Name: tobacco hornworm as an insect model system for cannabinoid pre-clinical studies
Uploaded: 2021-12-29T15:00:00
Description: Watch this Scientific Journal Video about Tobacco Hornworm as an Insect Model System for Cannabinoid Pre-clinical Studies at JoVE.com

This research was supported by the Institute of Cannabis Research at Colorado State University-Pueblo and the Ministry of Science and ICT (2021-DD-UP-0379), and Chuncheon city (Hemp R&#38;D and industrialization, 2020-2021).

1. Hornworm preparation and cannabidiol treatment
<ol>
	<li>Obtain 150-200 viable M. sexta eggs and wheat germ-based artificial diets (see Table of Materials).</li>
	<li>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 &#176;C with 40%-60% relative humidity.</li>
	<li>Allow tobacco hornworm eggs for 1-3 day...

<ol>
	<li>Kaplan, J. S., Stella, N., Catterall, W. A., Westenbroek, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cannabidiol+attenuates+seizures+and+social+deficits+in+a+mouse+model+of+Dravet+syndrome.">Cannabidiol attenuates seizures and social deficits in a mouse model of Dravet syndrome.</a> Proceedings of the National Academy of Sciences of the United States of America. 114 (42), 11229-11234 (2017).</li><li>Leehey, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+and+tolerability+of+cannabidiol+in+Parkinson+Disease:+An+open+label,+dose-escalation+study.">Safety and tolerability of cannabidiol in Parkinson Disease: An open label, dose-escalation study.</a> Cannabis and Cannabinoid Research. 5 (4), 326-336 (2020).</li><li>Al-Ghezi, Z. Z., Miranda, K., Nagarkatti, M., Nagarkatti, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combination+of+cannabinoids,+delta+9-+tetrahydrocannabinol+and+cannabidiol,+ameliorates+experimental+multiple+sclerosis+by+suppressing+neuroinflammation+through+regulation+of+miRNA-mediated+signaling+pathways.">Combination of cannabinoids, delta 9- tetrahydrocannabinol and cannabidiol, ameliorates experimental multiple sclerosis by suppressing neuroinflammation through regulation of miRNA-mediated signaling pathways.</a> Frontiers in Immunology. 10, 1921 (2019).</li><li>Seltzer, E. S., Watters, A. K., MacKenzie, D., Granat, L. M., Zhang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cannabidiol+(CBD)+as+a+promising+anti-cancer+drug.">Cannabidiol (CBD) as a promising anti-cancer drug.</a> Cancers (Basel). 12 (11), 3203 (2020).</li><li>Garcia-Morales, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CBD+reverts+the+mesenchymal+invasive+phenotype+of+breast+cancer+cells+induced+by+the+inflammatory+cytokine+IL-1beta).">CBD reverts the mesenchymal invasive phenotype of breast cancer cells induced by the inflammatory cytokine IL-1beta).</a> International Journal of Molecular Sciences. 21 (7), 2429 (2020).</li><li>Jeong, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cannabidiol+promotes+apoptosis+via+regulation+of+XIAP/Smac+in+gastric+cancer.">Cannabidiol promotes apoptosis via regulation of XIAP/Smac in gastric cancer.</a> Cell Death and Disease. 10 (11), 846 (2019).</li><li>Devinsky, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open-label+use+of+highly+purified+CBD+(Epidiolex&#174;)+in+patients+with+CDKL5+deficiency+disorder+and+Aicardi,+Dup15q,+and+Doose+syndromes.">Open-label use of highly purified CBD (Epidiolex&#174;) in patients with CDKL5 deficiency disorder and Aicardi, Dup15q, and Doose syndromes.</a> Epilepsy &amp; Behavior. 86, 131-137 (2018).</li><li>de la Harpe, A., Beukes, N., Frost, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CBD+activation+of+TRPV1+induces+oxidative+signaling+and+subsequent+ER+stress+in+breast+cancer+cell+lines.">CBD activation of TRPV1 induces oxidative signaling and subsequent ER stress in breast cancer cell lines.</a> Biotechnology and Applied Biochemistry. , (2021).</li><li>Gasparyan, A., Navarrete, F., Manzanares, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cannabidiol+and+sertraline+regulate+behavioral+and+brain+gene+expression+alterations+in+an+animal+model+of+PTSD.">Cannabidiol and sertraline regulate behavioral and brain gene expression alterations in an animal model of PTSD.</a> Frontiers in Pharmacology. 12, 694510 (2021).</li><li>Aso, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cannabidiol-enriched+extract+reduced+the+cognitive+impairment+but+not+the+epileptic+seizures+in+a+Lafora+disease+animal+model.">Cannabidiol-enriched extract reduced the cognitive impairment but not the epileptic seizures in a Lafora disease animal model.</a> Cannabis and Cannabinoid Research. 5 (2), 150-163 (2020).</li><li>Kadam, R. A., Borde, S. U., Madas, S. A., Salvi, S. S., Limaye, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+in+recruitment+and+retention+of+clinical+trial+subjects.">Challenges in recruitment and retention of clinical trial subjects.</a> Perspectives in Clinical Research. 7 (3), 137-143 (2016).</li><li>Kaur, G., Dufour, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+lines:+Valuable+tools+or+useless+artifacts.">Cell lines: Valuable tools or useless artifacts.</a> Spermatogenesis. 2 (1), 1-5 (2012).</li><li>Alves, P., Amaral, C., Teixeira, N., Correia-da-Silva, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cannabidiol+disrupts+apoptosis,+autophagy+and+invasion+processes+of+placental+trophoblasts.">Cannabidiol disrupts apoptosis, autophagy and invasion processes of placental trophoblasts.</a> Archives of Toxicology. , (2021).</li><li>. Trophoblast Available from: <a target="_blank" href="https://en.wikipedia.org/wiki/Trophoblast">https://en.wikipedia.org/wiki/Trophoblast</a> (2021)</li><li>Yang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cannabinoids+inhibited+pancreatic+cancer+via+P-21+activated+kinase+1+mediated+pathway.">Cannabinoids inhibited pancreatic cancer via P-21 activated kinase 1 mediated pathway.</a> International Journal of Molecular Sciences. 21 (21), 8035 (2020).</li><li>Jeong, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cannabidiol-induced+apoptosis+is+mediated+by+activation+of+Noxa+in+human+colorectal+cancer+cells.">Cannabidiol-induced apoptosis is mediated by activation of Noxa in human colorectal cancer cells.