Name: drosophila melanogaster larva injection protocol
Uploaded: 2021-10-19T14:45:00
Description: Watch this Scientific Journal Video about Drosophila melanogaster Larva Injection Protocol at JoVE.com

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>
	<li>Takehana, 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=Overexpression+of+a+pattern-recognition+receptor,+peptidoglycan-recognition+protein-LE,+activates+imd/relish-mediated+antibacterial+defense+and+the+prophenoloxidase+cascade+in+Drosophila+larvae.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GATA+factors+participate+in+tissue-specific+immune+responses+in+Drosophila+larvae.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+mode+of+induction+of+the+humoral+innate+immune+response+in+Drosophila+larvae.">A novel mode of induction of the humoral innate immune response in Drosophila larvae.</a> Disease Models &amp; Mech.anisms. 10, 271-281 (2017).</li><li>Kenney, E., Hawdon, J. 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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Independent+effects+on+cellular+and+humoral+immune+responses+underlie+genotype-by-genotype+interactions+between+Drosophila+and+parasitoids.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parasitoid+wasp+of+Drosophila+employs+preemptive+and+reactive+strategies+to+deplete+its+host's+blood+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+blood+cells+and+their+role+in+immune+responses.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+as+a+model+for+infectious+diseases.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+host+defense+of+Drosophila+melanogaster.">The host defense of Drosophila melanogaster.</a> Annual Reviews of Immunology. 25, 697-743 (2007).</li><li>Garriga, A., Mastore, M., Morton, A., Pino, F. G., Brivio, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+response+of+Drosophila+suzukii+larvae+to+infection+with+the+nematobacterial+complex+Steinernema+carpocapsae-Xenorhabdus+nematophila.">Immune response of Drosophila suzukii larvae to infection with the nematobacterial complex Steinernema carpocapsae-Xenorhabdus nematophila.</a> Insects. 11 (4), 210 (2020).</li><li>Trienens, M., Kraaijeveld, K., Wertheim, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defensive+repertoire+of+Drosophila+larvae+in+response+to+toxic+fungi.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+immunity+against+natural+and+nonnatural+viral+pathogens.">Drosophila immunity against natural and nonnatural viral pathogens.</a> Virology. 540, 165-171 (2020).</li><li>Gold, K. S., Br&#252;ckner, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages+and+cellular+immunity+in+Drosophila+melanogaster.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+innate+immunity:+regional+and+functional+specialization+of+prophenoloxidases.">Drosophila innate immunity: regional and functional specialization of prophenoloxidases.</a> BMC Biology. 13, 81 (2015).</li><li>Honti, V., Csord&#225;s, G., Kurucz, &. #. 2. 0. 1. ;., M&#225;rkus, R., And&#243;, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell-mediated+immunity+of+Drosophilamelanogaster:+hemocyte+lineages,+immune+compartments,+microanatomy+and+regulation.">The cell-mediated immunity of Drosophilamelanogaster: hemocyte lineages, immune compartments, microanatomy and regulation.</a> Developmental and Comparative Immunology. 42 (1), 47-56 (2014).</li><li>Siva-Jothy, J. A., Prakash, A., Vasanthakrishnan, R. B., Monteith, K. M., Vale, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+bacterial+infection+and+shedding+in+Drosophila+melanogaster.">Oral bacterial infection and shedding in Drosophila melanogaster.</a> Journal of Visualized Experiments: JoVE. (135), e57676 (2021).</li><li>Zabihihesari, A., Hilliker, A. J., Rezai, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localized+microinjection+of+intact+Drosophila+melanogaster+larva+to+investigate+the+effect+of+serotonin+on+heart+rate.">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><li>Flatt, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life-history+evolution+and+the+genetics+of+fitness+components+in+Drosophila+melanogaster.">Life-history evolution and the genetics of fitness components in Drosophila melanogaster.</a> Genetics. 214 (1), 3-48 (2020).</li><li>Ciche, T. A., Sternberg, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postembryonic+RNAi+in+Heterorhabditis+bacteriophora:+a+nematode+insect+parasite+and+host+for+insect+pathogenic+symbionts.">Postembryonic RNAi in Heterorhabditis bacteriophora: a nematode insect parasite and host for insect pathogenic symbionts.</a> BMC Developmental Biology. 7, 101 (2007).</li><li>Joyce, S. A., Watson, R. J., Clarke, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+regulation+of+pathogenicity+and+mutualism+in+Photorhabdus.">The regulation of pathogenicity and mutualism in Photorhabdus.</a> Current Opinion in Microbiology. 9 (2), 127-132 (2006).</li><li>Yang, G., Waterfield, N. 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+role+of+TcdB+and+TccC+subunits+in+secretion+of+the+Photorhabdus+Tcd+toxin+complex.">The role of TcdB and TccC subunits in secretion of the Photorhabdus Tcd toxin complex.</a> PLoS Pathogens. 9 (10), 1003644 (2013).</li><li>Shokal, U., 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=Effects+of+co-occurring+Wolbachia+and+Spiroplasma+endosymbionts+on+the+Drosophila+immune+response+against+insect+pathogenic+and+non-pathogenic+bacteria.">Effects of co-occurring Wolbachia and Spiroplasma endosymbionts on the Drosophila immune response against insect pathogenic and non-pathogenic bacteria.</a> BMC Microbiology. 16, 16 (2016).</li><li>Tomoyasu, Y., Denell, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Larval+RNAi+in+Tribolium+(Coleoptera)+for+analyzing+adult+development.">Larval RNAi in Tribolium (Coleoptera) for analyzing adult development.</a> Development Genes and Evolution. 214 (11), 575-578 (2004).</li></ol>

