J. Chua, N.A. Fisher, D. DeShazer and A.M. Friedlander designed the procedures described in the manuscript. J. Chua, N.A. Fisher, S.D. Falcinelli and D. DeShazer performed the experiments. J. Chua wrote the manuscript.
The authors thank Joshua J. W. Roan, Nora D. Doyle, Nicholas R. Carter and Steven A. Tobery for excellent technical assistance and David P. Fetterer and Steven J. Kern for statistical analysis.
The work was supported by the Defense Threat Reduction Agency Proposal #CBCALL12-THRB1-1-0270 to A.M.F and #CBS.MEDBIO.02.10.RD.034 to D.D.
Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the U.S. Army.
The content of this publication does not necessarily reflect the views or policies of the Department of Defense, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.

1. Preparations for Maintaining a Hissing Cockroach Colony
<ol>
	<li>Prepare cages for the hissing cockroaches to live in. Apply a thin layer of petroleum jelly, approximately 20 to 30 mm in width, to the circumference of the inner walls near the top of the cage to prevent the hissing cockroaches from climbing out of the cage and escaping. 
	NOTE: Hissing cockroaches can be housed in a variety of containers that have a large floor space, are of sufficient height, and have lids. Use mouse cages (~43 cm x 23 cm x 20 cm). For cages destined for 37 &#176;C, do not apply petroleum jelly.</li>
	<li>Include a cardboard egg carton placed upside down inside the cage to provide a hiding place for the naturally shy insects. Do not substitute the cardboard egg carton with one made of polystyrene foam to prevent ingestion of plastic.</li>
	<li>Do not provide bedding for easier clean-up and to increase the visibility of newly hatched nymphs.</li>
	<li>Obtain dry dog food which contains a composition of ~20% crude protein. Coarsely grind the dog food with a food processor or blender and store at 4 &#176;C.</li>
	<li>Obtain several shallow dishes and aquarium stones or a sponge for food and water. 
	NOTE: Petri dishes are recommeded.</li>
</ol>
2. Hissing Cockroach Care and Breeding
<ol>
	<li>Obtain outbred Madagascar hissing cockroaches (~5 cm) from a commercial breeder. The species used in this protocol is Gromphadorhina laevigata. Unpack the shipping box containing the hissing cockroaches immediately upon receipt.</li>
	<li>Follow appropriate institutional guidelines for the use of personal protective equipment in handling animals. 
	NOTE: Use thick disposable gloves for handling cockroaches as the tarsal claws of a hissing cockroach are sharp.</li>
	<li>Transfer up to 75 large hissing cockroaches (&#62;3 cm) per large cage. Transfer the newly hatched and smaller nymphs (&#60;3 cm) to a separate cage. 
	NOTE: Keeping significantly more than 75 large hissing cockroaches per mouse cage could lead to deteriorating health of the colony and could also result in deaths.</li>
	<li>Handle a newly molted cockroach, which is off-white in color, gently and avoid squeezing it. Alternatively, allow the exoskeleton to darken and harden before handling. 
	NOTE: It is not necessary to remove the mites, Gromphadorholaelaps schaeferi, that often accompany hissing cockroaches upon receipt from breeders. The beneficial mites keep the hissing cockroaches clean and are harmless to humans.</li>
	<li>Feed hissing cockroaches with ground dry dog food in a shallow dish once or twice a week. Provide enough dog food to ensure that the hissing cockroaches have sufficient food until the next feeding. Discard any food that has become wet and moldy.</li>
	<li>In addition to dog food, provide cut-up fruits and vegetables, such as apples, potatoes, or lettuce, in a shallow dish. Discard moldy or rotten food.</li>
	<li>Provide drinking water once or twice a week in a shallow dish but do not overfill the dish to prevent spillage in the cage. Place either small aquarium stones or a sponge in the dish to provide a landing pad for small nymphs. Use reverse osmosis water, if available.</li>
	<li>Keep hissing cockroaches in the dark at temperatures ranging from 21 &#176;C to 30 &#176;C. Keep large hissing cockroaches destined for experimentation at a lower temperature (~21 &#176;C) for ~2 months to decrease breeding and pregnancy. In contrast, keep nymphs at higher temperatures (28 &#176;C to 30 &#176;C) to hasten growth. 
	NOTE: Keep cages at room temperature (21 &#176;C) in a dark cabinet.</li>
	<li>Provide humidity by including a separate pan of water if hissing cockroaches are kept in an incubator.</li>
	<li>Clean the cage by scooping out dry feces regularly or by transferring hissing cockroaches to a clean cage every 2 to 3 weeks. Keep the bottom of the cage dry. Clean the cage immediately if excess water is allowed to accumulate and mix with the excrement at the bottom of the cage.</li>
</ol>
3. Cockroach Preparation for Experimentation
<ol>