</a> Cancer Letters. 447, 12-23 (2019).</li><li>Capes-Davis, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+lines+as+biological+models:+Practical+steps+for+more+reliable+research.">Cell lines as biological models: Practical steps for more reliable research.</a> Chemical Research in Toxicology. 32 (9), 1733-1736 (2019).</li><li>Chuang, S. H., Westenbroek, R. E., Stella, N., Catterall, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+antiseizure+efficacy+of+cannabidiol+and+clonazepam+in+a+conditional+mouse+model+of+Dravet+syndrome.">Combined antiseizure efficacy of cannabidiol and clonazepam in a conditional mouse model of Dravet syndrome.</a> Journal of Experimental Neurology. 2 (2), 81-85 (2021).</li><li>Orvos, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+electrophysiological+effect+of+cannabidiol+on+hERG+current+and+in+guinea-pig+and+rabbit+cardiac+preparations.">The electrophysiological effect of cannabidiol on hERG current and in guinea-pig and rabbit cardiac preparations.</a> Scientific Reports. 10 (1), 16079 (2020).</li><li>Verrico, C. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+randomized,+double-blind,+placebo-controlled+study+of+daily+cannabidiol+for+the+treatment+of+canine+osteoarthritis.">A randomized, double-blind, placebo-controlled study of daily cannabidiol for the treatment of canine osteoarthritis.</a> Pain. 161 (9), 2191-2202 (2020).</li><li>Barata, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroprotection+by+cannabidiol+and+hypothermia+in+a+piglet+model+of+newborn+hypoxic-ischemic+brain+damage.">Neuroprotection by cannabidiol and hypothermia in a piglet model of newborn hypoxic-ischemic brain damage.</a> Neuropharmacology. 146, 1-11 (2019).</li><li>Beardsley, P. M., Scimeca, J. A., Martin, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+agonistic+activity+of+delta+9-11-tetrahydrocannabinol+in+mice,+dogs+and+rhesus+monkeys+and+its+interactions+with+delta+9-tetrahydrocannabinol.">Studies on the agonistic activity of delta 9-11-tetrahydrocannabinol in mice, dogs and rhesus monkeys and its interactions with delta 9-tetrahydrocannabinol.</a> Journal of Pharmacology and Experimental Therapeutics. 241 (2), 521-526 (1987).</li><li>Ryan, D., McKemie, D. S., Kass, P. H., Puschner, B., Knych, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacokinetics+and+effects+on+arachidonic+acid+metabolism+of+low+doses+of+cannabidiol+following+oral+administration+to+horses.">Pharmacokinetics and effects on arachidonic acid metabolism of low doses of cannabidiol following oral administration to horses.</a> Drug Testing and Analysis. 13 (7), 1305-1317 (2021).</li><li>Bryda, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Mighty+Mouse:+The+impact+of+rodents+on+advances+in+biomedical+research.">The Mighty Mouse: The impact of rodents on advances in biomedical research.</a> Missouri Medicine. 110 (3), 207-211 (2013).</li><li>Qi, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CBD+promotes+oral+ulcer+healing+via+inhibiting+CMPK2-mediated+inflammasome.">CBD promotes oral ulcer healing via inhibiting CMPK2-mediated inflammasome.</a> Journal of Dental Research. , (2021).</li><li>Mastinu, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prosocial+effects+of+nonpsychotropic+Cannabis+sativa+in+mice.">Prosocial effects of nonpsychotropic Cannabis sativa in mice.</a> Cannabis and Cannabinoid Research. , (2021).</li><li>Justice, M. J., Dhillon, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+the+mouse+to+model+human+disease:+increasing+validity+and+reproducibility.">Using the mouse to model human disease: increasing validity and reproducibility.</a> Disease Models &amp; Mechanisms. 9 (2), 101-103 (2016).</li><li>Andre, R. G., Wirtz, R. A., Das, Y. T., An, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Insect Models for Biomedical Research. , 61-72 (1989).</li><li>He, J., Tan, A. M. X., Ng, S. Y., Rui, M., Yu, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cannabinoids+modulate+food+preference+and+consumption+in+Drosophila+melanogaster.">Cannabinoids modulate food preference and consumption in Drosophila melanogaster.</a> Scientific Reports. 11 (1), 4709 (2021).</li><li>Kanost, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multifaceted+biological+insights+from+a+draft+genome+sequence+of+the+tobacco+hornworm+moth,+Manduca+sexta.">Multifaceted biological insights from a draft genome sequence of the tobacco hornworm moth, Manduca sexta.</a> Insect Biochemistry and Molecular Biology. 76, 118-147 (2016).</li><li>Park, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contrasting+roles+of+cannabidiol+as+an+insecticide+and+rescuing+agent+for+ethanol-induced+death+in+the+tobacco+hornworm+Manduca+sexta.">Contrasting roles of cannabidiol as an insecticide and rescuing agent for ethanol-induced death in the tobacco hornworm Manduca sexta.</a> Scientific Reports. 9 (1), 10481 (2019).</li><li>Tukey, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparing+individual+means+in+the+analysis+of+variance.">Comparing individual means in the analysis of variance.</a> Biometrics. 5 (2), 99-114 (1949).</li><li>Mantel, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+survival+data+and+two+new+rank+order+statistics+arising+in+its+consideration.">Evaluation of survival data and two new rank order statistics arising in its consideration.</a> Cancer Chemotherapy Reports. 50 (3), 163-170 (1966).</li><li>Watts, S., Kariyat, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Picking+sides:+Feeding+on+the+abaxial+leaf+surface+is+costly+for+caterpillars.">Picking sides: Feeding on the abaxial leaf surface is costly for caterpillars.</a> Planta. 253 (4), 77 (2021).</li><li>McPartland, J. M., Agraval, J., Gleeson, D., Heasman, K., Glass, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cannabinoid+receptors+in+invertebrates.">Cannabinoid receptors in invertebrates.</a> Journal of Evolutionary Biology. 19 (2), 366-373 (2006).</li></ol>

