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: 24 h, and L3: 48 h). The first instar larvae begin to feed immediately on the surface of the medium. Second instar larvae burrow into the medium, whereas third instar larvae leave the medium and wander up the vial walls, looking for a place to pupariate for 24-48 h. The D. melanogaster line used for this protocol is Oregon R (FBsn0000276).
<ol>
	<li>Add dry food containing a cornmeal-soy-based diet to a narrow polystyrene vial (25 mm x 95 mm) to aboue 1/5 to 2/5 volume. Then add 9 mL of water and allow the vial to sit for 1 min until the diet is completely hydrated.</li>
	<li>Add about 10 granules of dry baker&#39;s yeast to the vial and place a mixture of at least 20 newly emerged male and female adult flies.</li>
	<li>Incubate the vial at 25 &#176;C on a 12:12-h light: dark photoperiodic cycle.</li>
	<li>To ensure life cycle progression, check the fly vials daily and record the initial development stages.</li>
</ol>
2. Larvae selection for infection
<ol>
	<li>Select larvae with a fine paintbrush (camel&#39;s hair is best) once they reach the wandering third instar stage on the day the infection will be carried out (Figure 1).</li>
	<li>Wash the selected larvae with Ringer&#39;s solution (100 mM NaCl, 1.8 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 5 mM HEPES pH 6.9) in a small Petri dish (60 mm x 15 mm) upon removal from their original vial23.</li>
	<li>Place the larvae on filter paper (150 mm diameter) that is slightly moistened with around 5 mL of Ringer&#39;s solution in a Petri dish (100 mm x 15 mm) (Figure 2A).</li>
	<li>Add ~1 mL of Ringer&#39;s solution daily to the filter paper as needed to avoid dryness and larval desiccation.</li>
</ol>
3. Bacterial preparation
<ol>
	<li>Culture P. asymbiotica bacteria on Luria Bertani (LB) agar media at 28 &#176;C for 48 h.</li>
	<li>Use a biosafety level 2 cabinet to transfer a single colony of P. asymbiotica to inoculate a liquid culture in 10 mL of LB media.</li>
	<li>Incubate the P. asymbiotica liquid culture overnight at 28 &#176;C in a rotary shaker set to 220 rpm.</li>
	<li>Culture E. coli bacteria in a similar fashion, but carry out the initial growth on agar media and liquid culture incubation at 37 &#176;C overnight.</li>
	<li>Centrifuge each overnight bacterial culture for 3 min at 13,000 x g and 4 &#176;C.</li>
	<li>Discard the supernatant and wash the resulting bacterial pellets three times with 10 mL of sterile PBS.</li>
	<li>Centrifuge the pellets again for 3 min at 13,000 x g and 4 &#176;C.</li>
	<li>Dilute each pellet with sterile PBS to adjust bacterial concentrations to the optical density (600 nm) of 0.25 for P. asymbiotica and 0.015 for E. coli, which correspond to 100-300 colony forming units per larva of each bacterial preparation, using a spectrophotometer.</li>
</ol>
4. Injector preparation
<ol>
	<li>Prepare capillaries using 3.5&#8243; glass capillary tubes and a micropipette puller. Set the instrument as follows: heat: 580; pull: 143; velocity: 25; delay: 1; pressure: 500.</li>
	<li>Use straight serrated medium point forceps to open the glass capillary to the degree that will allow the delivery of the experimental treatments while causing minimal damage to the larvae (Figure 2B). To optimize this procedure while practicing injections, use Trypan Blue solution (dilute the stock 0.4% with PBS to 0.2%) to easily track leakage while injecting and the survival of the blue larvae.</li>
	<li>Fill the capillary with mineral oil using a plastic, disposable 20 mL syringe with a hypodermic needle (22 G, 25 mm length).</li>
	<li>Set up the nanoliter injector and eject the oil out of the capillary (Figure 2C).</li>
	<li>Fill the capillary with the desired bacterial preparation for injection. Take the solution from a drop (~20 &#181;L) that is placed on parafilm. In this protocol, 50.2 nL of two bacterial stocks were injected.</li>
</ol>
5. Larvae injection
<ol>
	<li>Anesthetize D. melanogaster larvae using carbon dioxide for ~2 min prior to the procedure.</li>
	<li>Gently transfer the anesthetized larvae to a filter paper moistened in Ringer&#39;s solution in preparation for injection under a stereomicroscope. The larvae will be lethargic or passive at this point.</li>
	<li>To inject larvae, apply firm pressure on the dorsal side of the posterior end (the tracheal spiracles are apparent at the posterior end versus black mouthparts at the anterior end) using forceps (Figure 2D).</li>
	<li>Insert the needle horizontally towards the posterior end of the larvae, near the cuticle. A successful injection will not result in a leakage of the injected solution from the larvae (Figure 3).</li>
	<li>Remove the forceps that were used to apply pressure on the larvae&#39;s tail before withdrawing the needle. If not removed first, the forceps can force hemolymph and/or the intestine out of the larvae from the wound site.</li>
	<li>Infect the appropriate number of larvae for the study being performed. In this particular protocol, 20 larvae for each experimental condition were injected.</li>
	<li>Using forceps, gently transfer the injected larvae to a separate moist filter paper in a Petri dish (10 larvae per dish) or a food vial (depending on the purpose of the experiment) to allow for recovery.</li>
	<li>Place the Petri dishes or food vials in an incubator at 25 &#176;C. If larvae are kept in a Petri dish, add Ringer&#39;s solution as necessary to prevent desiccation.</li>
</ol>
6. Recording survival/mortality
<ol>
	<li>Record the number of dead and live D. melanogaster larvae according to the set experimental time points. Live larvae will continue to develop into pupae and adults.</li>
	<li>Use statistical programs, such as Prism, to enter the raw survival/mortality data, analyze them with the log-rank (Mantel-Cox) test, and represent the results in figures.</li>
</ol>

