Effect of Hemp Seed Oil in Drosophila melanogaster Behavior on Predatory Sounds

Introduction 

Hemp seed oil refers to a fully constituted oil from the seeds of the Cannabis sativa plant. 

It is appreciated and recognized for its nutritional characteristics and health benefits. Despite its well-known composition of fatty acids, which range from 23-35%, hemp oil also contains proteins (20-25%), carbohydrates (20-30%), and fiber (10-15%). Moreover, it has some trace minerals. Since it is composed of all major fatty and amino acids, it is considered an excellent nutritional source. Hemp oil has also been seen to have desirable pharmacological effects. It has been useful for normal cerebral functioning because of its fatty acid composition (Gulluni, 2018) . In this study it was found that Cannabis sativa slows down the progression of Alzheimer’s disease by reducing the degeneration of aging brain cells. 

The oil has linoleic acid (LA) and a-linolenic acid (LNA) as its major omega-6 and omega-3 polyunsaturated (PUFA) fatty acids, respectively. The fatty acids make up the most desired contents of hemp oil. The fatty acids ratios of 3:1 (LA: LNA) are considered optimal for better nutrition (Leizer et al., 2000). Its nutritional benefits are considered exceptional compared to other seed oils because of the presence of gamma-linolenic acid (GLA). Some of the benefits associated with omega-3, PUFA, are anti-cancer, anti-thrombotic, and anti-inflammatory properties. Moreover, dietary omega-3 PUFA increases metabolic rates and enhances fat burning. The important role of LNA and LA is related to the end and intermediary products that result after several biochemical pathways. LA is metabolized to GLA and later converted to arachidonic acid (AA). On the other hand, LNA is metabolized into docosahexaenoic acid (DHA) and eicosa-pentanoic acid (EPA). AA and EPA are metabolized by the body to form eicosanoids. Eventually, these compounds become prostaglandins that affect various functions like blood clotting, immunoregulation, and inflammation response. 

Hemp seed oil has been reported to have cannabidiol (CBD). The contamination with cannabidiol traces has been identified to result from pressing the oil. Contamination from cannabinoids has been focusing mainly on delta-9-tetrahydrocannabinol (THC). The levels of THC are approximately 50ppm. THC and CBD are produced and stored in the plant’s glandular structures. CBD has a higher concentration than THC in the most hemp oil and fiber varieties (Leizer et al., 2000). Therefore, CBD concentration as a contaminant in hemp seed oil usually is more than THC concentration. The significance of CBD presence is from the reported anti-epileptic, antimicrobial, and anticonvulsive properties. Despite low levels of CBD in the oil, there are notable health benefits attributed to its presence. THC is responsible for the psychoactive effects (Atakan, 2012). The interaction between CBD and THC affects the brain's neurotransmitter release. Neurotransmitters send messages between the brain cells. THC binds to Cannabinoid 1 receptors (CBD1R), found at the terminals of peripheral and central neurons. After binding, THC acts in a nonselective manner to inhibit neurotransmitter’s release. Besides, it can enhance the release of acetylcholine, dopamine, and glutamate in some parts of the brain. This happens after preventing the release of inhibitory neurotransmitters, such as GABA, onto neurons that release acetylcholine, dopamine, and glutamate. THC produces hypothermia, verbal and spatial impairment of the memory, and hypoactivity. 

Terpenoid compounds are also present in hemp seed oil. Despite being in low amounts like CBD, these compounds have several health benefits. The terpene's presence is probably the result of contamination from glandular hairs when the oil is being processed. Some of the health benefits associated with the major terpenes are having anti-allergenic, cytoprotective, and anti-inflammatory properties (Leizer et al., 2000). 

