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 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.
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.
1. Preparations for Maintaining a Hissing Cockroach Colony
2. Hissing Cockroach Care and Breeding
3. Cockroach Preparation for Experimentation
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.
5. Drug Preparations
6. Assembly of the Injector
7. Cockroach Injections
8. Recording Hissing Cockroach Morbidity and Mortality
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 (Δhcp1) than in groups that were infected with wildtype B. pseudomallei K96243, parental B. mallei SR1, or B. thailandensis DW503 (Figure 1). Conversely, infection with virulent mutants (Δhcp2 or Δhcp3) killed the hissing cockroaches similarly to the wildtype B. pseudomallei (Figure 1). Infection with the mammalian avirulent Burkholderia species, B. thailandensis E264, and its aminoglycoside sensitive derivative DW503, show that the hissing cockroach model is particularly suitable for elucidating mutations in B. thailandensis that lead to attenuation (Figure 2). Thus, it is a more fitting animal model for B. thailandensis studies than rodent models. Increasing concentrations or multiple injections of CLQ did not kill the hissing cockroaches; this illustrates that drug toxicity can also be tested in the hissing cockroach model (Figure 3). Further, the efficacy of CLQ against B. thailandensis infection is shown in Figure 4. Important aspects of hissing cockroach care and infection are shown in Figure 5. Table 1 can be used to score the morbidity of hissing cockroaches during experiments.
Figure 1: Hissing cockroach survival after injection with virulent and attenuated Burkholderia. Eight hissing cockroaches per group were injected with 25 µL of bacterial suspension. Hissing cockroaches were checked for survival once a day for 5 days. (A) Hissing cockroaches were injected with parental B. mallei SR1 (open square) or the Δhcp1 mutant (closed square) at 100 CFUs. (B) Hissing cockroaches were injected with wild type B. pseudomallei K96243 (open square), Δhcp1 (closed square), Δhcp2 (open triangle), or Δhcp3 (open circle) mutant at 10 CFUs. (C) Hissing cockroaches were injected with parental B. thailandensis DW503 (open square) or Δhcp1 mutant (closed square) at 100 CFUs. Figure originally published in reference 11. Please click here to view a larger version of this figure.
Figure 2: Hissing cockroach survival after injection of increasing concentrations of B. thailandensis for LD50 determination. Eight hissing cockroaches per group were injected with wildtype B. thailandensis E264 (A) or the aminoglycoside sensitive derivative DW503 (B) and survival was scored for 7 days. The LD50 is 3 CFUs for E264 and 6 CFUs for DW503. Please click here to view a larger version of this figure.
Figure 3: Hissing cockroach survival after injection with chloroquine. Five hissing cockroaches per group were injected once (A) or twice on two consecutive days (B) with 250 (diamond), 500 (square), or 1,000 µg (triangle) CLQ or PBS (circle) and survival was scored for 7 days. Please click here to view a larger version of this figure.
Figure 4: Hissing cockroach survival after infection with B. thailandensis and treatment with chloroquine. 10 to 12 hissing cockroaches per group were infected with B. thailandensis DW503 and not treated (square), infected with B. thailandensis DW503 and treated with CLQ (triangle), treated with CLQ alone (diamond), or were uninfected and untreated (circle). Survival was recorded for 7 days. The survival curve, a composite of 4 separate experiments, is expressed as a percentage equal to the total number of survivors divided by the total number of hissing cockroaches for each treatment on the days indicated. The CFU inoculum given ranged from 10 to 20 LD50. Figure originally published in reference 10. Please click here to view a larger version of this figure.
Figure 5: Images related to the hissing cockroach model. (A) A female hissing cockroach lacks protrusions on its head. (B) A male hissing cockroach can be identified by the presence of horns. (C) A hissing cockroach must molt out of its exoskeleton to grow. The emerging insect is white in color but gradually darkens as the new exoskeleton hardens. (D) Hissing cockroaches may be housed in a snap cap plastic container with ventilation holes during an experiment. (E) Under BSL3 conditions, hissing cockroaches are housed in screw cap plastic containers. (F) A large mouse cage is used to house a hissing cockroach colony. It should contain food, water and a cardboard egg carton for hiding. (G) Hissing cockroaches are injected with a 1 mL syringe attached to a repetitive pipette. (H) A hissing cockroach is inoculated by injection through the cutaneous membrane between the abdominal terga. Please click here to view a larger version of this figure.