<ol><li>Takehana, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Overexpression+of+a+pattern-recognition+receptor%2C+peptidoglycan-recognition+protein-LE%2C+activates+imd%2Frelish-mediated+antibacterial+defense+and+the+prophenoloxidase+cascade+in+Drosophila+larvae%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Overexpression of a pattern-recognition receptor, peptidoglycan-recognition protein-LE, activates imd/relish-mediated antibacterial defense and the prophenoloxidase cascade in Drosophila larvae.</a> Proceedings of the National Academy of Sciences of the United States of America. 99 (21), 13705-13710 (2002).</li>
<li>Senger, K., Harris, K., Levine, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((GATA+factors+participate+in+tissue-specific+immune+responses+in+Drosophila+larvae%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">GATA factors participate in tissue-specific immune responses in Drosophila larvae.</a> Proceedings of the National Academy of Sciences of the United States of America. 103 (43), 15957-15962 (2006).</li>
<li>Kenmoku, H., Hori, A., Kuraishi, T., Kurata, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+mode+of+induction+of+the+humoral+innate+immune+response+in+Drosophila+larvae%5BTitle%5D)+AND+%22Disease+Models+%26+Mech.anisms%22%5BJournal%5D)">A novel mode of induction of the humoral innate immune response in Drosophila larvae.</a> Disease Models & Mech.anisms. 10, 271-281 (2017).</li>
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<li>Kenney, E., Hawdon, J. M., O'Halloran, D. M., Eleftherianos, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Secreted+virulence+factors+from+Heterorhabditis+bacteriophora+highlight+its+utility+as+a+model+parasite+among+Clade+V+nematodes%5BTitle%5D)+AND+%22International+Journal+for+Parasitology%22%5BJournal%5D)">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>
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<li>Leitão, A. B., Bian, X., Day, J. P., Pitton, S., Demir, E., Jiggins, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Independent+effects+on+cellular+and+humoral+immune+responses+underlie+genotype-by-genotype+interactions+between+Drosophila+and+parasitoids%5BTitle%5D)+AND+%22PLoS+Pathogens%22%5BJournal%5D)">Independent effects on cellular and humoral immune responses underlie genotype-by-genotype interactions between Drosophila and parasitoids.</a> PLoS Pathogens. 15 (10), 1008084(2019).</li>
<li>Ramroop, J. R., Heavner, M. E., Razzak, Z. H., Govind, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Parasitoid+wasp+of+Drosophila+employs+preemptive+and+reactive+strategies+to+deplete+its+host%27s+blood+cells%5BTitle%5D)+AND+%22PLoS+Pathogens%22%5BJournal%5D)">Parasitoid wasp of Drosophila employs preemptive and reactive strategies to deplete its host's blood cells.</a> PLoS Pathogens. 17 (5), 1009615(2021).</li>
<li>Vlisidou, I., Wood, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Drosophila+blood+cells+and+their+role+in+immune+responses%5BTitle%5D)+AND+%22The+FEBS+Journal%22%5BJournal%5D)">Drosophila blood cells and their role in immune responses.</a> The FEBS Journal. 282 (8), 1368-1382 (2015).</li>
<li>Harnish, J. M., Link, N., Yamamoto, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Drosophila+as+a+model+for+infectious+diseases%5BTitle%5D)+AND+%22International+Journal+of+Molecular+Sciences%22%5BJournal%5D)">Drosophila as a model for infectious diseases.</a> International Journal of Molecular Sciences. 22 (5), 2724(2017).</li>
<li>Lemaitre, B., Hoffmann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+host+defense+of+Drosophila+melanogaster%5BTitle%5D)+AND+%22Annual+Reviews+of+Immunology%22%5BJournal%5D)">The host defense of Drosophila melanogaster.</a> Annual Reviews of Immunology. 25, 697-743 (2007).</li>
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<li>Trienens, M., Kraaijeveld, K., Wertheim, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Defensive+repertoire+of+Drosophila+larvae+in+response+to+toxic+fungi%5BTitle%5D)+AND+%22Molecular+Ecology%22%5BJournal%5D)">Defensive repertoire of Drosophila larvae in response to toxic fungi.</a> Molecular Ecology. 26 (19), 5043-5057 (2017).</li>
<li>Tafesh-Edwards, G., Eleftherianos, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Drosophila+immunity+against+natural+and+nonnatural+viral+pathogens%5BTitle%5D)+AND+%22Virology%22%5BJournal%5D)">Drosophila immunity against natural and nonnatural viral pathogens.</a> Virology. 540, 165-171 (2020).</li>
<li>Gold, K. S., Brückner, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Macrophages+and+cellular+immunity+in+Drosophila+melanogaster%5BTitle%5D)+AND+%22Seminars+in+Immunology%22%5BJournal%5D)">Macrophages and cellular immunity in Drosophila melanogaster.</a> Seminars in Immunology. 27 (6), 357-368 (2015).</li>
<li>Dudzic, J. P., Kondo, S., Ueda, R., Bergman, C. M., Lemaitre, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Drosophila+innate+immunity%3A+regional+and+functional+specialization+of+prophenoloxidases%5BTitle%5D)+AND+%22BMC+Biology%22%5BJournal%5D)">Drosophila innate immunity: regional and functional specialization of prophenoloxidases.</a> BMC Biology. 13, 81(2015).</li>
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<li>Zabihihesari, A., Hilliker, A. J., Rezai, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Localized+microinjection+of+intact+Drosophila+melanogaster+larva+to+investigate+the+effect+of+serotonin+on+heart+rate%5BTitle%5D)+AND+%22Lab+on+a+Chip%22%5BJournal%5D)">Localized microinjection of intact Drosophila melanogaster larva to investigate the effect of serotonin on heart rate.</a> Lab on a Chip. 20 (2), 343-355 (2020).</li>
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<li>Ciche, T. A., Sternberg, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Postembryonic+RNAi+in+Heterorhabditis+bacteriophora%3A+a+nematode+insect+parasite+and+host+for+insect+pathogenic+symbionts%5BTitle%5D)+AND+%22BMC+Developmental+Biology%22%5BJournal%5D)">Postembryonic RNAi in Heterorhabditis bacteriophora: a nematode insect parasite and host for insect pathogenic symbionts.</a> BMC Developmental Biology. 7, 101(2007).</li>
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</ol>

