The authors wish to thank past members of the Goodrich-Blair lab: Samantha Orchard, Kimberly Cowles, Erin Herbert-Tran, Greg Richards, Megan Menard, and Youngjin Park for their contributions to the development of this protocol. This work was funded by the National Science Foundation grant IOS-0950873 and the National Institutes of Health NRSA fellowship FAI084441Z.

1. Insect Egg Sterilization and Rearing
<ol>
	<li>Prepare diet by first autoclaving 15 g of the provided agar in 900-1,000 ml H2O. Immediately after autoclaving, mix with 166 g wheat germ diet and blend well in a laboratory blender. Pour into a dish (or dishes) to cool, then transfer diet to aluminum foil, wrap tightly, and store at 4 &deg;C.</li>
	<li>Upon arrival, sterilize M. sexta eggs with 250 ml of 0.6% bleach solution for 2-3 min in a glass filter holder and vacuum flask apparatus with a 90 mm filter paper, stirring occasionally.</li>
	<li>Turn on vacuum to drain bleach solution and wash eggs 3-4 times with 250 ml sterile distilled H2O per wash.</li>
	<li>Transfer eggs to fresh 90 mm filter paper placed in a Petri plate lid and allow them to dry until they no longer stick together (about 20-30 min).</li>
	<li>Transfer eggs to the bottom of plastic containers with insect diet, placing approximately 40 eggs in each container (Figure 1A). Eggs are separated from diet to prevent moisture-induced fungal contamination. Maintain eggs at 26 &deg;C with a 16 hr light: 8 hr dark photoperiod. Eggs will lighten to a yellow-white color prior to hatching.</li>
	<li>When hatching is complete, carefully transfer approximately 25 larvae each to new containers with diet on the bottom (Figure 1B). It is important to pick up each larva by the long black horn using forceps to avoid harming them during their fragile early developmental stages. Incubate for 2 days as above. While larval death is unusual at this stage, some dead or, more likely, developmentally stunted larvae may be observed. Such insects should be excluded from further study and sacrificed by freezing.</li>
	<li>To avoid cannibalistic behaviors, transfer larvae to individual containers with small pieces of food (Figure 1C) and incubate as above. Monitor insect development, replacing food and cleaning feces out of containers every other day until they undergo the third larval molt (usually 6-9 days after hatching). This is the 4th instar larval stage, characterized by the appearance of black hook-like crochets on each proleg and increased prominence of stripes along the insect body (Figure 1D). The larvae may vary considerably in size, but typically weigh 0.15-0.4 g upon entering the 4th instar stage. Larvae of similar size should be chosen for injection.</li>
</ol>
2. Preparation of Bacteria for Injection
<ol>
	<li>Grow the bacterial strain(s) to be tested according to standard procedures to the growth phase you wish to test.</li>
	<li>Pellet 500 &mu;l of each bacterial strain to be tested by spinning for 2 min in a microcentrifuge at 13,000 rpm at room temperature.</li>
	<li>Resuspend cells in 1 ml sterile 1x phosphate buffered saline (PBS) and pellet again as above.</li>
	<li>Resuspend cells in 0.5 ml sterile 1x PBS and measure the optical density (OD) of the suspension. Dilute all suspensions to the same OD, if necessary. Sterile growth media may also be used for cell resuspension and dilution in place of 1x PBS, if desired.</li>
</ol>
3. Injection of 4th Instar Larvae
<ol>
	<li>Prepare serial 10-fold dilutions of the first bacterial strain in 6 wells of a 96-well microtiter plate in sterile 1x PBS, using a fresh pipette tip for each dilution (Figure 2A). The final volume of cell suspension in each well should exceed the volume required for injection (10 &mu;l per insect) plus 20 &mu;l to plate the inoculum (see steps 3.2 and 3.7).</li>
	<li>Spot 10 &mu;l each for 5 dilutions (10-2 through 10-6) along the top of a Petri plate containing an appropriate growth medium. Tilt the plate so that the spots spread to the center (Figure 2B).</li>
	<li>Remove insect diet and feces and place insects in containers on ice for approximately 5 min.</li>
	<li>Sterilize the syringe needle by rinsing 3 times each in 2 tubes of 70-100% ethanol, followed by one tube of sterile H2O (Figure 2C). The volume of liquid must be sufficient to submerge the needle.</li>
	<li>Quantitate the dilute cell suspensions under a microscope using a hemocytometer. Depending on the desired inoculum, draw 10 &mu;l from the appropriate microtiter well (Figure 2D).</li>
	<li>Swab the insect surface with 95% ethanol, and inject the 10 ml cell suspension into the insect at an angle (less than 45&deg;) behind the one of the abdominal prolegs. Take care not to puncture the gut and inject the contents just underneath the epidermal layer (Figure 2E). While some loss of hemolymph (clear, green-blue liquid) is normal, particulate matter and yellow liquid emerging from the injection site are indicative of gut puncture. If this occurs, sacrifice the insect and obtain a new larva to replace it.</li>
	<li>Repeat step 3.6 for each remaining insect in the first injection group. Needle sterilization is not necessary between each insect injected with the same dilution and strain of bacteria unless contamination occurs. Alternatively, a repeating dispenser may be used for greater consistency in injection volume among samples.</li>
	<li>After injecting each insect with the first strain of bacteria, repeat step 3.2, this time spotting cell suspensions in the middle of the plate and letting them flow toward the bottom (Figure 2F). Incubate plates at appropriate temperature and count colonies to enumerate the inoculum. This second plating step is used to verify that the samples were not contaminated during the injection process, as this would result in disparate colony numbers between the first and second platings.</li>
	<li>Repeat steps 3.1 through 3.8 for each strain of bacteria in the experiment. Finally, inject 3-5 insects with sterile 1x PBS using a sterile needle as a negative control group. Incubate insects at 26 &deg;C with a 16 hr light: 8 hr dark photoperiod.</li>
	<li>Monitor insect survival over time after injection. Insect death is characterized as a lack of voluntary movement upon stimulation. Insects often exhibit appetite loss, diarrhea (watery, yellow excrement), and/or water loss ("sweating") during infection prior to death; these characteristics may be worth noting as well.</li>
</ol>