1 | Live, Normal | -actively mobile -able to grasp and hold onto fingers when hissing cockroach is picked up |
2 | Live, Lethargic | -immobile but crawls when prodded |
3 | Live, Moribund | -immobile with legs tucked in -does not move when prodded -antennae and/ or legs move when prodded |
4 | Dead | -immobile with legs tucked in -antennae do not move when prodded -legs do not move when prodded |
Table 1: Hissing cockroach morbidity scoring system. The overall score for a group of hissing cockroaches is based on the hissing cockroach with the highest score in the group.
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 cockroaches die and at a faster rate at higher temperatures when containers were not cleaned daily.
The key to consistent dosing or inoculation of the hissing cockroach is to press the dispenser button on the repetitive pipette firmly. We recommend practicing this technique, loading of the syringe onto the repetitive pipette, and performing blank injections. The most time-consuming step of the procedure for an operator new to the technique is holding or immobilizing the hissing cockroach during injection. Therefore, we also highly recommend practicing the technique of holding and injecting multiple hissing cockroaches before tackling a more ambitious project. This can be achieved by maintaining a small group of hissing cockroaches that is used exclusively for injection practices. Although we have found that injection can be performed quickly when holding the hissing cockroach on its side, other techniques for holding hissing cockroaches (e.g. immobilizing a hissing cockroach on a smooth curved surface; perching the hissing cockroach on the middle finger while the index finger and thumb immobilize it) may be preferred and should be explored by different operators.
The use of the hissing cockroach model affords several advantages over other insect models (e.g. the wax worm larva Galleria mellonella and the fruit fly Drosophila melanogaster) that have been previously used as animal models with Burkholderia infection 16,17,18. For example, the experimental window for a hissing cockroach ranges from months to years allowing flexibility to the researchers, whereas that for a wax worm larva is only five days 19,20. For a wax worm larva, the five day period also coincides with cocoon encasement; removal of cocoons is a labor intensive process that may cause physical trauma to the larvae 20. More importantly, a B. thailandensis T6SS-1 mutant that is attenuated in both the Syrian hamster and the hissing cockroach 11, was virulent in Galleria, suggesting that Galleria is not a good model for the study of some mutants such as T6SS in B. thailandensis (data not shown).
The use of the hissing cockroach presents several advantages over the fruit fly. The hissing cockroach is large and of a substantial body mass with a tough exoskeleton that allows it to be easily handled during injections. In contrast, the fruit fly is small and requires specialized equipment for inoculation. Also, whereas the hissing cockroach naturally lives in temperatures that are similar to or exceed human body temperature, the optimal temperature for the fruit fly is between 22 to 28 °C. This makes the fruit fly of limited use in the context of studying processes that are dependent on human body temperature (such as multi-nucleated giant cell formation in Burkholderia 10).
Some disadvantages to the use of hissing cockroaches do exist. The genetics of the hissing cockroach are not as well studied as those of Drosophila or even Galleria. The hissing cockroach also has a substantial "ick" or gross factor. However, the hissing cockroach remains an attractive and viable surrogate host for Burkholderia by providing clear advantages to its use in research that are unique to the species. As we have illustrated that the Madagascar hissing cockroach is a tractable surrogate host for Burkholderia, it very likely can also serve as a surrogate host for other bacterial pathogens and we are currently utilizing it in such studies.
The authors have nothing to disclose.
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.
Madagascar hissing cockroach |
Carolina Biological Supply Co, Burlington, NC | 143668 | |
Kibbles n Bits, any flavor | Big Heart Pet Brands, San Francisco, CA | UPC #079100519378 | |
Snap on disposable plastic containers or equivalent | Rubbermaid, Huntersville, NC | UPC #FG7F71RETCHIL | |
Screw on disposable plastic containers or equivalent | Rubbermaid, Huntersville, NC | UPC #FG7J0000TCHIL | |
Tridak STEPPER series repetitive pipette | Dymax Corporation www.dymax.com |
T15469 | |
Syringe (1 mL) | Becton Dickinson, Franklin Lakes, NJ | 309659 | |
Needle (26 or 27G x 1/2) | Becton Dickinson, Franklin Lakes, NJ | 305109, 305111 | |
Chloroquine diphosphate | Sigma-Aldrich, St. Louis, MO | C6628 | |
Phosphate buffered saline | Gibco/ Thermo Fisher Scientific, Gaithersburg, MD | 10010023 | |
Difco Luria- Bertani (Lennox) | Becton Dickinson, Sparks, MD | 240230 | |
Agar | Sigma-Aldrich, St. Louis, MO | A1296 | |
Glycerol | Sigma-Aldrich, St. Louis, MO | G6279 |