	<li>Transfer the appropriate number of hissing cockroaches in a cage to a 37 &#176;C humidified incubator 1 to 3 weeks prior to an experiment for acclimation. Include a control group for injection. This acclimation period is critical for avoiding temperature shock during experimentation.</li>
	<li>Do not apply petroleum jelly to cages destined for 37 &#176;C because the higher temperature melts the jelly.</li>
	<li>Check the hissing cockroaches every 1 to 2 days to replace food and water and clean the cage.</li>
	<li>Obtain clear disposable plastic food containers with lids for grouping the hissing cockroaches during experimentation. For ventilation, punch holes in the lid or the side of the container with a nail and hammer. 
	NOTE: Use screw cap containers over snap cap containers in the biosafety level 3 setting. Snap cap containers can be reinforced with tape, if necessary.</li>
	<li>On the day of injection, distribute 6 to 12 acclimatized hissing cockroaches into groups per container, ensuring equal distribution by sex and body mass.</li>
	<li>Determine the sex of individual hissing cockroaches. Determine the sex by the prominence of horns found on males and the lack thereof on females.</li>
	<li>Weigh the individual hissing cockroaches. To facilitate the weighing of a hissing cockroach on the balance, enclose it within two weigh boats that have been tared. 
	NOTE: For consistency between experiments, use hissing cockroaches with a weight of 4 to 8 g.&#160;However, no difference in survival after infection have been found between small (1.5 to 2 g) and large (6 to 8 g) hissing cockroaches. For drug studies, use hissing cockroaches with a weight of ~5 g to obtain a more consistent drug concentration per body mass.</li>
	<li>In lieu of a water dish, include high water content fruit or vegetables, such as a slice of apple or potato. Discard rotten food. Do not provide additional water to keep the container dry.</li>
	<li>Return hissing cockroaches to 37 &#176;C until injections.</li>
</ol>
4. Bacterial Culture and Preparations
NOTE: The bacterial species used in this protocol are B. mallei, B. pseudomallei, and B. thailandensis. All manipulations with B. mallei and B. pseudomallei must be performed in Class II or Class III biological safety cabinets located in a biosafety level (BSL) 3 laboratory. Perform manipulations with B. thailandensis in similar biological safety cabinets located either in a BSL2 or BSL3 laboratory. Follow institutional standard operating procedure for BSL3 work. Follow institutional guidelines for use of personal protective equipment when handling bacteria.
<ol>
	<li>Prepare a master plate of Burkholderia at least 3 days prior to infection. Use Luria-Bertani (Lennox) (LB) agar for B. pseudomallei or B. thailandensis and use LB agar supplemented with 4% glycerol for B. mallei. Streak bacteria from a 25% glycerol stock stored at -80 &#176;C. 
	NOTE: Always streak the master plate directly from the frozen glycerol stock; avoid serial passage of the bacteria from plate to plate as this may cause reduced virulence.</li>
	<li>Inoculate 10 to 20 mL LB broth with several colonies of B. pseudomallei or B. thailandensis from the master plate. Similarly inoculate LB broth supplemented with 4% glycerol for B. mallei.</li>
	<li>Shake the broth culture at 175 to 250 rpm at 37 &#176;C for ~18 h.</li>
	<li>Centrifuge 2 to 3 mL of culture at 5,000 x g for 10 min.</li>
	<li>Discard the supernatant and resuspend the bacterial pellet in sterile phosphate buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2PO4, 1.8 mM KH2PO4, pH 7.4).</li>
	<li>Dilute the bacteria with PBS to achieve an optical density (OD) of 0.5, measured at an absorbance at 600 nm in a spectrophotometer.</li>
	<li>Serially dilute the bacteria ten-fold in PBS from the starting OD of 0.5. Use these suspensions for infection.</li>
	<li>Determine the colony forming units (CFU) of the inoculum by serial dilution and plating of 100 &#181;L aliquots on agar media. Incubate plates at 37 &#176;C for 24 to 48 h.</li>
</ol>
5. Drug Preparations
<ol>
	<li>Determine the appropriate amount of drug to be given per body mass. The CLQ dosage given to the hissing cockroach was based on the standard dosage for mammals, which is 50 mg/kg/day.</li>
	<li>Resuspend or dilute the drug of interest in vehicle. For example, dilute CLQ in PBS at a concentration of 12 mg/mL. This concentration provides 300 &#181;g of drug to a ~6 g hissing cockroach in a 25 &#181;L injected volume.</li>
	<li>If necessary, sterilize the drug solution by passing it through a 0.22 &#181;m syringe filter.</li>
	<li>Store the drug solution at 4 &#176;C until use. Warm the drug solution to at least 21 &#176;C (room temperature) prior to injections.</li>
</ol>
6. Assembly of the Injector
<ol>
	<li>Set the pointer to the desired volume (25 &#181;L) by rotating the adjustment screw on a repetitive pipette.</li>