Many studies have based the nutritional benefits of hemp seed oil as a factor in fatty acids' presence. However, other beneficial constituents like methyl salicylate and b - sitosterol complement the oil's nutritional benefits and increase its appreciation as a functional food. β-sitosterol reduces hypercholesterolemia and has antifungal, anti-inflammatory, and antiviral properties. Moreover, it has been identified that plasma cholesterol levels are affected by plant sterols. Cholesterol solubility in the intestinal lumen is reduced by phytosterols which exclude cholesterol from the micelles. This reduces absorption. Moreover, there is increased uptake competition between the cholesterol and sterols into the intestinal mucosa. Tocopherols are also a major constituent of the oil, and they have antioxidant properties. 

The research on hemp seed oil is still new, and therefore, many studies are underway, particularly in the United States. This research is being done to find more practical uses beyond the medical or pharmacological applications currently available. This can be attributed to the laws that have prevented the cultivation and utilization of Cannabis sativa in the country. This has also affected its research endeavors on its various other applications. A recent area of research is the application of Cannabis sativa to pesticide and insect repellent pharmacology. There has also been an increasing trust by the agricultural sector to botanical insecticides in eradicating insects and agricultural pests (Ona et al., 2021) . This is a significant topic of research. It has also received tremendous interest and support from Integrated Pest Management programs. Scientists have been working hard to understand the role of cannabinoids present in hemp seed oil as potential insecticide and repellent. This is because there has been a growing resistance of pests and insects to the conventional insecticides and pesticides currently in the market. This resistance can be partly explained by the mutation in RDL gene present in a majority of these pests. The RDL gene and encodes for GABA ligand-gated chloride channels. The mechanism of action of these insecticides is the inhibition of the central nervous system of various insects with structures such as cyclodienes and fipronil. The AsRDL protein in the RDL gene has 557 amino acid residues. This RDL subunit has four segments M1, M2, M3 and M4. Point mutations in the M2 and M3 provide a low sensitivity of the Binding site. This is the basis of insecticide resistance. This type of resistance has been manifested in Drosophila species and has resulted in resistance to cyclodiene and phenyl pyrazole insecticides in several species. The increase in resistance to the conventional insecticides by this mutation has led to the development of additional research on other ways of controlling agricultural pests and insects. 

This has led to increased research on the insect repellant properties of Cannabis Sativa. It has been used as an effective insect repellant when planted along with typical agricultural produce as a companion plant. Therefore, there is great interest in understanding the mechanism with which Cannabis sativa achieved this and which particular extracts were responsible. This is being studied in this report. This report aims to determine the significance of hemp seed oil from the Cannabis sativa plant on the physiology of insects, particularly Drosophila melanogaster fruit flies. 

Drosophila melanogaster is an excellent organism for genetics, genomics, and neurobiology research studies. They have many predators, including zebra jumping spiders and the Chinese praying mantids. The main anti-predatory mechanisms of Drosophila melanogaster species include abdominal lifting, stopping, and retreating ( Larkin, 2020). In the presence of predators, particularly jumping spiders, there is a significant increase in their activity. Wasps attack through an ambush. Wasps feed on Drosophila melanogaster fruit fly larva and feed on its thorax as meat. Parasitic wasps cause a high number of Drosophila melanogaster fruit fly deaths. Several mechanisms that the Drosophila melanogaster fruit flies employ to avoid wasp attack. These include retaining the eggs and laying them in a non-wasp infected area. The jumping spider actively hunts its prey and uses vision. The vision is extensive because of their simple eyes, which are anterior medial. This increases the field of view and makes it easy to spot the prey. In contrast, mantis ambushes their prey. It is hypothesized that Drosophila melanogaster carefully analyses the predatory mechanism, either reducing its activity with ambush predators or increasing with active predators. 

