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 exponentially increased. Additionally, clinical-grade isolates of single cannabinoids and cannabinoid mixtures have been successfully tested in human subjects⁷, cell lines^5,8, and diverse animal model systems^9,10.

A clinical trial would be ideal for validating the efficacy and adverse effects of cannabinoids on a specific disease. However, there are numerous challenges in clinical trials, including ethical/IRB approval, recruitment, and retention of the subjects¹¹. To overcome these hurdles, various human cell lines were used because human-derived cell lines are cost-effective, easy to handle, can bypass the ethical issues, and provide consistent and reproducible results as the cell lines are a 'pure population of cells that have no cross-contamination of other cells and chemicals'¹².

Alves et al. (2021)¹³ tested CBD in a dose-dependent manner in the placental trophoblasts, which are specialized cells of the placenta that play an essential role in embryo implantation and interaction with the decidualized maternal uterus¹⁴. Their results showed that CBD caused cell viability loss, cell cycle progression disruption, and apoptosis induction. These observations demonstrate the potential negative impacts of Cannabis use by pregnant women¹³. Likewise, a series of cell lines were also used to examine the pharmacological effects of CBD in human diseases, in particular, various forms of cancer. The in vitro studies successfully demonstrated anti-cancer effects in pancreatic¹⁵, breast⁸, and colorectal cancer cells¹⁶. However, while being widely available and easy to handle, specific cell lines such as HeLa, HEK293 are prone to genetic and phenotypic changes due to alterations in their growth conditions or handling¹⁷.

In Cannabis research, various animal model systems, ranging from small animals such as mouse¹⁸, guinea-pig¹⁹, and rabbit¹⁹ to large animals such as canine²⁰, piglet²¹, monkey²², horse²³, have been used to explore unknown therapeutic effects. Mice have been the most preferred animal model system for cannabinoid research due to their anatomical, physiological, and genetic similarity to humans²⁴. Most significantly, mice have CB1/2 receptors in their nervous system, which are present in humans. They also have a shorter life cycle than human subjects, with easier maintenance and abundant genetic resources, thus making it much easier to monitor the effects of cannabinoids throughout an entire life cycle. The mammalian system is widely used and has successfully demonstrated that CBD relieves seizure disorders¹, post-traumatic stress disorder⁹, oral ulcers²⁵, and dementia-like symptoms¹⁰. The mouse model has also enabled a social interaction study of individuals within a community which is extremely difficult in large animals and humans²⁶.

Despite all the advantages of the animal model system, it is still costly and requires intensive care during drug administration and data collection. Additionally, there is scrutiny of using mice in research because of irreproducibility and poor recapitulation of human conditions due to limitations in experimental design and rigor²⁷.

With the increasing demand for medical/preclinical studies of cannabinoids, a non-mammalian model system is needed. Invertebrate models traditionally conferred distinctive benefits over vertebrate models. The significant benefits include the ease and low cost of rearing many specimens and enabling researchers to monitor multiple generations of genetically homogeneous populations²⁸. A recent study proved the fruit fly, Drosophila melanogaster, to be an effective insect model system to investigate pharmacological functions of cannabinoids in modulating feeding behaviors²⁹. Among the insect model systems, the authors focused on the tobacco hornworm, Manduca sexta, also known as Carolina sphinx moth or hawk moth, as a novel insect model system for cannabinoid research.

Manduca sexta belongs to the family of Sphingidae. The insect is the most common plant pest in the southern United States, where they feed on solanaceous plants. The insect model has a long history in research in insect physiology, biochemistry, neurobiology, and drug interaction studies. Manduca sexta's research portfolio includes a draft genome sequence, allowing for a molecular-level understanding of essential cellular processes³⁰. Another crucial benefit of this model system is its large size, reaching more than 100 mm in length and 10 g in weight in the 18-25 days of larval development. The large size enables researchers to easily monitor morphological and behavioral changes in real-time in response to the CBD treatment. Also, due to the size, electrophysiological responses were examined with the abdominal nervous system, including ganglia dissected from the larvae without high-resolution microscope settings. The unique feature allows researchers to readily investigate acute and long-term responses to the administered cannabinoid(s).