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 costly21,27.&#160;In the current protocol, to expand the use of D. melanogaster, a simple injection technique that represents an efficient and rapid method to deliver bacteria into the hemocoel of D. melanogaster larvae is outlined. The essential part of the technique described here is the actual injection of the desired pathogen or other liquid substances using a microinjector. In addition to describing this essential operation, we also describe accessory methods such as growing and culturing bacteria and handling materials.
The principal advantage of this method is that the needle is held approximately parallel to the larva, and the holding is done with very little pressure on the larva, thus reducing the possibility of hemolymph leakage, fatal injury, or inadequate delivery of the desired substance. The needle is then inserted toward the posterior end of the larva to complete the injection. The precise way of holding the larva, the manner of inserting the needle, the speed at which the desired substance is injected, and the direction of withdrawal of the needle are all matters that can best be improved by practice. The most challenging step to overcome is withdrawing the needle out of the larva without any resulting leakage of essential organs. However, with preciseness and experience, the difficulty encountered during this step becomes less.
This technique is also an excellent tool in multiple applications for introducing a variety of substances in a uniform manner that can be repeated several times, thus allowing for consistent results. The rapid mortality observed in P. asymbiotica-infected larvae reflects the high virulence of this bacterial strain to insects24,25,26. P. asymbiotica is well documented for expressing virulence genes during infection that increase bacterial survival and pathogenicity against insect hosts by inhibiting hemocyte migration and phagocytosis24,25. Also expectedly, larvae are resistant to injections with the non-pathogenic bacterial strains of E. coli as well as injections with PBS, thus confirming previous results in this research field26. Hence, microinjection is achieved without any adverse effect on animal viability, allowing its use in molecular and immunological studies to explore the pathogenesis of a wide range of microbial infections.