	<li>Push the release bar of the repetitive pipette inward and pull the pusher outward. The repetitive pipette is now ready to accommodate a loaded syringe. 
	NOTE: Calibrate the volume ejected by the repetitive pipette by measuring the amount of water ejected on a balance.</li>
	<li>Fill a 1 mL syringe with suspension containing either bacteria or drug.</li>
	<li>Attach syringe to a sterile 26 or 27 G x &#189; inch (or shorter) needle.</li>
	<li>Tap the syringe to float the air bubbles to the top and expel the bubbles and some suspension into a container filled with 10% bleach.</li>
	<li>Snap the syringe onto the syringe clip of the repetitive pipette with the needle bevel facing up.</li>
	<li>Pull the release bar outward and press the dispenser button firmly to perform blank injections into a container filled with 10% bleach until the syringe plunger is against the pusher.</li>
	<li>Perform 1 or 2 additional blank injections to ensure liquid is being ejected out of the syringe. The repetitive pipette is ready for injections.</li>
</ol>
7. Cockroach Injections
<ol>
	<li>Perform all cockroach injections in a Class II or Class III biological safety cabinet in a BSL2 or BSL3 setting using institutional recommended personal protective equipment.</li>
	<li>Clean work surfaces in the safety cabinet that may come in contact with the hissing cockroaches. Use 10% bleach followed by 70% ethanol to remove residual bleach. Allow the surface to air dry.</li>
	<li>With one hand, grip the hissing cockroach by its side. Immobilize a hissing cockroach such that it is unable to recoil during injection. Bend the hissing cockroach slightly so that the cutaneous membranes between the abdominal terga are exposed.</li>
	<li>With the other hand, hold the repetitive pipette such that the needle is at a 0&#176; to 30&#176; angle from the dorso-ventral midline of the hissing cockroach. Puncture the cutaneous membrane adjacent to the 3rd, 4th, or 5th tergum from the posterior end. 
	NOTE: Entry of the needle into the hissing cockroach at the angle indicated ensures that the needle does not go through and exit out of the hissing cockroach. The injection site ensures that the ejected material is contained within the abdominal cavity filled with hemolymph. Practice holding the hissing cockroaches and injecting with water before undertaking injections with live bacteria or drug.</li>
	<li>Push the dispense button firmly to inject the volume. Gently withdraw the needle at the same angle as used for entry.</li>
	<li>Place the injected hissing cockroach in a separate container to distinguish it from the hissing cockroaches that have not yet been injected. Wipe off any hemolymph that may have oozed out from the injection site.</li>
	<li>Continue injecting other hissing cockroaches within a group using the same syringe and needle. Stop injecting before the syringe plunger reaches the bottom of the barrel to prevent incomplete dosing in the last injection.</li>
	<li>For multiple injections in a single hissing cockroach, such as those with bacteria and drug, inject at different sides of a tergum on the dorsal side of the hissing cockroaches. Alternatively, perform multiple injections by injecting at different terga (3rd to 5th).</li>
	<li>Ensure that the lids of the containers are securely fastened. Reinforce the lids with tape, if necessary.</li>
	<li>Incubate hissing cockroaches in a humidified 37 &#176;C incubator.</li>
</ol>
8. Recording Hissing Cockroach Morbidity and Mortality
<ol>
	<li>Perform all hissing cockroach examinations in a Class II or Class III biological safety cabinet with institutional recommended personal protective equipment. Clean the surfaces in the safety cabinet with 10% bleach followed by 70% ethanol. Allow the work area to air dry.</li>
	<li>Score hissing cockroaches once or twice daily over a period of 1 to 2 weeks using the morbidity scoring table (Table 1).</li>
	<li>Remove dead hissing cockroaches from the container and record the number of survivors.</li>
	<li>Transfer remaining live hissing cockroaches to a clean container if excess moisture has accumulated at the bottom of the container. Alternatively, use disposable paper towels to wipe off excess moisture.</li>
	<li>Replace rotten food daily and return hissing cockroaches to the humidified 37 &#176;C incubator.</li>
	<li>At the end of study, place remaining survivors in a biohazard bag and freeze at -80 &#176;C to euthanize.</li>
	<li>Statistically analyze the data14 to determine LD50 and by Kaplan-Meier and Log-Rank analysis for survival. As with all insect experimentation, some deaths may occur in the control group. These deaths are attributed to the natural mortality of insects 15. For a more detailed discussion on how to account for deaths in the control group, see reference15.</li>
</ol>