<ol>
	<li>Bintrim, S. B., Ensign, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insertional+inactivation+of+genes+encoding+the+crystalline+inclusion+proteins+of+Photorhabdus+luminescens+results+in+mutants+with+pleiotropic+phenotypes.">Insertional inactivation of genes encoding the crystalline inclusion proteins of Photorhabdus luminescens results in mutants with pleiotropic phenotypes.</a> J. Bacteriol. 180, 1261-1269 (1998).</li><li>Schesser, J. H., Kramer, K. J., Bulla, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioassay+for+homogeneous+parasporal+crystal+of+Bacillus+thuringiensis+using+the+tobacco+hornworm,+Manduca+sexta.">Bioassay for homogeneous parasporal crystal of Bacillus thuringiensis using the tobacco hornworm, Manduca sexta.</a> Appl. Environ. Microbiol. 33, 878-880 (1977).</li><li>P&#233;chy-Tarr, M., Bruck, D. J., Maurhofer, M., Fischer, E., Vogne, C., Henkels, M. D., Donahue, K. M., Grunder, J., Loper, J. E., Keel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+analysis+of+a+novel+gene+cluster+encoding+an+insect+toxin+in+plant-associated+strains+of+Pseudomonas+fluorescens.">Molecular analysis of a novel gene cluster encoding an insect toxin in plant-associated strains of Pseudomonas fluorescens.</a> Environ. Microbiol. 10, 2368-2386 (2008).</li><li>Nu&#241;ez-Valdez, M. E., Calder&#243;n, M. A., Aranda, E., Hern&#225;ndez, L., Ram&#237;rez-Gama, R. M., Lina, L., Rodr&#237;guez-Segura, Z., Guti&#233;rrez Mdel, C., Villalobos, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+putative+Mexican+strain+of+Serratia+entomophila+pathogenic+against+root-damaging+larvae+of+Scarabaeidae+(Coleoptera).">Identification of a putative Mexican strain of Serratia entomophila pathogenic against root-damaging larvae of Scarabaeidae (Coleoptera).</a> Appl. Environ. Microbiol. 74, 802-810 (2008).</li><li>Forst, S. A., Tabatabai, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+histidine+kinase,+EnvZ,+in+the+production+of+outer+membrane+proteins+in+the+symbiotic-pathogenic+bacterium+Xenorhabdus+nematophilus.">Role of the histidine kinase, EnvZ, in the production of outer membrane proteins in the symbiotic-pathogenic bacterium Xenorhabdus nematophilus.</a> Appl. Environ. Microbiol. 63, 962-968 (1997).</li><li>Kanost, M. R., Jiang, H., Yu, X. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+immune+responses+of+a+lepidopteran+insect,+Manduca+sexta.">Innate immune responses of a lepidopteran insect, Manduca sexta.</a> Immunol. Rev. 198, 97-105 (2004).</li><li>Yu, X. Q., Zhu, Y. F., Ma, C., Fabrick, J. A., Kanost, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pattern+recognition+proteins+in+Manduca+sexta+plasma.">Pattern recognition proteins in Manduca sexta plasma.</a> Insect Biochem. Mol. Biol. 32, 1287-1293 (2002).</li><li>Eleftherianos, I., ffrench-Constant, R. H., Clarke, D. J., Dowling, A. J., Reynolds, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+the+immune+response+to+the+entomopathogen+Photorhabdus.">Dissecting the immune response to the entomopathogen Photorhabdus.</a> Trends Microbiol. 18, 552-560 (2010).</li><li>Herbert, E. E., Goodrich-Blair, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Friend+and+foe:+the+two+faces+of+Xenorhabdus+nematophila.">Friend and foe: the two faces of Xenorhabdus nematophila.</a> Nat. Rev. Microbiol. 5, 634-646 (2007).