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><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 survivals24,25,26. Particularly, in comparison to larvae infected with P. asymbiotica, which exhibit a 57% survival rate 24 h following injection, larvae injected with E. coli show an 85% survival rate at the same time point.
<img alt="Fly life cycle stages; diagram showing adults, pupae, and larval stages in container; entomology." class="xfigimg" src="/files/ftp_upload/63144/63144fig01.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1: Selection of Drosophila melanogaster larvae for injection. The life cycle of Drosophila melanogaster, from egg fertilization to adult life, takes approximately 10 days. During larval growth, larvae feed until ready to pupate and change to adults. For the purpose of injection experiments, third instar larvae, which leave the culture medium and wander up the walls of the vial, are selected. <a href="https://www.jove.com/files/ftp_upload/63144/63144fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Larvae preparation and injection; capillary setup, microscope injector for gene editing experiment." class="xfigimg" src="/files/ftp_upload/63144/63144fig02.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2: Depiction of Drosophila melanogaster larval injection procedure. (A) Wandering third instar larvae are selected, washed with Ringer's solution, and placed on filter paper in a Petri dish in preparation for injection. (B) Using forceps, the glass capillary is broken to allow the delivery of the experimental treatments. (C) The programmable nanoliter injector is set up in preparation for injection under a stereomicroscope. (D) To inject larvae, pressure is applied on the dorsal side of the larvae's tail using forceps. <a href="https://www.jove.com/files/ftp_upload/63144/63144fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Larvae injection setup diagram with microscope illustrating anterior and posterior injection points." class="xfigimg" src="/files/ftp_upload/63144/63144fig03.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3: Illustration of delivering bacterial cells into Drosophila melanogaster larvae through microinjection. The dorsal side of the posterior end is stabilized using forceps. Then, the capillary is inserted horizontally towards the posterior end of the larvae, near the cuticle. Following the injection, the forceps are removed before carefully withdrawing the capillary to prevent hemolymph leakage from the wound site. <a href="https://www.jove.com/files/ftp_upload/63144/63144fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Survival rate graph, PBS vs. E. coli, P. asymbiotica, over 72 hours post-injection, line chart." class="xfigimg" src="/files/ftp_upload/63144/63144fig04.jpg" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
Figure 4: Survival of Drosophila melanogaster larvae following injection of pathogenic and non-pathogenic bacteria. Oregon R larvae of D. melanogaster were injected with 50.2 nL of Photorhabdus asymbiotica (ATCC43943), Escherichia coli (K12), or PBS. Whereas PBS and E. coli controls showed no significance in survival ratios, P. asymbiotica injections compromised fly survival rapidly. Each survival curve is comprised of measurements from three independent trials, each including 20 larvae (*** p &lt; 0.001). <a href="https://www.jove.com/files/ftp_upload/63144/63144fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