Drosophila melanogaster uses the antennae to hear. The approaching predator's sounds influence vibrations of the fly's distal antennal segment, and the fly has specialized mechanoreceptors that transduce the vibrations. The left and right antennae vibrate because of sounds from many azimuthal angles. The fly compares the signal from both antennae to know the sound source. Moreover, the flies have a non-tympanal auditory organ, arista, which helps them in directional hearing (Batchelor & Wilson, 2019). Natural and artificial predators initiate different reactions from Drosophila melanogaster . They mainly depend on visual cues to detect the predator and have different responses for different predators. For instance, in the presence of a pantropical jumping spider, the fly's activity increases. However, as the fly explores the surrounding, the activity reduces. If the fly detects a moving mock spider, the fly's activity remains high. This is an indication of anxiety responses that result after detecting a predator's threat. Drosophila melanogaster adults tend to run away after noticing a twin-flagged jumping spider ( de la Flor et al., 2017) . Moreover, Drosophila melanogaster visually detects a wasp's presence. The fly responds by changing the oviposition substrate presence or through apoptosis of oocytes. The fly uses the wings to alert the rest of the population about the wasp's presence. 

Some animals have exploratory behaviors that allow them to receive information about their surroundings. Many preys have natural reactions to defend themselves from the threats imposed by the predators. The responses depend on environmental cues, such as the availability of an escape and the predator's type and proximity. The perception of a particular threat initiates the escape behavior. Threat perception changes with experience. Drosophila melanogaster is a lower trophic level animal, and it is a prey of many predators like ants, wasps, spiders, among others. It is prey in the adult and larva growth stages. Being aware of the predators, the fly always leaves any area they deem dangerous. Typically, the general response of the fly after detecting a predator is typically freezing or startle-flight. Drosophila melanogaster is found in all continents and is the most common pest in homes, restaurants, and other places where food is served. It belongs to the family of Trephritidae , also called fruit flies. It is a model insect for the laboratory analysis of many other insects. It is also widely used to understand higher eukaryotes' molecular and development patterns ( Lemaitre & Hoffmann, 2007). It has been established that nearly 75% of diseases we have as humans have similar manifestations in Drosophila melanogaster and, therefore, have been widely used in biomedical research for cancer, cardiac, neurological, and kidney pathologies. 

Recently it has been the topic of interest to establish whether cannabinoids found in Canabis sativa plant can be used to formulate insect repellent substances. For a long period, Cannabis sativa used as a companion plant because of its repellent and pesticide properties. Other examples of such plants include garlic, castor, marigold, neem, and pyrethrum. Cannabis sativa was planted along with agricultural produce and have been considered effective as insect repellents. The Drosophila melanogaster fruit flies were preferred to carry out this experiment. The reasons for the choice of Drosophila melanogaster for this experiment are many. These fruit flies are small and can be managed easily. The care and maintenance are relatively cheap because they use little space and have simple nutritional requirements such as spoilt fruit. They are also anesthetized and manipulated with no specialized equipment ( Nørby, 2009). They are sexually dimorphic, with males significantly different from females, allowing easy differentiation. They also have a short generation time and do well at normal room temperature. 

This experiment establishes the effect that hemp seed oil has on the activity of fruit flies in response to predation. It seeks to show that hemp seed oil can reduce the activity of Drosophila melanogaster fruit flies even in the presence of both active predators and those that ambush. It will investigate whether the psychoactive compound cannabidiol can effectively increase the susceptibility of Drosophila melanogaster fruit flies to predation. It also seeks to examine how these cannabinoids in the diet of these insects affected their growth and development. 

Method 

The principle behind this experiment is the role of psychoactive compound cannabidiol present in hempseed oil. Cannabidiol has insecticidal properties and therefore is expected to reduce the activity even in the presence of predators. Different genotypic varieties of Drosophila melanogaster fruit fly express contrasting affinities for hemp seed oil in the diet. These flies will be expected to relax in response to predatory sounds. This study examines how Drosophila melanogaster species react under the influence of hemp seed oil. 

The experiment utilized 7.5 ml scoop of dry food, 12.5 ml of water and a dash of baker's yeast. Hemp seed oil was used in different proportions i.e., 0.02 ml, 0.1 ml, 0.2 ml and a control without any hemp seed oil. Initial ratios of 0.2 ml and 1 ml of hempseed oil were used. When 1 ml of hempseed oil was used, the flies died instantly, so the amount was limited. Wasps, yellow jackets, jumping spiders, and Chinese mantis were used as predators of flies. Because of limited time, only one predator from each experiment was used i.e., wasps and Chinese mantis. A slice record was obtained and constantly computed to see how they survived or reproduced. 