</li><li>D'Argenio, D. A., Gallagher, L. A., Berg, C. A., Manoil, C. <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+host+for+Pseudomonas+aeruginosa+Infection.">Drosophila as a model host for Pseudomonas aeruginosa Infection.</a> J. Bacteriol. 183, 1466-1471 (2001).</li><li>Waterfield, N., Dowling, A., Sharma, S., Daborn, P. J., Potter, U., Ffrench-Constant, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+toxicity+of+Photorhabdus+luminescens+W14+toxin+complexes+in+Escherichia+coli.">Oral toxicity of Photorhabdus luminescens W14 toxin complexes in Escherichia coli.</a> Appl. Environ. Microbiol. 67, 5017-5024 (2001).</li><li>Park, Y., Herbert, E. E., Cowles, C. E., Cowles, K. N., Menard, M. L., Orchard, S. S., Goodrich-Blair, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+variation+in+Xenorhabdus+nematophila+virulence+and+suppression+of+Manduca+sexta+immunity.">Clonal variation in Xenorhabdus nematophila virulence and suppression of Manduca sexta immunity.</a> Cell. Microbiol. 9, 645-656 (2007).</li><li>Park, Y., Kim, Y., Putnam, S. M., Stanley, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bacterium+Xenorhabdus+nematophilus+depresses+nodulation+reactions+to+infection+by+inhibiting+eicosanoid+biosynthesis+in+tobacco+hornworms,+Manduca+sexta.">The bacterium Xenorhabdus nematophilus depresses nodulation reactions to infection by inhibiting eicosanoid biosynthesis in tobacco hornworms, Manduca sexta.</a> Arch. Insect Biochem. Physiol. 52, 71-80 (2003).</li><li>Cowles, K. N., Cowles, C. E., Richards, G. R., Martens, E. C., Goodrich-Blair, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+global+regulator+Lrp+contributes+to+mutualism,+pathogenesis+and+phenotypic+variation+in+the+bacterium+Xenorhabdus+nematophila.">The global regulator Lrp contributes to mutualism, pathogenesis and phenotypic variation in the bacterium Xenorhabdus nematophila.</a> Cell. Microbiol. 9, 1311-1323 (2007).</li><li>Cowles, K. N., Goodrich-Blair, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+activity+of+a+Xenorhabdus+nematophila+haemolysin+required+for+full+virulence+towards+Manduca+sexta+insects.">Expression and activity of a Xenorhabdus nematophila haemolysin required for full virulence towards Manduca sexta insects.</a> Cell. Microbiol. 7, 209-219 (2005).</li><li>Goodrich-Blair, H., 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=Mutualism+and+pathogenesis+in+Xenorhabdus+and+Photorhabdus:+two+roads+to+the+same+destination.">Mutualism and pathogenesis in Xenorhabdus and Photorhabdus: two roads to the same destination.</a> Mol. Microbiol. 64, 260-268 (2007).</li><li>Eleftherianos, I., Baldwin, H., ffrench-Constant, R. H., Reynolds, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+modulation+of+immunity:+changes+within+the+feeding+period+of+the+fifth+larval+stage+in+the+defence+reactions+of+Manduca+sexta+to+infection+by+Photorhabdus.">Developmental modulation of immunity: changes within the feeding period of the fifth larval stage in the defence reactions of Manduca sexta to infection by Photorhabdus.</a> J. Insect Physiol. 54, 309-318 (2008).</li><li>Kavanagh, K., Reeves, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploiting+the+potential+of+insects+for+in+vivo+pathogenicity+testing+of+microbial+pathogens.">Exploiting the potential of insects for in vivo pathogenicity testing of microbial pathogens.</a> FEMS Microbiol. Rev. 28, 101-112 (2004).</li></ol>