drosophila-melanogaster-larva-injection-protocol

Universidad Científica del Sur

Research

JoVE Journal

Immunology and Infection

6.1K Views.  The George Washington University. 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. 

Video: Drosophila melanogaster Larva Injection Protocol

Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster

In multicellular organisms, a small group of cells is endowed with a specialized function in their biogenic activity, inducing a systemic response to&#160;growth and reproduction. In insects, the larval prothoracic gland (PG) and the adult female ovary play essential roles in biosynthesizing the principal steroid hormones called ecdysteroids. These ecdysteroidogenic organs are innervated from the nervous system, through&#160;which the timing of biosynthesis is affected by environmental cues. Here we describe a protocol for visualizing ecdysteroidogenic organs and their interactive organs in larvae and adults of the fruit fly Drosophila melanogaster, which provides a suitable model system for studying steroid hormone biosynthesis and its regulatory mechanism. Skillful dissection allows us to maintain the positions of ecdysteroidogenic organs and their interactive organs including the brain, the ventral nerve cord, and other tissues. Immunostaining with antibodies against ecdysteroidogenic enzymes, along with transgenic fluorescence proteins driven by tissue-specific promoters, are available to label ecdysteroidogenic cells. Moreover, the innervations of the ecdysteroidogenic organs can also be labeled by specific antibodies or a collection of GAL4 drivers in various types of neurons. Therefore, the ecdysteroidogenic organs and their neuronal connections can be&#160;visualized simultaneously&#160;by immunostaining and transgenic techniques. Finally, we describe how to visualize germline stem cells, whose proliferation and maintenance are controlled by ecdysteroids. This method contributes to comprehensive understanding of steroid hormone biosynthesis and its neuronal regulatory mechanism.

Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster

We describe a protocol for dissection, fixation, and immunostaining of steroidogenic organs in Drosophila larvae and adult females to study steroid hormone biosynthesis and its regulatory mechanism. In addition to steroidogenic organs, we visualize the innervation of steroidogenic organs as well as steroidogenic target cells such as germline stem cells.

In multicellular organisms, a small group of cells is endowed with a specialized function in their biogenic activity, inducing a systemic response ...

Developmental Biology

Quantitative Measurement of the Immune Response and Sleep in Drosophila

A complex interaction between the immune response and host behavior has been described in a wide range of species. Excess sleep, in particular, is known to occur as a response to infection in mammals 1 and has also recently been described in Drosophila melanogaster2. It is generally accepted that sleep is beneficial to the host during an infection and that it is important for the maintenance of a robust immune system3,4. However, experimental evidence that supports this hypothesis is limited4, and the function of excess sleep during an immune response remains unclear. We have used a multidisciplinary approach to address this complex problem, and have conducted studies in the simple genetic model system, the fruitfly Drosophila melanogaster. We use a standard assay for measuring locomotor behavior and sleep in flies, and demonstrate how this assay is used to measure behavior in flies infected with a pathogenic strain of bacteria. This assay is also useful for monitoring the duration of survival in individual flies during an infection. Additional measures of immune function include the ability of flies to clear an infection and the activation of NF&kappa;B, a key transcription factor that is central to the innate immune response in Drosophila. Both survival outcome and bacterial clearance during infection together are indicators of resistance and tolerance to infection. Resistance refers to the ability of flies to clear an infection, while tolerance is defined as the ability of the host to limit damage from an infection and thereby survive despite high levels of pathogen within the system5. Real-time monitoring of NF&kappa;B activity during infection provides insight into a molecular mechanism of survival during infection. The use of Drosophila in these straightforward assays facilitates the genetic and molecular analyses of sleep and the immune response and how these two complex systems are reciprocally influenced.