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The authors have nothing to disclose.

Optimal experimental conditions begin with a healthy hissing cockroach colony, which requires a minimal but consistent time commitment. Although hissing cockroaches can go for a relatively long period of time (~weeks) without food and water, weekly or bi-weekly cage maintenance must be provided. This includes checking the food and water supply and ensuring that the cage is dry. Maintaining dry living conditions is especially important during acclimation and incubation at higher temperatures; we find that more hissing coc...

The use of insects as alternative non-mammalian animal models to study bacterial pathogenesis and innate host defense has been gaining momentum in recent years. Logistically, this is due to their relatively inexpensive cost and the ease in obtaining, handling, and caring for insects compared to mammals. There is also no regulatory policy governing the use of insects in research; it is not subject to the purview or restrictions set forth by any animal use committee or government agency. Insects as surrogate animal models are particularly amenable to comprehensive screening studies for virulence factors, host-pathogen interactions, and assessments of anti-microbial drug efficacy. Their use can reduce the number of mammals used for research thereby overcoming some of the ethical dilemmas inherent to the conduct of animal experimentation 1,2.
Insects may serve as surrogate hosts because there is a high degree of commonality between the innate immune systems of insects and mammals 1,3. Both insect plasmatocytes and mammalian macrophages phagocytose microorganisms 4. The insect counterpart to the neutrophil is the hemocyte 5,6. Intracellular oxidative burst pathways in insect and mammalian cells are similar; reactive oxygen species in both are produced by orthologous p47phox and p67phox proteins 5. The signaling cascades downstream of Toll receptors in insects and Toll-like receptors and Interleukin-1 in mammals are also remarkably similar; both result in production of antimicrobial peptides, such as defensins 7. Thus, insects can be utilized to study general innate immune mechanisms that are shared by metazoans.
An insect called the Madagascar hissing cockroach from the genus Gromphadorhina, is one of the largest cockroach species that exists, typically reaching 5 to 8 cm at maturity. It is native only to the island of Madagascar and is characterized by the hissing sound it makes - a sound that is produced when the hissing cockroach expels air through respiratory openings called spiracles 8. The distinctive hiss serves as a form of social communication among hissing cockroaches for courtship and aggression 9 and can be heard when a male is disturbed in its habitat. The Madagascar hissing cockroach is slow moving compared to the American cockroach and other urban pest species. It is easy to care for and breed; a pregnant hissing cockroach can produce 20 to 30 offspring at a time. A baby hissing cockroach, called a nymph, reaches sexual maturity in 5 months after undergoing 6 molts and can live up to 5 years both in the wild and in captivity 8.
We have utilized the Madagascar hissing cockroach as a surrogate host for infection with the intracellular pathogens Burkholderia mallei, B. pseudomallei, and B. thailandensis 10,11. The virulence of these pathogens in hissing cockroaches was compared to their virulence in the benchmark animal model for Burkholderia, the Syrian hamster. We found that the 50% lethal dose (LD50) of B. pseudomallei and B. mallei was similar in both models 11. Interestingly, B. thailandensis, although avirulent in the rodent model, is lethal in the hissing cockroach 11. This difference with respect to B. thailandensis infection underscores the utility of the hissing cockroach model; B. thailandensis attenuating mutants can be more readily resolved in the hissing cockroach than in rodent models. Furthermore, as B. thailandensis is often used as the model organism for B. pseudomallei and B. mallei 10,12,13, identifying attenuating mutations in it could lead to similar targets in its more virulent relatives.
Despite the difference in virulence of B. thailandensis in the hissing cockroach versus the Syrian hamster, mutations in critical virulence factors, such as those in the type 6 secretion system-1 (T6SS-1), which are attenuating in B. mallei and B. pseudomallei, are similarly attenuating for B. thailandensis 11. The hissing cockroach model is further validated in that individual T6SS mutants (T6SS-2 to T6SS-6) in B. pseudomallei, which have no bearing on virulence in Syrian hamsters, remain virulent in the hissing cockroaches 11. Thus, the hissing cockroach is a viable surrogate animal model for the three Burkholderia species. We recently utilized the hissing cockroach as a surrogate animal model to examine the efficacy of the anti-malarial drug chloroquine (CLQ) against Burkholderia infection 10 and its toxicity.
Here, we describe the rearing and care of the Madagascar hissing cockroach and provide details on how to infect this insect with three Burkholderia species. Furthermore, we illustrate that the hissing cockroach is a viable surrogate model to study virulence and drug efficacy in Burkholderia infections and that it likely can also serve as a surrogate host for other bacterial pathogens in similar studies.