Despite such versatility, M. sexta has only recently been explored for its suitability as an experimental model for Cannabis and cannabinoid studies. In 2019, the authors used the insect model system for the first time to address the hypothesis that Cannabis has evolved to produce Cannabidiol to protect itself from insect herbivores^30,31. The result clearly showed that the plants exploited CBD as a feeding deterrent and inhibited the growth of the pest insect M. sexta caterpillar, as well as causing increased mortality³¹. The study also demonstrated the rescuing effects of CBD to intoxicated ethanol larvae, identifying the potential vehicle effect of ethanol as a carrier of the CBD. As shown, the insect model system effectively investigated the therapeutic effects of cannabinoids within 3-4 weeks with less labor and costs than other animal systems. Although the insect model lacks cannabinoid receptors (i.e., no CB1/2 receptors), the model system provides a valuable tool to understand the pharmacological roles of cannabinoids through a cannabinoid receptor-independent manner.

The authors of this study have previously worked with the tobacco hornworm as a model system for cannabinoid research³¹. After careful consideration of the benefits and risks of using M. sexta, we have provided a method involving the proper care and preparation of diet for preclinical trials that allow for opportunities for future preclinical laboratory use.

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 days to hatch inside the insect rearing chamber maintained at 25 °C with 40%-60% relative humidity.
4. Prepare cannabidiol (CBD) stock solution (200 mM) by adding 1.26 g of >98% purity CBD isolate in 20 mL of EtOH (200 proof) or 100% medium chain triglycerides (MCT) oil (see Table of Materials).
   NOTE: CBD isolate is light-sensitive, so handle at dark.
5. Add 5 mL and 10 mL of the 200 mM CBD stock solution to the 1,000 g of AD to bring the final concentrations of the diets 1 mM and 2 mM of CBD, respectively.
   NOTE: Ensure the diet and CBD stock solution are well-blended until a completely homogeneous mixture is formed. Blend the AD containing stock of CBD in a plastic bag for at least 45 min by hand.
   CAUTION: Coffee mixer or any other metal grinder appeared to be ineffective.
6. Dispense 20 g of the three media, control (AD), vehicle (AD + 0.1% of EtOH or MCT oil), and CBD containing media (AD + 0.1% of EtOH or MCT oil + 1 mM/2 mM of CBD) to the bottom of the 50 mL tube.
7. Randomly distribute 1st instar larvae (~2 mm long) individually in a 50 mL test tube and cover with a perforated lid or cheesecloth (see Table of Materials).
   NOTE: Place the tube upside down and grow insects at an insect rearing chamber maintained 25 °C with 40%-60% relative humidity.
8. Grow them inside an insect rearing chamber (see Table of Materials) maintained at 25 °C with a 12 h light/dark cycle.

2. M. sexta larval growth, diet consumption, and mortality measurements

1. Measure the larval growth (i.e., size and weight) with an analytical balance and mortality at 2-day intervals after being transferred to individual containers until pupation is recognized as the dark brown coloration of a hardened exocuticle layer.
   1. Record the initial mass (in grams) of each group of larvae before introducing the larvae to their respective diets and subtract the mass of the larvae at each measurement from the initial mass to determine mass gains between larvae developmental stages until the larvae complete the pupation stage.
   2. Record the number of days between the instar developmental stages to understand differences in the developmental timeframe between stages of larvae growth until pupation on each diet.
      NOTE: Scrape off the fecal matter from the container to avoid any mold contamination. Collect the matter for future testing dependent on experiment purposes (e.g., CBD accumulation rate calculation, microbial profiling). It is important to carefully handle the insect during the fragile periods of apolysis or ecdysis. When taking out of the larvae from a container, gently grab the main body of the insect with a flat-tip and wide forceps and do not force to remove the outer layer of skin when an insect is in the process of shedding.
2. Measure the diet consumption³¹ by weighing the diet loss of the container between 1^st instar larvae and pupation. Record the initial grams of diet at the beginning of the experiment and subtract the initial amount from the remaining amount of diet when the larvae entered the complete pupation stage.
   NOTE: The fecal matter should be excluded from the diet measurement. The fecal matter and other debris (i.e., skin sheds) can be easily removed from the media by placing the container upside down.
3. For mobility measurements, allow the subjected insect to acclimate the chamber environment for at least 5 min and track the distances³¹ that three groups of 5^th instar insects (80-100 mm in length) traveled using an automated, computerized fear conditioning chamber (see Table of Materials).
4. Analyze the mobility response³¹ through video recorded 60 frames/s for 5 min using a motion detection software (see Table of Materials) which generates a motion index.