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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Immunology and Infection

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

Manduca sexta, commonly known as the tobacco hornworm, is considered a significant agricultural pest, feeding on solanaceous plants including tobacco and tomato. The susceptibility of M. sexta larvae to a variety of entomopathogenic bacterial species1-5, as well as the wealth of information available regarding the insect's immune system6-8, and the pending genome sequence9 make it a good model organism for use in studying host-microbe interactions during pathogenesis. In addition, M. sexta larvae are relatively large and easy to manipulate and maintain in the laboratory relative to other susceptible insect species. Their large size also facilitates efficient tissue/hemolymph extraction for analysis of the host response to infection.
The method presented here describes the direct injection of bacteria into the hemocoel (blood cavity) of M. sexta larvae. This approach can be used to analyze and compare the virulence characteristics of various bacterial species, strains, or mutants by simply monitoring the time to insect death after injection. This method was developed to study the pathogenicity of Xenorhabdus and Photorhabdus species, which typically associate with nematode vectors as a means to gain entry into the insect. Entomopathogenic nematodes typically infect larvae via natural digestive or respiratory openings, and release their symbiotic bacterial contents into the insect hemolymph (blood) shortly thereafter10. The injection method described here bypasses the need for a nematode vector, thus uncoupling the effects of bacteria and nematode on the insect. This method allows for accurate enumeration of infectious material (cells or protein) within the inoculum, which is not possible using other existing methods for analyzing entomopathogenesis, including nicking11 and oral toxicity assays12. Also, oral toxicity assays address the virulence of secreted toxins introduced into the digestive system of larvae, whereas the direct injection method addresses the virulence of whole-cell inocula.
The utility of the direct injection method as described here is to analyze bacterial pathogenesis by monitoring insect mortality. However, this method can easily be expanded for use in studying the effects of infection on the M. sexta immune system. The insect responds to infection via both humoral and cellular responses. The humoral response includes recognition of bacterial-associated patterns and subsequent production of various antimicrobial peptides7; the expression of genes encoding these peptides can be monitored subsequent to direct infection via RNA extraction and quantitative PCR13. The cellular response to infection involves nodulation, encapsulation, and phagocytosis of infectious agents by hemocytes6. To analyze these responses, injected insects can be dissected and visualized by microscopy13, 14.

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

The method described here utilizes direct injection of entomopathogenic bacteria into the hemocoel of Manduca sexta insect larvae. M. sexta is a commercially available and well-studied insect. Thus, this method represents a simple approach to analyzing host-bacterial interactions from the perspective of one or both partners.

Manduca sexta, commonly known as the tobacco hornworm, is considered a significant agricultural pest, feeding on solanaceous plants including tobacco and ...