<table><tbody><tr><td>Madagascar hissing cockroach&lt;br/&gt; &amp;nbsp;&amp;nbsp;&lt;br/&gt; &amp;nbsp;&lt;br/&gt; &amp;nbsp;&lt;br/&gt; &amp;nbsp;</td><td>Carolina Biological Supply Co, Burlington, NC&amp;nbsp;</td><td>143668</td><td></td></tr><tr><td>Kibbles n Bits, any flavor</td><td>Big Heart Pet Brands, San Francisco, CA</td><td>UPC #079100519378</td><td></td></tr><tr><td>Snap on disposable plastic containers or equivalent</td><td>Rubbermaid, Huntersville, NC</td><td>UPC #FG7F71RETCHIL</td><td></td></tr><tr><td>Screw on disposable plastic containers or equivalent</td><td>Rubbermaid, Huntersville, NC</td><td>UPC #FG7J0000TCHIL</td><td></td></tr><tr><td>Tridak STEPPER series repetitive pipette</td><td>Dymax Corporation&lt;br/&gt; www.dymax.com</td><td>T15469</td><td></td></tr><tr><td>Syringe (1 mL)&amp;nbsp;</td><td>Becton Dickinson, Franklin Lakes, NJ</td><td>309659</td><td></td></tr><tr><td>Needle (26 or 27G x 1/2)</td><td>Becton Dickinson, Franklin Lakes, NJ</td><td>305109, 305111</td><td></td></tr><tr><td>Chloroquine diphosphate</td><td>Sigma-Aldrich, St. Louis, MO</td><td>C6628</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Gibco/ Thermo Fisher Scientific, Gaithersburg, MD</td><td>10010023</td><td></td></tr><tr><td>Difco Luria- Bertani (Lennox)</td><td>Becton Dickinson, Sparks, MD</td><td>240230</td><td></td></tr><tr><td>Agar&amp;nbsp;</td><td>Sigma-Aldrich, St. Louis, MO</td><td>A1296</td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich, St. Louis, MO</td><td>G6279</td><td></td></tr></tbody></table>

the madagascar hissing cockroach as an alternative non-mammalian animal model to investigate virulence, pathogenesis, and drug efficacy

Many aspects of innate immunity are conserved between mammals and insects. An insect, the Madagascar hissing cockroach from the genus Gromphadorhina, can be utilized as an alternative animal model for the study of virulence, host-pathogen interaction, innate immune response, and drug efficacy. Details for the rearing, care and breeding of the hissing cockroach are provided. We also illustrate how it can be infected with bacteria such as the intracellular pathogens Burkholderia mallei, B. pseudomallei, and B. thailandensis. Use of the hissing cockroach is inexpensive and overcomes regulatory issues dealing with the use of mammals in research. In addition, results found using the hissing cockroach model are reproducible and similar to those obtained using mammalian models. Thus, the Madagascar hissing cockroach represents an attractive surrogate host that should be explored when conducting animal studies.

This section illustrates the results that were obtained when Madagascar hissing cockroaches were infected with B. mallei, B. pseudomallei, or B. thailandensis; the results show that this insect is a tractable animal model for different species of Burkholderia in studying virulence, drug toxicity, and drug efficacy against bacterial infection. More hissing cockroaches survived in groups that were infected with the attenuated mutants (&#916;hcp1) than in ...

Watch this Scientific Journal Video about The Madagascar Hissing Cockroach as an Alternative Non-mammalian Animal Model to Investigate Virulence, Pathogenesis, and Drug Efficacy at JoVE.com

The Madagascar Hissing Cockroach as an Alternative Non-mammalian Animal Model to Investigate Virulence, Pathogenesis, and Drug Efficacy

We present a protocol to utilize the Madagascar hissing cockroach as an alternative non-mammalian animal model to conduct bacterial virulence, pathogenesis, drug toxicity, drug efficacy, and innate immune response studies.

Many aspects of innate immunity are conserved between mammals and insects. An insect, the Madagascar hissing cockroach from the genus Gromphadorhina, can ...

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Research

JoVE Journal

Immunology and Infection

10.9K Views.  United States Army Medical Research Institute of Infectious Diseases. Presenteren we een protocol voor het gebruik van de Madagaskar Sissende kakkerlak als een alternatieve bij niet-zoogdieren diermodel uit te voeren van bacteriële virulentie, pathogenese, drug toxiciteit, werkzaamheid van de drug en aangeboren immuunrespons studies.