3. Statistical analysis

1. Analyze the differences in the larval growth (i.e., size and weight) and the motion index by one-way ANOVA with Tukey's post-test³².
2. Use the log-rank (Mantel-Cox) test³³ for survival curve comparisons.
   NOTE: All the statistical analyses were performed using statistical analysis software (see Table of Materials).

Representative Results

Manduca sexta as a model system to examine cannabinoids toxicity
Figure 1 depicts the key components of the CBD experiment using tobacco hornworm Manduca sexta. Large numbers of insects (>20) were individually reared at 25 °C on a 12 h:12 h = light: dark cycle. The insects' size, weight, and mortality were measured at 2-day intervals to monitor for short-and long-term responses after high-dose CBD (2 mM) treatment.

Figure 2 shows the adverse effects of CBD on the insect's growth and development. The insects reared on an artificial diet (AD) showed the best growth performance. The vehicle control that used 0.1% medium-chain triglyceride (MCT) oil as a dissolving agent for CBD isolate also showed normal growth without any detrimental effects. However, a high dose of CBD (2 mM) induced weight loss (Figure 2C) and led to a higher mortality rate than those of control and vehicle groups (Figure 2D).

On day 24, the average size of the larvae fed on AD was 63.9 mm (n = 20-22). However, the size of larvae reared on AD containing 2 mM of CBD was 50.7 mm, which was ~21% smaller than the larvae grown on AD (red line in Figure 2C)³¹. On day 24, the average weight of larva reared on AD was 6.5 g, which was 2.2-fold greater than those of larvae reared on AD with 2 mM of CBD (n = 12-16, p < 0.00001)³¹. Notably, the high dose of CBD (2 mM) significantly increased the mortality rate up to 40%, while the control and vehicle groups showed only a 20% mortality rate (Figure 2D)³¹. The results indicated that the high dose of CBD (2 mM) in the diet is detrimental to insect development and correlates to increased mortality.

Manduca sexta as a model system to explore unknown therapeutic functions of cannabinoids
Figure 2 showed that the insect model system effectively monitors any detrimental effects of CBD by monitoring their morphological and physiological changes. The preliminary result indicated that >1% ethanol (EtOH) is negatively related to their growth, mobility, diet consumption, and survival rate. To examine whether CBD improves insect's mobility and feeding behavior in the EtOH-intoxicated M. sexta larvae, the total amount of diet consumed by insects and the distance they traveled for 10 min were measured from insects grown under three feeding conditions (AD, AD + 1% EtOH, and AD + 1% EtOH + 1 mM CBD). Figure 3A shows that M. sexta larvae reared on AD containing 1 mM of CBD consumed at least 3.1-times greater diet mass than those reared on EtOH-added diet³¹. However, the diet consumption of the insects reared on 2 mM of CBD-added media was not significantly different than those of larvae reared on EtOH-only diets (p > 0.05)³¹.

