The protocol proposes using tobacco hornworm as an insect model system for cannabinoid preclinical studies. This system allows researchers to investigate acute and long-term responses to cannabinoids readily. The insect model system enables researchers to investigate cannabinoid therapeutic effects in a large population within three to four weeks with less labor and costs than other animal systems.
Obtain 150 to 200 viable M.sexta eggs and wheat germ-based artificial diets. Place the hornworm eggs in a polystyrene Petri dish with a wheat germ-based artificial diet layer and transfer the eggs to an insect rearing chamber maintained at 25 degree Celsius with 40 to 60%relative humidity. Allow the eggs to hatch for one to three days inside the insect rearing chamber.
To prepare a 200 millimolar cannabidiol stock solution, add 1.26 grams of greater than 98%purity cannabidiol isolate in 20 milliliters of ethanol or 100%medium chain triglyceride oil. Add five milliliters and 10 milliliters of the 200 millimolar cannabidiol stock solution to the 1, 000 grams of an artificial diet for one millimolar and two millimolar of cannabidiol, respectively. Dispense 20 grams of control vehicle and cannabidiol-containing media to the bottom of the 50-milliliter tube.
Randomly distribute approximately two millimeter-long first instar larvae individually in a 50-milliliter test tube and cover with a perforated lid or cheesecloth. Grow them inside an insect rearing chamber maintained at 25 degree Celsius with a 12-hours light-dark cycle. To measure the larval growth, record each group's initial mass and size with an analytical balance and ruler, respectively, and introduce the larvae to their respective diets.
Subtract the mass of the larvae at each measurement at two-day intervals from the initial mass to determine mass gains between larvae developmental stages. Also, record the number of days between the instar developmental stages to understand differences in the developmental timeframe until pupation. To measure the diet consumption, record the initial grams of diet at the beginning at the experiment and subtract it from the remaining amount of diet when the larvae entered the complete pupation stage.
For the larval mobility measurement, allow the subjected insect to acclimate to the fear conditioning chamber for at least five minutes and track the distances the three groups of fifth instar insects traveled for five minutes. Then, run the motion detection software to analyze the mobility response. The insects reared on an artificial diet showed the best growth performance.
The vehicle control that used 0.1%medium chain triglyceride oil as a dissolving agent for CBD isolate also showed normal growth without any detrimental effects. However, a high dose of CBD of two millimeter induced weight loss and led to a higher mortality rate than control and vehicle groups. M.sexta larvae reared on AD with one millimeter CBD consumed at least 3.1 times greater diet than those reared on an ethanol-added diet.
However, the diet consumption of the insects reared on two millimolar CBD-added media was not significantly different from the larvae reared on ethanol-only diets. The mobile index of M.sexta larva reared on different conditions shows that 1%ethanol and one millimolar CBD-treated larvae did not affect mobility. The 2%ethanol treatments turned out to be lethal.
Adding a high dose of CBD into AD containing 2%ethanol restored low mobility. Once CBD is added to diets, completely homogenize the diet CBD mixture and grow insects in the insect rearing chamber maintained at 25 degrees Celsius with 40 to 60%humidity to avoid a fungal outbreak. Electrophysiological responses can be also monitored with the ventral chain ganglia dissected from Manduca sexta to determine whether CBD affects the tobacco hornworm's central nervous system.
The insect model system will enable researchers to explore the pharmaceutical roles of other major and minor cannabinoids by testing over multiple generations, allowing for an experimental design in higher mammals.