Video: The Madagascar Hissing Cockroach as an Alternative Non-mammalian Animal Model to Investigate Virulence, Pathogenesis, and Drug Efficacy

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative without tools to study gene function. The development and sharing of detailed protocols for the establishment of new model systems in laboratories, and for tools to carry out functional studies, is thus crucial for leveraging the power of genomics. Coleoptera (beetles) are the largest clade of insects and occupy virtually all types of habitats on the planet. In addition to providing ideal models for fundamental research, studies of beetles can have impacts on pest control as they are often pests of households, agriculture, and food industries. Detailed protocols for rearing and maintenance of D. maculatus laboratory colonies and for carrying out dsRNA-mediated interference in D. maculatus are presented. Both embryonic and parental RNAi procedures-including apparatus set up, preparation, injection, and post-injection recovery-are described. Methods are also presented for analyzing embryonic phenotypes, including viability, patterning defects in hatched larvae, and cuticle preparations for unhatched larvae. These assays, together with in situ hybridization and immunostaining for molecular markers, make D. maculatus an accessible model system for basic and applied research. They further provide useful information for establishing procedures in other emerging insect model systems.

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Here, we present protocols for rearing an intermediate-germ beetle, Dermestes maculatus (D. maculatus) in the lab. We also share protocols for embryonic and parental RNAi and methods for analyzing embryonic phenotypes to study gene function in this species.

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

Genetics

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the intestine the primary location for disease. The nematode intestine structurally and functionally mimics mammalian intestines and is transparent making it amenable to microscopic study of colonization. Here we show that pathogens can cause disease and death. We are able to identify microbial mutants that show altered virulence. Its conserved innate response to biotic stresses makes C. elegans an excellent system to probe facets of host innate immune interactions. We show that hosts with mutations in the dual oxidase gene cannot produce reactive oxygen species and are unable to resist microbial insult. We further demonstrate the versatility of the presented survival assay by showing that it can be used to study the effects of inhibitors of microbial growth. This assay may also be used to discover fungal virulence factors as targets for the development of novel antifungal agents, as well as provide an opportunity to further uncover host-microbe interactions. The design of this assay lends itself well to high throughput whole-genome screens, while the ability to cryo-preserve worms for future use makes it a cost-effective and attractive whole animal model to study.

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

Here, we present the nematode Caenorhabditis elegans as a versatile host model to study microbial interaction.

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the ...

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

The study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of insects as infection models provides a valuable alternative. Compared to other non-vertebrate model hosts such as nematodes, insects have a relatively advanced system of antimicrobial defenses and are thus more likely to produce information relevant to the mammalian infection process. Like mammals, insects possess a complex innate immune system1. Cells in the hemolymph are capable of phagocytosing or encapsulating microbial invaders, and humoral responses include the inducible production of lysozyme and small antibacterial peptides2,3. In addition, analogies are found between the epithelial cells of insect larval midguts and intestinal cells of mammalian digestive systems. Finally, several basic components essential for the bacterial infection process such as cell adhesion, resistance to antimicrobial peptides, tissue degradation and adaptation to oxidative stress are likely to be important in both insects and mammals1. Thus, insects are polyvalent tools for the identification and characterization of microbial virulence factors involved in mammalian infections.
Larvae of the greater wax moth Galleria mellonella have been shown to provide a useful insight into the pathogenesis of a wide range of microbial infections including mammalian fungal (Fusarium oxysporum, Aspergillus fumigatus, Candida albicans) and bacterial pathogens, such as Staphylococcus aureus, Proteus vulgaris, Serratia marcescens Pseudomonas aeruginosa, Listeria monocytogenes or Enterococcus faecalis4-7. Regardless of the bacterial species, results obtained with Galleria larvae infected by direct injection through the cuticle consistently correlate with those of similar mammalian studies: bacterial strains that are attenuated in mammalian models demonstrate lower virulence in Galleria, and strains causing severe human infections are also highly virulent in the Galleria model8-11. Oral infection of Galleria is much less used and additional compounds, like specific toxins, are needed to reach mortality.
G. mellonella larvae present several technical advantages: they are relatively large (last instar larvae before pupation are about 2 cm long and weight 250 mg), thus enabling the injection of defined doses of bacteria; they can be reared at various temperatures (20 &deg;C to 30 &deg;C) and infection studies can be conducted between 15 &deg;C to above 37 &deg;C12,13, allowing experiments that mimic a mammalian environment. In addition, insect rearing is easy and relatively cheap. Infection of the larvae allows monitoring bacterial virulence by several means, including calculation of LD5014, measurement of bacterial survival15,16 and examination of the infection process17. Here, we describe the rearing of the insects, covering all life stages of G. mellonella. We provide a detailed protocol of infection by two routes of inoculation: oral and intra haemocoelic. The bacterial model used in this protocol is Bacillus cereus, a Gram positive pathogen implicated in gastrointestinal as well as in other severe local or systemic opportunistic infections18,19.

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

Oral and intra haemocolic infection of larvae of the greater wax moth Galleria mellonella is described. This insect can be used to study virulence factors of entomopathogenic as well as mammalian opportunistic bacteria. Rearing of the insects, methods of infection and examples of in vivo analysis are described.