Quantitative Measurement of the Immune Response and Sleep in Drosophila

To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NF&kappa;B.

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of the Drosophila melanogaster syncytial embryo for studying mitosis1. Drosophila has useful genetics with a sequenced genome, and it can be easily maintained and manipulated. Many mitotic mutants exist, and transgenic flies expressing functional fluorescently (e.g. GFP) - tagged mitotic proteins have been and are being generated. Targeted gene expression is possible using the GAL4/UAS system2. 

The Drosophila early embryo carries out multiple mitoses very rapidly (cell cycle duration, &asymp;10 min). It is well suited for imaging mitosis, because during cycles 10-13, nuclei divide rapidly and synchronously without intervening cytokinesis at the surface of the embryo in a single monolayer just underneath the cortex. These rapidly dividing nuclei probably use the same mitotic machinery as other cells, but they are optimized for speed; the checkpoint is generally believed to not be stringent, allowing the study of mitotic proteins whose absence would cause cell cycle arrest in cells with a strong checkpoint. Embryos expressing GFP labeled proteins or microinjected with fluorescently labeled proteins can be easily imaged to follow live dynamics (Fig. 1). In addition, embryos can be microinjected with function-blocking antibodies or inhibitors of specific proteins to study the effect of the loss or perturbation of their function3. These reagents can diffuse throughout the embryo, reaching many spindles to produce a gradient of concentration of inhibitor, which in turn results in a gradient of defects comparable to an allelic series of mutants. Ideally, if the target protein is fluorescently labeled, the gradient of inhibition can be directly visualized4. It is assumed that the strongest phenotype is comparable to the null phenotype, although it is hard to formally exclude the possibility that the antibodies may have dominant effects in rare instances, so rigorous controls and cautious interpretation must be applied. Further away from the injection site, protein function is only partially lost allowing other functions of the target protein to become evident.

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of microinjection and high resolution imaging in the Drosophila melanogaster syncytial embryo to study mitosis.

This protocol describes the use of the Drosophila melanogaster syncytial embryo  for studying mitosis1.  Drosophila has useful genetics with a sequenced ...

Biology

phiC31 Integrase-Mediated Embryo Transgenesis in Drosophila

Embryo Microinjection for Transgenesis in Drosophila

Transgenesis in Drosophila is an essential approach to studying gene function at the organism level. Embryo microinjection is a crucial step for the construction of transgenic flies. Microinjection requires some types of equipment, including a microinjector, a micromanipulator, an inverted microscope, and a stereo microscope.&#160;Plasmids isolated with a plasmid miniprep kit are qualified for microinjection. Embryos at the pre-blastoderm or syncytial blastoderm stage, where nuclei share a common cytoplasm, are subjected to microinjection. A cell strainer eases the process of dechorionating embryos. The optimal time for dechorionation and desiccation of embryos needs to be determined experimentally. To increase the efficiency of embryo microinjection, needles prepared by a puller need to be beveled by a needle grinder. In the process of grinding needles, we utilize a foot air pump with a pressure gauge to avoid the capillary effect of the needle tip. We routinely inject 120-140 embryos for each plasmid and obtain at least one transgenic line for around 85% of plasmids. This article takes the phiC31 integrase-mediated transgenesis in Drosophila as an example and&#160;presents a detailed protocol for embryo microinjection for transgenesis in Drosophila.

Author Spotlight: Optimizing Embryo Microinjection for Transgenesis in Drosophila

This article takes the phiC31 integrase-mediated transgenesis in Drosophila as an example and presents an optimized protocol for embryo microinjection,&#160;a crucial step for creating transgenic flies.

Transgenesis in Drosophila is an essential approach to studying gene function at the organism level. Embryo microinjection is a crucial step for the ...

