Tobacco Hornworm as an Insect Model System for Cannabinoid Pre-clinical Studies

Summary

The present protocol provides instructional information for using tobacco hornworm Manduca sexta in cannabinoid research. The method described here includes all necessary supplies and protocols to monitor physiological and behavioral changes of the insect model in response to cannabidiol (CBD) treatment.

Abstract

With increased attention on cannabinoids in medicine, several mammalian model organisms have been used to elucidate their unknown pharmaceutical functions. However, many difficulties remain in mammalian research, which necessitates the development of non-mammalian model organisms for cannabinoid research. The authors suggest the tobacco hornworm Manduca sexta as a novel insect model system. This protocol provides information on preparing the artificial diet with varying amounts of cannabidiol (CBD), setting up a cultivation environment, and monitoring their physiological and behavioral changes in response to CBD treatment. Briefly, upon receiving hornworm eggs, the eggs were allowed 1-3 days at 25 °C on a 12:12 light-dark cycle to hatch before being randomly distributed into control (wheat germ-based artificial diet; AD), vehicle (AD + 0.1% medium-chain triglyceride oil; MCT oil) and treatment groups (AD + 0.1% MCT + 1 mM or 2 mM of CBD). Once the media was prepared, 1^st instar larvae were individually placed in a 50 mL test tube with a wooden skewer stick, and then the test tube was covered with a cheesecloth. Measurements were taken in 2-day intervals for physiological and behavioral responses to the CBD administration. This simple cultivation procedure allows researchers to test large specimens in a given experiment. Additionally, the relatively short life cycles enable researchers to study the impact of cannabinoid treatments over multiple generations of a homogenous population, allowing for data to support an experimental design in higher mammalian model organisms.

Introduction

Over the past years, public attention has been centered on cannabinoids due to their therapeutic potential, including the treatment of epilepsy¹, Parkinson's disease², multiple sclerosis³, and various forms of cancer^4,5,6 with cannabidiol (CBD). Since Cannabis is legalized as an agricultural commodity in the Agricultural Improvement Act of 2018, Public Law 115-334 (the 2018 Farm Bill), Cannabis and its cannabinoid derivatives in the food, cosmetic, and pharmaceutical industries have ....

Protocol

1. Hornworm preparation and cannabidiol treatment

1. Obtain 150-200 viable M. sexta eggs and wheat germ-based artificial diets (see Table of Materials).
2. Place the hornworm eggs in a polystyrene Petri dish with a wheat germ-based artificial diet (AD) layer and transfer the eggs to an insect rearing chamber (see Table of Materials) maintained at 25 °C with 40%-60% relative humidity.
3. Allow tobacco hornworm eggs for 1-3 day.......

Discussion

The feeding study demonstrated that high doses of CBD (2 mM) inhibited the insect's growth and increased mortality³¹. The insect model also showed sensitivity to ethanol; however, CBD effectively detoxicated the ethanol toxicity, increasing their survival rate, diet consumption, and food searching behaviors to similar levels to the control group (Figure 3A,B)³¹. The described insect model system is composed of three critica.......

Materials

Name	Company	Catalog Number	Comments
Analytic balance	Mettler Instrument Corp.	AE100S
Cannabidiol isolate (>99.4%)	Lilu's Garden
Cheesecloth	VWR INTERNATIONAL	470150-438
Corning 50mL clear polypropylene (PP) centrifuge tubes	VWR	89093-192
Ethyl Alcohol, 200 Proof	Sigma-Aldrich	EX0276-1
Fear conditioning chamber	Coulbourn Instruments
Insect rearing chamber	Darwin Chambers	INR034
Medium chain triglycerides (MCT) oil	Walmart
Motion detection software (Actimetrics)	Coulbourn Instruments
Polystyrene petri dish (120 mm x 120 mm x 17mm)	VWR INTERNATIONAL	688161
Tobacco hormworm artificial diet	Carolina Biological Supply Company	Item # 143908	Ready-To-Use-Hornworm-Diet
Tobacco hormworm eggs	Carolina Biological Supply Company	Item # 143880	Unit of 30-50