The study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of ...

Two Infection Assays to Study Non-Lethal Virulence Phenotypes in C. Albicans using C. Elegans

While pathogens can be deadly to humans, many of them cause a range of infection types with non-lethal phenotypes. Candida albicans, an opportunistic fungal pathogen of humans, is the fourth most common cause of nosocomial infections which results in ~40% mortality. However, other C. albicans infections are less severe and rarely lethal and include vulvovaginal candidiasis, impacting ~75% of women, as well as oropharyngeal candidiasis, predominantly impacting infants, AIDS patients and cancer patients. While murine models are most frequently used to study C. albicans pathogenesis, these models predominantly assess host survival and are costly, time consuming, and limited in replication. Therefore, several mini-model systems, including Drosophila melanogaster, Danio rerio, Galleria mellonella, and Caenorhabditis elegans, have been developed to study C. albicans. These mini-models are well-suited for screening mutant libraries or diverse genetic backgrounds of C. albicans. Here we describe two approaches to study C. albicans infection using C. elegans. The first is a fecundity assay which measures host reproduction and monitors survival of individual hosts. The second is a lineage expansion assay which measures how C. albicans infection affects host population growth over multiple generations. Together, these assays provide a simple, cost-effective way to quickly assess C. albicans virulence.

Two Infection Assays to Study Non-Lethal Virulence Phenotypes in C. Albicans using C. Elegans

Fungal opportunist pathogens can cause life-threatening as well as minor infections, but non-lethal phenotypes are frequently ignored when studying virulence. Therefore, we developed a nematode model that monitors both the survival and reproduction aspects of host to investigate fungal virulence.

While pathogens can be deadly to humans, many of them cause a range of infection types with non-lethal phenotypes. Candida albicans, an opportunistic ...

Applications of RNA Interference in American Cockroach

Cockroaches, a sanitary pest, are essential species in insect developmental and metamorphic studies due to their easy feeding and hemimetabolous characteristics. Altogether with well-annotated genome sequences, these advantages have made American cockroach, Periplaneta americana, an important hemimetabolous insect model. Limited by the shortage of knockout strategy, effective RNA interference (RNAi)-based gene knockdown becomes an indispensable technique in functional gene research of P. americana. The present protocol describes the RNAi operation techniques in P. americana. The protocol includes (1) selection of the P. americana at proper developmental stages, (2) preparation for the injection setting, (3) dsRNA injection, and (4) gene knockdown efficiency detection. RNAi is a powerful reverse genetic tool in P. americana. The majority of P. americana tissues are sensitive to extracellular dsRNA. Its simplicity allows researchers to quickly obtain dysfunctional phenotypes under one or multiple targeting dsRNA injections, enabling researchers to better use the P. americana for developmental and metamorphic studies.

The present protocol describes step-by-step guidelines for the RNAi operation techniques in P. americana.

Cockroaches, a sanitary pest, are essential species in insect developmental and metamorphic studies due to their easy feeding and hemimetabolous ...

Developmental Biology

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or A. baumannii

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, preferably in models which mimic the intended clinical indication. A method for inducing robust lung infections in immunocompetent rats and mice is described which allows for the assessment of treatments in a model of serious pneumonia caused by S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae or A. baumannii. Animals are anesthetized, and an agar-based inoculum is deposited deep into the lung via nonsurgical intratracheal intubation. The resulting infection is consistent, reproducible, and stable for at least 48 h and up to 96 h for most isolates. Studies with marketed antibacterials have demonstrated good correlation between in vivo efficacy and in vitro susceptibility, and concordance between pharmacokinetic/pharmacodynamic targets determined in this model and clinically accepted targets has been observed. Although there is an initial time investment when learning the technique, it can be performed quickly and efficiently once proficiency is achieved. Benefits of the model include elimination of the neutropenic requirement, increased robustness and reproducibility, ability to study more pathogens and isolates, improved flexibility in study design and establishment of a challenging infection in an immunocompetent host.

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or  A. baumannii

A method for establishing lung infections in immunocompetent rodents is described. With proficiency, this method can be performed quickly and easily to induce stable infection with many isolates of S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae and A. baumannii.

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, ...