Genetics

Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

The fruit fly Drosophila melanogaster is one of the premier model organisms for studying the function and evolution of immune defense. Many aspects of innate immunity are conserved between insects and mammals, and since Drosophila can readily be genetically and experimentally manipulated, they are powerful for studying immune system function and the physiological consequences of disease. The procedure demonstrated here allows infection of flies by introduction of bacteria directly into the body cavity, bypassing epithelial barriers and more passive forms of defense and allowing focus on systemic infection. The procedure includes protocols for the measuring rates of host mortality, systemic pathogen load, and degree of induction of the host immune system. This infection procedure is inexpensive, robust and quantitatively repeatable, and can be used in studies of functional genetics, evolutionary life history, and physiology.

Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

Drosophila melanogaster is an outstanding model organism for studying innate immune systems and the physiological consequences of infection and disease. This protocol describes how to deliver robust and quantitatively repeatable bacterial infections to D. melanogaster, and how to subsequently measure infection severity and quantify the host immune response.

The fruit fly Drosophila melanogaster is one of the premier model organisms for studying the function and evolution of immune defense. Many aspects of ...

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Proper nervous system development includes the formation of the blood-brain barrier, the diffusion barrier that tightly regulates access to the nervous system and protects neural tissue from toxins and pathogens. Defects in the formation of this barrier have been correlated with neuropathies, and the breakdown of this barrier has been observed in many neurodegenerative diseases. Therefore, it is critical to identify the genes that regulate the formation and maintenance of the blood-brain barrier to identify potential therapeutic targets. In order to understand the exact roles these genes play in neural development, it is necessary to assay the effects of altered gene expression on the integrity of the blood-brain barrier. Many of the molecules that function in the establishment of the blood-brain barrier have been found to be conserved across eukaryotic species, including the fruit fly, Drosophila melanogaster. Fruit flies have proven to be an excellent model system for examining the molecular mechanisms regulating nervous system development and function. This protocol describes a step-by-step procedure to assay for blood-brain barrier integrity during the embryonic and larval stages of D. melanogaster development.

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Blood-brain barrier integrity is critical for nervous system function. In Drosophila melanogaster, the blood-brain barrier is formed by glial cells during late embryogenesis. This protocol describes methods to assay for blood-brain barrier formation and maintenance in D. melanogaster embryos and third instar larvae.

Proper nervous system development includes the formation of the blood-brain barrier, the diffusion barrier that tightly regulates access to the nervous ...

Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos

The purpose of this protocol is to visualize intranuclear actin rods that assemble in live Drosophila melanogaster embryos following heat stress. Actin rods are a hallmark of a conserved, inducible Actin Stress Response (ASR) that accompanies human pathologies, including neurodegenerative disease. Previously, we showed that the ASR contributes to morphogenesis failures and reduced viability of developing embryos. This protocol allows the continued study of mechanisms underlying actin rod assembly and the ASR in a model system that is highly amenable to imaging, genetics and biochemistry. Embryos are collected and mounted on a coverslip to prepare them for injection. Rhodamine-conjugated globular actin (G-actinRed) is diluted and loaded into a microneedle. A single injection is made into the center of each embryo. After injection, embryos are incubated at elevated temperature and intranuclear actin rods are then visualized by confocal microscopy. Fluorescence recovery after photobleaching (FRAP) experiments may be performed on the actin rods; and other actin-rich structures in the cytoplasm can also be imaged. We find that G-actinRed polymerizes like endogenous G-actin and does not, on its own, interfere with normal embryo development. One limitation of this protocol is that care must be taken during injection to avoid serious injury to the embryo. However, with practice, injecting G-actinRed into Drosophila embryos is a fast and reliable way to visualize actin rods and can easily be used with flies of any genotype or with the introduction of other cellular stresses, including hypoxia and oxidative stress.

Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos

The goal of this protocol is to inject Rhodamine-conjugated globular actin into Drosophila embryos and image intranuclear actin rod assembly following heat stress.

The purpose of this protocol is to visualize intranuclear actin rods that assemble in live Drosophila melanogaster embryos following heat stress. Actin ...

