by describing a precise method that will allow the use of Drosophila melanogaster larva as a model for various immune and molecular studies. This is a simple injection technique that presents an efficient, cost-effective and rapid method to repeatedly deliver a variety of substances in a uniform manner, which allows for consistent and reliable results. Begin by selecting third instar stage larvae and washing them with Ringer's solution in a small Petri dish.Place a filter paper in a Petri dish and moisten it with 5 ml of Ringer's solution. Then place the larvae on the filter paper. Prepare capillaries using 3.5"glass capillary tubes in a micropipette puller.Open the glass capillary with the help of straight serrated medium point forceps. Fill the open capillary with mineral oil using a plastic disposable syringe. Then, eject the oil out of the capillary tube with a nanoliter injector.Fill the capillary with the desired bacterial preparation for injection. Transfer the anesthetized larvae to a filter paper moistened with Ringer's solution. Using forceps, apply firm pressure on the dorsal side of the posterior end of the larvae, where the tracheal spiracles are apparent.Then, insert the needle horizontally, towards the posterior end of the larvae, and inject the bacterial stocks. Remove forceps to avoid spillage of hemolymph or intestine. Then, withdraw the needle previously inserted into larvae.Infect 20 larvae for each experimental condition. Using a paintbrush, gently transfer the injected larvae to a separate moist filter paper. Add Ringer's solution as necessary to prevent desiccation.Incubate the Petri dish in an incubator at 25 C.Drosophila melanogaster larvae infected with Photorhabdus asymbiotica exhibited a 57%survival rate for 24 hours following injection, while larva injected with Escherichia coli showed an 85%survival rate at the same time point. The importance of this method is that the needle is held approximately parallel to the larva. The pressure applied to hold the larva is very little, which reduces the possibility of hemolymph leakage, fatal injury, or inadequate delivery of the desired substance.This technique can be perfected with practice.

drosophila-melanogaster-larva-injection-protocol

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JoVE Journal

Immunology and Infection

5.6K visualizações.  The George Washington University. Este protocolo aborda as dificuldades e desafios com a larva de microinjeção, descrevendo um método preciso que permitirá o uso da larva de melanogaster Drosophila como modelo para vários estudos imunológicos e moleculares. Esta é uma técnica de injeção simples que apresenta um método eficiente, econômico e rápido para fornecer repetidamente uma variedade de substâncias de forma uniforme, o que permite resultados consistentes e confiáveis. Comece selecionando o terceiro estági...