Four test tubes for the four genotypes types R+, R-, S+, and S – were used. 0.1 ml, 0.2 ml, and HSO 7.5 ml dry food and 12.5 ml water, and baker's yeast were added to the test tubes. Drosophila melanogaster flies were placed in a petri dish with predators close to the dish. Data such as the number of flies that died, the number of flies added, and the rate of reproduction were recorded roughly every minute with the predator sound with hemp seed oil and predator sounds without hemp seed oil. In this experiment, R- represents no hemp seed oil, R+ indicates the presence of hemp seed oil, S- indicates that there is no hemp seed oil and S+ indicates the presence of hemp seed oil. 

The results showed that the S+ is genotype did not live for more than a week. On the other hand, R-, S -, and R+ are reproduced and continued to survive. R- which had no hemp seed oil displayed little or no movement. As the concentration of hemp seed oil was increased, the flies' activity also increased correspondingly. The fly moved quickly around the dish in R+, 0.2 ml of hempseed oil, and attempted to escape. They responded similarly as they moved towards the sound and retracted and froze. Due to the little results of the S genotype, the flies did not move much and subsequently remain frozen for the rest of the duration. R(-) showed little or no movement whereas R(+) showed a variation of activity level based on the concentration of hemp seed oil that was added. When 0.02ml was added to R(+), there was a slight increase in movement with one moving towards the sound, then away and stayed still while the others stood still the entire duration of time When 0.1 ml was added to R(+), one moved towards the sound, and then eventually moved away. The other fly was slowly but steadily moving away from the sound. When 0.2 ml was added, They quickly moved away from the sound and try to escape the petri dish. In the S (+), it was observed that these flies were moving in circles. 

Discussion 

From the results, we can see the variation in the Drosophila melanogaster species responded differently with the administration of hempseed oil. We hypothesized that the cholesterol in hemp seed oil affected the S genotype's survival and reproduction, posing massive death and reduction in their number. Its genetic makeup could have caused it not to utilize the hemp seed oil-infused diet or could result from errors during the procedure. 

Hemp seed oil increased the activity of Drosophila melanogaster fruit fly as a response to predatory sounds, particularly R+. They were more anxious than when no hemp seed oil was added. A hypothesis proposed why the address of Drosophila melanogaster fruit flies became anxious when exposed to hemp seed oil is that this fly has its endogenous cannabinoids that made it responsive to the phytocannabinoids from the hemp seed oil ( Gómez et al., 2019). These endogenous cannabinoids are present in their hemolymph. However, the endogenous cannabinoids present in Drosophila melanogaster serve an immunological role. The consequence of cannabinoid intake was an increased rate of cardiac contractility in Drosophila melanogaster . This mechanism is synonymous to the effect of cannabis on the hearts of mammals. It is also noted that the mechanisms of calcium function did not change. However, an explanation could be the influence of these cannabinoids on the interaction between calcium and myofilaments (Khaliullina et al., 2015) This caused increased cardiac contractility and excitability, resulting in Drosophila melanogaster being anxious. The tubes where no hemp seed oil was added served as a control to explain the increase or decrease in the behavior of Drosophila melanogaster houseflies in the experiment. Our hypothesis in this experiment that Drosophila melanogaster fruit flies would be relaxed when a portion of hemp seed oil was added to their diet was proved wrong. They ran away from the sound, aggressively rubbing their wings together. Some even froze and did not move from the petri dish. All flies died when they were exposed to 1ml of hemp seed oil. Exposure to these levels became fatally toxic to its survival. 