Larval mobility was also tracked to examine if CBD affected their mobility when intoxicated with EtOH. The mobile index is presented as the percentage (%) of freeze. Figure 3B compares the mobile index of M. sexta larvae reared on different conditions. The results show that 1% EtOH-treated larvae did not affect mobility (p > 0.05). The 1 mM CBD administration also did not affect mobility (p > 0.05)³¹. The 2% EtOH treatments turned out to be lethal to M. sexta larvae; therefore, no mobility index was recorded. With the addition of the high dose of CBD (2 mM) into AD containing 2% EtOH, the mobility remained low (80% freeze)³¹.

Figure 1: The summarized process of using tobacco hornworm Manduca sexta caterpillars in cannabidiol study. (A) Hornworm eggs hatched in a separate large container with a layer of artificial diet. (B) A syringe was used to fill the container to prevent any diet from sticking to the sides of the containers. (C) A 2^nd instar tobacco hornworm in a 50 mL test tube with cheesecloth. (D) A 3^rd instar tobacco hornworm. (E) Hornworm length (mm) and weight (g) were measured on a scale. (F) 5^th instar tobacco hornworm which undergoes ecdysis and ready for pupation. Please click here to view a larger version of this figure.

Figure 2: Effects of Cannabidiol (CBD) on the growth and mortality of tobacco hornworm Manduca sexta. (A) Tobacco hornworm caterpillars at 5^th, 3^rd instar, and early pupation. The size (B), weight (C), and mortality (D) of M. sexta when fed on artificial diet (AD), AD + 0.1% of medium-chain triglyceride (MCT), and AD + 0.1% of MCT + 2 mM of CBD. For statistical analyses on insect growth and survival rate, a one-way ANOVA with Tukey's multiple comparisons test (n = 20-22, p < 0.05) and Mantel-Cox test (n = 20-22, p < 0.05) were used, respectively. The figure is adapted from Reference³¹. Please click here to view a larger version of this figure.

Figure 3: The effects of Cannabidiol (CBD) on insect feeding behavior and mobility. (A) Diet consumption of tobacco hornworm caterpillars reared on artificial diet (AD), AD + 1-2% of ethanol (EtOH), and AD + 1-2% of EtOH + 1-2mM of CBD (one-way ANOVA, Tukey's multiple comparison at p < 0.05). (B) Insect mobility. The mobility is depicted as freeze %. *** indicates p < 0.01. The figure is adapted from Reference³¹. Please click here to view a larger version of this figure.

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 critical steps: (1) ensuring M. sexta's eggs are hatched uniformly in size and timing, (2) preparing the growth media that are homogeneously blended with cannabinoids to a targeted concentration, and (3) maintaining the growth media to be free of fungal contamination while maintaining ideal humidity level at 40%-60%. The insect model system enabled us to address the research question within 25 days, from media preparation to data collection and interpretation. Most importantly, the insect system produced consistent results from large specimens.

To ensure the success of the cultivated M. sexta larvae, maintaining the relative humidity at 40%-60% inside the container is essential. If a container fails to hold the high humidity, an artificial diet containing the cannabinoids will be desiccated rapidly, causing early experiment termination due to the insects' death. However, in a closed system, the high humidity provides an ideal condition for the fungal outbreak, which is difficult to eradicate. The authors suggest using a perforated lid or cheesecloth to supply sufficient air circulation while minimizing water loss from the media. In a natural environment, the caterpillars prefer to feed on the abaxial side of a leaf where humidity is higher while presenting fewer trichomes than the leaf's surface area³⁴. Thus, placing a container upside down was exceptionally helpful while providing a refuge area or crawling wood stick. This also helps to remove fecal matter from the media area and makes it easy to collect the waste for further assays.

As cannabinoids receptors are absent in invertabrates³⁵, the tobacco hornworm M. sexta might not be suitable for therapeutic studies mediated by the endocannabinoid system. However, with the numerous benefits demonstrated in our pilot study, the insect should be considered a new model system to investigate the pharmacological functions of cannabinoids, particularly studies involving non-CB receptor-mediated pharmacokinetics. The relatively short life cycle of M. sexta allows researchers to understand the impacts of a cannabinoid-containing diet over multiple generations, allowing for an experimental design in higher mammal model organisms.

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