RNAi Interference by dsRNA Injection into Drosophila Embryos

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements and tools for genetic manipulation have become available in Drosophila 2. Soon after the initial discovery by Frie and Mello 3 that double stranded RNA can be used to knockdown the activity of individual genes in Caenorhabditis elegans, RNA interference (RNAi) was shown to provide a powerful reverse genetic approach to analyze gene functions in Drosophila organ development 4, 5.
Many organs, including lung, kidney, liver, and vascular system, are composed of branched tubular networks that transport vital fluids or gases 6, 7. The analysis of Drosophila tracheal formation provides an excellent model system to study the morphogenesis of other tubular organs 8. The Berkeley Drosophila genome project has revealed hundreds of genes that are expressed in the tracheal system. To study the molecular and cellular mechanism of tube formation, the challenge is to understand the roles of these genes in tracheal development. Here, we described a detailed method of dsRNA injection into Drosophila embryo to knockdown individual gene expression. We successfully knocked down endogenous dysfusion(dys) gene expression by dsRNA injection. Dys is a bHLH-PAS protein expressed in tracheal fusion cells, and it is required for tracheal branch fusion 9, 10. dys-RNAi completely eliminated dys expression and resulted in tracheal fusion defect. This relatively simple method provides a tool to identify genes requried for tissure and organ development in Drosophila.

RNAi Interference by dsRNA Injection into Drosophila Embryos

RNA interference has been proven very effective to analyze gene function in Drosophila tracheal development. A detailed protocol used by Jiang lab to inject dsRNA into fly embryos to knockdown gene expression is illustrated. This technique has the potential for screening genes required for tissue and organ development in Drosophila.

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements ...

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function to maintain glucose homeostasis during developmental larval stages 1,2. Studies on largely post-mitotic adult flies have revealed perturbation of glucose homeostasis as the result of genetic ablation of insulin-like peptide (ILP) producing cells (IPCs) 3. Thus, adult fruit flies hold great promise as a suitable genetic model system for metabolic disorders including type II diabetes. To further develop the fruit fly system, comparable physiological assays used to measure glucose tolerance and insulin sensitivity in mammals must be established. To this end, we have recently described a novel procedure for measuring oral glucose tolerance response in the adult fly and demonstrated the importance of adult IPCs in maintaining glucose homeostasis 4,5. Here, we have modified a previously described procedure for insulin injection 6 and combined it with a novel hemolymph extraction method to measure peripheral insulin sensitivity in the adult fly. Uniquely, our protocol allows direct physiological measurements of the adult fly's ability to dispose of a peripheral glucose load upon insulin injection, a methodology that makes it feasible to characterize insulin signaling mutants and potential interventions affecting glucose tolerance and insulin sensitivity in the adult fly.

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Conserved insulin signaling pathways found in the fruit fly Drosophila melanogaster make this organism a potential tool for modeling metabolic disorders including type II diabetes. To this end, it is critical to establish physiological assays to effectively measure systemic insulin action in peripheral glucose disposal in the adult fly.

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function ...

Microinjection of Live Drosophila Embryos: Early Delivery of Reagents to the Developing Embryo

Source: Brust-Mascher, I., Scholey, J. M. <a href="https://www.jove.com/video/1382/" target="_blank">Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo</a>. J. Vis. Exp. (2009).
This video describes how to perform microinjections that deliver reagents into live Drosophila embryos. Researchers can use this method to introduce exogenous compounds like nucleic acids and proteins during the early stages of embryo development. The featured protocol demonstrates how to set up and perform the procedure for injection of any soluble reagent&#8211;here, a fluorescent protein for live imaging experiments.

Source: Brust-Mascher, I., Scholey, J. M. Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo. J. Vis. Exp. ...

Drosophila melanogaster Larva Injection Protocol

Summary

Explore More Videos

Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster

Quantitative Measurement of the Immune Response and Sleep in Drosophila

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

Embryo Microinjection for Transgenesis in Drosophila

Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos

RNAi Interference by dsRNA Injection into Drosophila Embryos

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Microinjection of Live Drosophila Embryos: Early Delivery of Reagents to the Developing Embryo