A Protocol for Analyzing Hepatitis C Virus Replication

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HCV is an enveloped RNA virus belonging to the family Flaviviridae. Current treatment is not fully effective and causes adverse side effects. There is no HCV vaccine available. Thus, continued effort is required for developing a vaccine and better therapy. An HCV cell culture system is critical for studying various stages of HCV growth including viral entry, genome replication, packaging, and egress. In the current procedure presented, we used a wild-type intragenotype 2a chimeric virus, FNX-HCV, and a recombinant FNX-Rluc virus carrying a Renilla luciferase reporter gene to study the virus replication. A human hepatoma cell line (Huh-7 based) was used for transfection of in vitro transcribed HCV genomic RNAs. Cell-free culture supernatants, protein lysates and total RNA were harvested at various time points post-transfection to assess HCV growth. HCV genome replication status was evaluated by quantitative RT-PCR and visualizing the presence of HCV double-stranded RNA. The HCV protein expression was verified by Western blot and immunofluorescence assays using antibodies specific for HCV NS3 and NS5A proteins. HCV RNA transfected cells released infectious particles into culture supernatant and the viral titer was measured. Luciferase assays were utilized to assess the replication level and infectivity of reporter HCV. In conclusion, we present various virological assays for characterizing different stages of the HCV replication cycle.

Hepatitis C Virus (HCV) is a major human pathogen that causes liver disorders, including cirrhosis and cancer. An HCV infectious cell culture system is essential for understanding the molecular mechanism of HCV replication and developing new therapeutic approaches. Here we describe a protocol to investigate various stages of the HCV replication cycle.

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and ...

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense against gram-negative bacterium Salmonella typhimurium. Salmonella establishes persistent infection in the intestine of C. elegans and results in early death of infected animals. A number of immunity mechanisms have been identified in C. elegans to defend against Salmonella infections. Autophagy, an evolutionarily conserved lysosomal degradation pathway, has been shown to limit the Salmonella replication in C. elegans and in mammals. Here, a protocol is described to infect C. elegans with Salmonella typhimurium, in which the worms are exposed to Salmonella for a limited time, similar to Salmonella infection in humans. Salmonella infection significantly shortens the lifespan of C. elegans. Using the essential autophagy gene bec-1 as an example, we combined this infection method with C. elegans RNAi feeding approach and showed this protocol can be used to examine the function of C. elegans host genes in defense against Salmonella infection. Since C. elegans whole genome RNAi libraries are available, this protocol makes it possible to comprehensively screen for C. elegans genes that protect against Salmonella and other intestinal pathogens using genome-wide RNAi libraries.

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

C. elegans has emerged as a new genetic model to study host-pathogen interactions. Here we describe a protocol to infect C. elegans with Salmonella typhimurium coupled with the double-strand RNAi interference technique to examine the role of host genes in defense against Salmonella infection.

In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense ...

Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their intercommunication, are paramount for the protection against infections. The interactions of epithelial and innate immune cells with a pathogen are best investigated in vivo, where complex behavior unfolds over time and space. However, existing models do not allow for easy spatio-temporal imaging of the battle with pathogens at the mucosal level.
The model developed here creates a mucosal infection by direct injection of the fungal pathogen, Candida albicans, into the swimbladder of juvenile zebrafish. The resulting infection enables high-resolution imaging of epithelial and innate immune cell behavior throughout the development of mucosal disease. The versatility of this method allows for interrogation of the host to probe the detailed sequence of immune events leading to phagocyte recruitment and to examine the roles of particular cell types and molecular pathways in protection. In addition, the behavior of the pathogen as a function of immune attack can be imaged simultaneously by using fluorescent protein-expressing C. albicans. Increased spatial resolution of the host-pathogen interaction is also possible using the described rapid swimbladder dissection technique.
The mucosal infection model described here is straightforward and highly reproducible, making it a valuable tool for the study of mucosal candidiasis. This system may also be broadly translatable to other mucosal pathogens such as mycobacterial, bacterial or viral microbes that normally infect through epithelial surfaces.

In vivo spatio-temporal interactions of pathogen and immune defenses at the mucosal level are not easily imaged in existing vertebrate hosts. The method presented here describes a versatile platform to study mucosal candidiasis in live vertebrates using the swimbladder of the juvenile zebrafish as an infection site.

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

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 ...

Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos

The molecular mechanisms underlying the phagocytosis of apoptotic cells need to be elucidated in more detail because of its role in immune and inflammatory intractable diseases. We herein developed an experimental method to investigate phagocytosis quantitatively using the fruit fly Drosophila, in which the gene network controlling engulfment reactions is evolutionally conserved from mammals. In order to accurately detect and count engulfing and un-engulfing phagocytes using whole animals, Drosophila embryos were homogenized to obtain dispersed cells including phagocytes and apoptotic cells. The use of dispersed embryonic cells enables us to measure in vivo phagocytosis levels as if we performed an in vitro phagocytosis assay in which it is possible to observe all phagocytes and apoptotic cells in whole embryos and precisely quantify the level of phagocytosis. We confirmed that this method reproduces those of previous studies that identified the genes required for the phagocytosis of apoptotic cells. This method allows the engulfment of dead cells to be analyzed, and when combined with the powerful genetics of Drosophila, will reveal the complex phagocytic reactions comprised of the migration, recognition, engulfment, and degradation of apoptotic cells by phagocytes.

Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos

We herein describe a phagocytosis assay using the dispersed embryonic cells of Drosophila. It enables us to easily and precisely quantify in vivo phagocytosis levels, and to identify new molecules required for the phagocytosis of apoptotic cells.

The molecular mechanisms underlying the phagocytosis of apoptotic cells need to be elucidated in more detail because of its role in immune and ...

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the goal of this protocol is to develop a method to visualize the pathogen invasion in a specific immune compartment of flies, namely hemocytes. Using the method presented here, up to 3 &#215; 106 live hemocytes can be obtained from 200 Drosophila 3rd instar larvae in 30 min for ex vivo infection. Alternatively, hemocytes can be infected in vivo through injection of 3rd instar larvae followed by hemocyte extraction up to 24 h post-infection. These infected primary cells were fixed, stained, and imaged using confocal microscopy. Then, 3D representations were generated from the images to definitively show pathogen invasion. Additionally, high-quality RNA for qRT-PCR can be obtained for the detection of pathogen mRNA following infection, and sufficient protein can be extracted from these cells for Western blot analysis. Taken together, we present a method for definite reconciliation of pathogen invasion and confirmation of infection using bacterial and viral pathogen types and an efficient method for hemocyte extraction to obtain enough live hemocytes from Drosophila larvae for ex vivo and in vivo infection experiments.

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

This method demonstrates how to visualize pathogen invasion into insect cells with three-dimensional (3D) models. Hemocytes from Drosophila larvae were infected with viral or bacterial pathogens, either ex vivo or in vivo. Infected hemocytes were then fixed and stained for imaging with a confocal microscope and subsequent 3D cellular reconstruction.

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the ...

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...

Induction of Mouse Lung Injury by Endotracheal Injection of Bleomycin

Pulmonary fibrosis is a hallmark of several human lung diseases with a different etiology. Since current therapies are rather limited, mouse models continue to be an essential tool for developing new antifibrotic strategies. Here we provide an effective method to investigate in vivo antifibrotic activity of human mesenchymal stromal cells obtained from whole umbilical cord (hUC-MSC) in attenuating bleomycin-induced lung injury. C57BL/6 mice receive a single endotracheal injection of bleomycin (1.5 U/kg body weight) followed by a double infusion of hUC-MSC (2.5 x 105) into the tail vein, 24 h and 7 days after the bleomycin administration. Upon sacrifice at days 8, 14, or 21, inflammatory and fibrotic changes, collagen content, and hUC-MSC presence in explanted lung tissue are analyzed. The injection of bleomycin into the mouse&#160;trachea allows the direct targeting of the lungs, leading to extensive pulmonary inflammation and fibrosis. The systemic administration of a double dose of hUC-MSC results in the early blunting of the bleomycin-induced lung injury. Intravenously infused hUC-MSC are transiently engrafted into the mouse lungs, where they exert their anti-inflammatory and antifibrotic activity. In conclusion, this protocol has been successfully applied for the preclinical testing of hUC-MSC in an experimental mouse model of human pulmonary fibrosis. However, this technique can be easily extended both to study the effect of different endotracheally administered substances on the pathophysiology of the lungs and to validate new anti-inflammatory and antifibrotic systemic therapies.

Here we present an effective method to investigate the antifibrotic activity of intravenously infused human mesenchymal stromal cells obtained from the whole umbilical cord following the induction of lung injury by an endotracheal injection of bleomycin in C57BL/6 mice. This protocol can be easily extended to the preclinical testing of other therapeutics.

Pulmonary fibrosis is a hallmark of several human lung diseases with a different etiology. Since current therapies are rather limited, mouse models ...