Abstract
Fifteen male Madagascar hissing cockroaches (Gromphadorhina portentosa) and 15 male orange-spotted cockroaches (Blaptica dubia) were used to determine a safe and efficacious dosage of alfaxalone and to evaluate its use in insects. Alfaxalone dosages of 100, 150, 200, 250, 300, 400, 500, and 750 mg/kg were evaluated in G. portentosa. One animal was used to evaluate each dosage with the exception of the 750 mg/kg trial. The first animal used had some leakage, so an additional G. portentosa was used for that trial. The remaining G. portentosa received no treatments and were used as controls. Alfaxalone dosages of 100, 250, 500, and 750 mg/kg were evaluated in B. dubia. One animal was used to evaluate each dosage, and the rest were used as controls. Gromphadorhina portentosa and B. dubia used in the trials were weighed and assessed for a righting response prior to each trial. Alfaxalone was delivered intracoelomically to an animal, and loss of movement and righting response were evaluated by direct observation. After 20 minutes post injection, unanesthetized animals were moved to a holding container to monitor for delayed effects. No signs of anesthesia were observed after six hours for G. portentosa and after two hours for B. dubia. Mortalities included five G. portentosa, three of which were in the control group. The other two received an alfaxalone dosage of 750 mg/kg. It is not known at this time whether those two mortalities were associated with the anesthetic protocol used. There were no mortalities in the B. dubia.
Introduction
Invertebrates are playing an increasing role in veterinary medicine as zoos, museums, and private collectors maintain large invertebrate collections. Insects are just one group of invertebrates that are of particular interest due to their diversity, potential to be captivating exhibit animals, and the conservation value of some species. The success of physical restraint in insects for examinations and/or procedures mainly depends on the size and species of insect being handled. In most cases, insects can be manually restrained without risk to animal or handler;7 however, physical restraint is not always possible. Many species are fast moving, can escape restraint, and be difficult to recover. Another challenge is that some species are fragile and easily damaged with restraint. Therefore, the development of anesthetic protocols is an important step in advancing invertebrate medicine. Historically, hypothermia and CO2 gas have been used for anesthesia in entomological research. The use of these methods is controversial due to multiple side effects, such as convulsion and excitation at induction and high mortality.3,7,8 Anesthetic inhalants are a more progressive approach and are commonly used in terrestrial species by placing the animal in an anesthetic chamber through which the agents are pumped. Anesthetic inhalants used include isoflurane, sevoflurane, and halothane.3,7,8 Drawbacks to using gas anesthesia include the uncertainty in the amount of drug the animal is receiving, and repeated exposure to the inhalant may be needed to keep the animal under anesthesia. The latter can result in release of the inhalant into the environment and unintended exposure to personnel. A safe, reliable, and effective injectable anesthetic protocol would allow for full access to the animal without requiring repeated or constant exposure to anesthetic gases.
Alfaxalone (Alfaxan®, 10 mg/ml, Jurox Inc., Kansas City, MO, USA) is an intravenous injectable anesthetic licensed for use in cats (Felis catus) and dogs (Canis familiaris) for the induction and maintenance of anesthesia.1 Alfaxalone induces anesthesia through activity at the gamma-aminobutyric acid subtype A receptor (GABAA) by enhancing the effects of GABA, a major inhibitory neurotransmitter in the central nervous system.2 Its use has been described in invertebrates such as the Chilean rose tarantula (Grammostola rosea) and blue crab (Callinectes sapidus).4,6 To the best of the authors’ knowledge, the efficacy of alfaxalone has not been evaluated in the Madagascar hissing cockroach (Gromphadorhina portentosa) and orange-spotted cockroach (Blaptica dubia).
Material and Methods
Fifteen male G. portentosa and 15 male B. dubia were provided by the Bug Zoo located in the Plant Sciences Building at Colorado State University. The range of weights for G. portentosa was 4–6 g, while B. dubia weighed 1 g. The animals were housed by species following the care instructions provided by the Bug Zoo. The G. portentosa colony was housed in a 28x18x16-cm container using cardboard egg cartons as substrate. The B. dubia colony was housed in a 21x13x13-cm container using the same substrate. Both containers were placed inside a 111x65x75-cm treatment cage (Snyder Manufacturing Company, Centennial, CO, USA) with the temperature set to 23.9°C (optimal temperature: 23.9–29.4°C) with 50% humidity (optimal humidity: >30% without water running down the sides of the cage). The treatment cage malfunctioned the day the animals were housed inside, causing the temperature to decrease to 12.8°C overnight. Climate control using the treatment cage thereafter was discontinued. The colonies were then maintained at room temperature (20.8–22.5°C) with a heating lamp provided for 10–12 hours. Humidity was provided by placing wet paper towels in the containers. The paper towels were changed daily and moistened as needed. The animals were fed an apple or orange slice three times a week, with the uneaten fruit removed the next day. The animals were acclimated for five days prior to initiation of the study. Although the approval of the Animal Care and Use Committee is not required by Colorado State University for invertebrates, all efforts were taken to minimize stress and discomfort to the animals used in this study. Animals at the end of the study were returned to the Bug Zoo.