Use of the Invertebrate Galleria mellonella as an Infection Model to Study the Mycobacterium tuberculosis Complex

Tuberculosis is the leading global cause of infectious disease mortality and roughly a quarter of the world&#8217;s population is believed to be infected with Mycobacterium tuberculosis. Despite decades of research, many of the mechanisms behind the success of M. tuberculosis as a pathogenic organism remain to be investigated, and the development of safer, more effective antimycobacterial drugs are urgently needed to tackle the rise and spread of drug resistant tuberculosis. However, the progression of tuberculosis research is bottlenecked by traditional mammalian infection models that are expensive, time consuming, and ethically challenging. Previously we established the larvae of the insect Galleria mellonella (greater wax moth) as a novel, reproducible, low cost, high-throughput and ethically acceptable infection model for members of the M. tuberculosis complex. Here we describe the maintenance, preparation, and infection of G. mellonella with bioluminescent Mycobacterium bovis BCG lux. Using this infection model, mycobacterial dose dependent virulence can be observed, and a rapid readout of in vivo mycobacterial burden using bioluminescence measurements is easily achievable and reproducible. Although limitations exist, such as the lack of a fully annotated genome for transcriptomic analysis, ontological analysis against genetically similar insects can be carried out. As a low cost, rapid, and ethically acceptable model for tuberculosis, G. mellonella can be used as a pre-screen to determine drug efficacy and toxicity, and to determine comparative mycobacterial virulence prior to the use of conventional mammalian models. The use of the G. mellonella-mycobacteria model will lead to a reduction in the substantial number of animals currently used in tuberculosis research.

Use of the Invertebrate Galleria mellonella as an Infection Model to Study the Mycobacterium tuberculosis Complex

Galleria mellonella was recently established as a reproducible, cheap, and ethically acceptable infection model for the Mycobacterium tuberculosis complex. Here we describe and demonstrate the steps taken to establish successful infection of G. mellonella with bioluminescent Mycobacterium bovis BCG lux.

Tuberculosis is the leading global cause of infectious disease mortality and roughly a quarter of the world&#8217;s population is believed to be infected ...

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.

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.

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

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

Emergent properties and external factors (population-level and ecosystem-level interactions, in particular) play important roles in mediating ecologically-important endpoints, though they are rarely considered in toxicological studies. D. melanogaster is emerging as a toxicology model for the behavioral, neurological, and genetic impacts of toxicants, to name a few. More importantly, species in the genus Drosophila can be utilized as a model system for an integrative framework approach to incorporate emergent properties and answer ecologically-relevant questions in&#160;toxicology research. The aim of this paper is to provide a protocol for exposing species in the genus Drosophila to pollutants to be used as a model system for a range of phenotypic outputs and ecologically-relevant questions. More specifically, this protocol can be used to 1) link multiple biological levels of organization and understand the impact of toxicants on both individual- and population-level fitness; 2) test the impact of toxicants at different stages of developmental exposure; 3) test multigenerational and evolutionary implications of pollutants; and 4) test multiple contaminants and stressors simultaneously.

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

In this paper, we provide a detailed protocol for exposing species in the genus Drosophila to pollutants with the goal of studying the impact of exposure on a range of phenotypic outputs at different developmental stages and for more than one generation.

Emergent properties and external factors (population-level and ecosystem-level interactions, in particular) play important roles in mediating ...

Biology

Establishment and Maintenance of Gnotobiotic American Cockroaches (Periplaneta americana)

Gnotobiotic animals are a powerful tool for the study of controls on microbiome structure and function. Presented here is a protocol for the establishment and maintenance of gnotobiotic American cockroaches (Periplaneta americana). This approach includes built-in sterility checks for ongoing quality control. Gnotobiotic insects are defined here as cockroaches that still contain their vertically transmitted endosymbiont (Blattabacterium) but lack other microbes that normally reside on their surface and in their digestive tract. For this protocol, egg cases (oothecae) are removed from a (nonsterile) stock colony and surface sterilized. Once collected and sterilized, the oothecae are incubated at 30 &#176;C for approximately 4&#8722;6 weeks on brain-heart infusion (BHI) agar until they hatch or are removed due to contamination. Hatched nymphs are transferred to an Erlenmeyer flask containing a BHI floor, sterile water, and sterile rat food. To ensure that the nymphs are not housing microbes that are unable to grow on BHI in the given conditions, an additional quality control measure uses restriction fragment-length polymorphism (RFLP) to test for nonendosymbiotic microbes. Gnotobiotic nymphs generated using this approach can be inoculated with simple or complex microbial communities and used as a tool in gut microbiome studies.

Establishment and Maintenance of Gnotobiotic American Cockroaches Periplaneta americana

This protocol is used in establishing and maintaining gnotobiotic American cockroaches (Periplaneta americana) by surface sterilizing the egg cases (oothecae) prior to hatching. These gnotobiotic insects contain their vertically transmitted Blattabacterium endosymbionts but have axenic guts.

Gnotobiotic animals are a powerful tool for the study of controls on microbiome structure and function. Presented here is a protocol for the establishment ...

The Madagascar Hissing Cockroach as an Alternative Non-mammalian Animal Model to Investigate Virulence, Pathogenesis, and Drug Efficacy

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