No conflicts of interest declared.

The direct injection of M. sexta larvae with entomopathogenic bacteria, as described here, serves as a simple and effective means to analyze bacterial virulence. The method is also highly adaptable to suit different experimental subjects and/or conditions. Bacteria can be prepared in various ways prior to injection. In the case of X. nematophila, wild type cells grown in nutrient-rich Luria-Bertani (LB) medium to mid-log phase are typically the most virulent, killing most or all insects within 30 hr sub...

<table border="1">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>90 mm filter paper</td>
			<td>Whatman</td>
			<td>1001 090</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glass filter holder</td>
			<td>Millipore</td>
			<td>XX1004700</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Manduca sexta eggs</td>
			<td>Carolina Biological Supply</td>
			<td>143880</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Gypsy Moth Diet + agar</td>
			<td>MP Biomedicals</td>
			<td>0296029301</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>5.5 oz. plastic containers and lids</td>
			<td>Solo Cup Company</td>
			<td>URC55-0090 Pl4-0090</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1 oz. plastic containers and lids</td>
			<td>DART Container Corporation</td>
			<td>100PC 100PCL25</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1x PBS</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>137 mm NaCl, 2.7 mM KCl, 8 mM Na2HPO4, 1.46 mM KH2PO4, pH 7.4</td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>Hamilton</td>
			<td>80208</td>
			<td>30 gauge, 0.375" length, point style 2</td>
		</tr>
	</tbody>
</table>

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.

A representative example of an insect mortality assay is depicted in Figure 3. In this experiment, insects were injected with approximately 50 colony forming units (CFU) of either wild type (ATCC19061) or an attenuated mutant strain (lrp13) of Xenorhabdus nematophila grown to mid-log phase (n=6 insects per strain). Insects were observed for approximately 72 hr, and the percent of injected insects still alive at each timepoint recorded. In this case, the attenuated strain exhi...

Watch this Scientific Journal Video about Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence at JoVE.com

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.

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 ...

rearing-injection-manduca-sexta-larvae-to-assess-bacterial

Research

JoVE Journal

Immunology and Infection

12.6K Views.  University of Wisconsin-Madison. 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.