Gromphadorhina portentosa and B. dubia used in the trials were weighed and assessed for a righting response prior to each trial. Alfaxalone dosages of 100, 150, 200, 250, 300, 400, 500, and 750 mg/kg were evaluated in G. portentosa. One animal was used to evaluate each dosage, with the exception of the 750 mg/kg trial. The first animal used had some leakage, so an additional G. portentosa was used for that trial. The remaining G. portentosa received no treatments and were used as controls. Alfaxalone dosages of 100, 250, 500, and 750 mg/kg were evaluated in B. dubia. One animal was used to evaluate each dosage, and the rest were used as controls. Alfaxalone was delivered intracoelomically to an animal, and then it was observed for loss of movement and righting response. Total immobility, absence of a righting response, and apparent lack of awareness to stimuli indicate full anesthesia in insects.3,7,8 A scoring system was designed to assess anesthesia in G. portentosa and B. dubia. Scored parameters included tactile stimulation of limbs, antennae, and cerci, as well as hissing in the case of G. portentosa.
After 20 minutes post injection, unanesthetized animals were moved to a holding container to monitor for delayed effects by visual evaluation of total immobility. These animals were assessed every 30 minutes for the duration of the trials: six hours for G. portentosa and two hours for B. dubia.
Results
No signs of anesthesia were observed in any of the cockroaches at any of the administered dosages. Mortalities included five G. portentosa, three of which were in the control group. Of the three in the control group, one died during the acclimation period. The other two died during the G. portentosa alfaxalone trial period. The two remaining mortalities received an alfaxalone dosage of 750 mg/kg. They died after returning to the Bug Zoo at the end of the study, which was one week after their respective trial date. These animals were found deceased in their enclosure. No necropsy or histopathology was performed on deceased animals. There were no mortalities in the B. dubia.
Discussion
This pilot study suggests that alfaxalone is not an effective anesthetic drug for G. portentosa and B. dubia. Although the function is similar, the pharmacology of the insect GABA receptor differs from that of the vertebrate GABAA receptor.5 Several steroids that are active on vertebrate GABAA receptors are much less effective on insect GABA receptors. This is the case for pregnane steroids.10 Alfaxalone is a neuroactive steroid molecule that is a synthetic analogue of progesterone, but it does not bind to sex hormone, glucocorticoid, or mineralocorticoid receptors.2 The authors suspect alfaxalone’s pharmacologic profile did not allow it to exert an effect on the steroid site on the insect GABA receptor when used at the dosages and route administered.
While the cause of death for the G. portentosa mortalities cannot be elucidated, it is likely that the cold shock experienced from the treatment cage malfunction played a role. Gromphadorhina portentosa, originally from Madagascar, evolved to be adapted to tropical climates. Spending the night at 12.8°C may have caused excessive stress in some individuals. Since G. portentosa in both the treatment and control groups experienced mortalities, it is less likely that the mortalities in the treatment group were solely due to the anesthetic protocol. This is further supported by the lack of mortality in the B. dubia. The two G. portentosa mortalities in the treatment group did receive 750 mg/kg, but they died one week after their trial date. The cause of death was not identified in this study.
The maximum dosage used in this study was 750 mg/kg, which equated to a maximum fluid volume of 375 µl for G. portentosa and 75 µl for B. dubia. Higher dosages were not used due to leakage that higher injection volumes would have caused. The licensed dosage range for alfaxalone is 2.2–9.7 mg/kg for F. catus and 1.5–4.5 mg/kg for C. familiaris.1 This is in stark contrast to the 750 mg/kg used in this study without an effect on cockroaches. This trend of higher dosages of alfaxalone in invertebrates is seen in G. rosea and C. sapidus. Alfaxalone administered at 200 mg/kg in G. rosea induced about 10 minutes of moderate anesthesia.4 The majority of G. rosea retained muscle tone and sensitivity to stimulation at this dosage. When used in C. sapidus, an alfaxalone dosage of 15 mg/kg provided adequate anesthesia for about 11 minutes, during which none of the animals responded to tactile stimulation. At 100 mg/kg, the length of anesthesia was prolonged to just over one hour.6 The reason for the various dosages in different invertebrate species is unknown at this time, but factors such as drug profile, differences in drug binding sites, metabolism, and elimination may play a role.
The invertebrates are a diverse group of animals and require special anesthetic considerations. In the case of alfaxalone, higher dosages are required for invertebrates compared to mammalian species. Higher dosages translate into higher injection volumes, which are difficult to administer given the size of some invertebrates. This requires a more concentrated formulation of a drug or new drugs/drug combinations. Since the steroid site is weakly effective in insects, future studies should target the other sites on the GABA receptor. One promising site is the benzodiazepine binding site, which shows similarities to the peripheral benzodiazepine sites of vertebrates.9 Once a safe and efficacious injectable drug is found for insects, the next step is to find a concentration for aerosolized delivery to facilitate ease of administration.
Acknowledgments
The authors would like to thank the Colorado State University Bug Zoo for providing the cockroaches used in this study. They also thank Jurox for its generous donation of one bottle of alfaxalone.
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