The feeding study demonstrated that high doses of CBD (2 mM) inhibited the insect&#39;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...

Over the past years, public attention has been centered on cannabinoids due to their therapeutic potential, including the treatment of epilepsy1, Parkinson&#39;s disease2, multiple sclerosis3, and various forms of cancer4,5,6 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 lines5,8, and diverse animal model systems9,10.
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 &#39;pure population of cells that have no cross-contamination of other cells and chemicals&#39;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&#39;s&#160;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&#160;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 herbivores30,31. 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.

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		<tr>
			<td>Analytic balance</td>
			<td>Mettler Instrument Corp.</td>
			<td>AE100S</td>
			<td></td>
		</tr>
		<tr>
			<td>Cannabidiol isolate (&#62;99.4%)</td>
			<td>Lilu&#39;s Garden</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cheesecloth</td>
			<td>VWR INTERNATIONAL</td>
			<td>470150-438</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 50mL clear polypropylene (PP) centrifuge tubes</td>
			<td>VWR</td>
			<td>89093-192</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl Alcohol, 200 Proof</td>
			<td>Sigma-Aldrich</td>
			<td>EX0276-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Fear conditioning chamber</td>
			<td>Coulbourn Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Insect rearing chamber</td>
			<td>Darwin Chambers</td>
			<td>INR034</td>
			<td></td>
		</tr>
		<tr>
			<td>Medium chain triglycerides (MCT) oil</td>
			<td>Walmart</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Motion detection software (Actimetrics)</td>
			<td>Coulbourn Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene petri dish (120 mm x 120 mm x 17mm)</td>
			<td>VWR INTERNATIONAL</td>
			<td>688161</td>
			<td></td>
		</tr>
		<tr>
			<td>Tobacco hormworm artificial diet</td>
			<td>Carolina Biological Supply Company</td>
			<td>Item # 143908</td>
			<td>Ready-To-Use-Hornworm-Diet</td>
		</tr>
		<tr>
			<td>Tobacco hormworm eggs</td>
			<td>Carolina Biological Supply Company</td>
			<td>Item # 143880</td>
			<td>Unit of 30-50</td>
		</tr>
	</tbody>
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tobacco hornworm as an insect model system for cannabinoid pre-clinical studies

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 &#176;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, 1st 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.

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 (&#62;20) were individually reared at 25 &#176;C on a 12 h:12 h = light: dark cycle. The insects&#39; 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.
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Tobacco Hornworm as an Insect Model System for Cannabinoid Pre-clinical Studies

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.

With increased attention on cannabinoids in medicine, several mammalian model organisms have been used to elucidate their unknown pharmaceutical ...

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.

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