The mechanism was that hemp seed oil provided nutrients that supported their growth and development. Steroids that are ecdysone's precursors are elevated in Drosophila melanogaster larva when exposed to hemp seed oil. Ecdysone is a steroid hormone secreted by the prothoracic gland that stimulates metamorphosis and regulates molting in insects in its active form. Other steroids from hemp seed oil, mainly hexane extracts, are also responsible for growth, reproduction, and development. 

Sterols found in hemp seed oil accelerate pupa development. They also increased the body weight and the length of their wings. It has also been established that hemp seed oil also contains components that affect insect development besides cholesterol. These include phytosterols and polyunsaturated fatty acids. However, phytosterol did not seem to increase the rate at which pupa developed, but it caused a delay. Phytosterols are usually converted to cholesterol in Drosophila melanogaster fruit flies. They contribute to their growth, development, and reproduction. Other studies have showed that phytosterols had more contribution to the growth and development of Drosophila melanogaster fruit flies than cholesterol (Lee, 2010) . This could explain the proliferation of Drosophila melanogaster fruit flies in the petri dish containing hemp seed oil. Therefore, it can be safely assumed that other substances present in Cannabis sativa were responsible for the repellant properties that it has. These substances were not present in hemp seed oil or in significantly lower amounts to cause the effect we were experimenting with. A recent study to explain this arrived at terpenoids as the major constituent of Cannabis sativa responsible for its insect repellant properties ( Benelli et al., 2018). This substance is present in hemp seed oil in extremely small quantities and, therefore, cannot influence these flies. 

We could have used carbon dioxide to put the flies to sleep to make it easier to transport them. Additionally, we could have used less water and separate it from the food we provided. If this experiment should be repeated, they could also test the effects of cholesterol on predatory sounds. Where possible, we could have included more Drosophila melanogaster fruit fly predators in order to determine whether it was a generalized response to all parasites, as opposed to only the test sample. We could have also allowed the food to dry properly before introducing the Drosophila melanogaster fruit flies to prevent any death not related to the tested parameters. 

Conclusion 

The effect of hemp seed oil on Drosophila melanogaster gave results that we had not hypothesized. Flies also became more anxious than relaxed with hemp seed oil administration. Also, the flies reacted to more wasps sounds than mantis sounds They also could not determine wild-type vs mutated flies since S+ kept dying with hemp seed oil in their food. Therefore, we agreed with the hypothesis that hemp seed oil helped in the growth and development of Drosophila melanogaster since R+ fruit flies reproduce tremendously, where is R- and S- also showed similar results but not to the same extent. This experiment establishes that hemp seed oil does not cause relaxation, especially in the presence of predator sounds. On the contrary, it increases their activity. Studies have also shown that it increases the rate of their growth and development (Lee et at.,2010). Therefore, the mechanism with which Cannabis sativa was presumed to repel various insects is not based on its cannabinoid content. Some studies suggest that it could be a result of terpenoids responsible for arthropod deterrence (Silva et al., 2021). However, aqueous and solvent distillations of cannabis contain lesser amounts of this substance and therefore explained why the results of our hypothesis were negative. It was impossible to determine the genotype variations because the results were inconclusive. The S+ genotype kept on dying when hemp seed oil was placed in their food. The reason could be that they have an inability to utilize the nutrients found in hemp seed oil for their growth and development. We also learnt that a maximum amount of 0.2ml of hemp seed oil can be used in this experiment. 

Tabulation of results. 

The diagram below shows variation in movement trajectories from the wasp assay. 

The diagram below shows variation in movement trajectories of flies to the sound of praying mantis.

The table below show the number of flies that counted that were present, died or reproduced in different concentrations of hemp seed oil in the presence of wasp sounds. 

0.2	R+	S+	R-	S-
11/11	3	4	4	5
11/16	Died replaced with 5	Died replaced with 3	4	5
11/18	4 one lost	3	4	5
11/23	3 one lost	Died replaced with 2	4	5
11/30	10+(more reproduced)	9 new flies put in	15+	15+
12/2	20+	6(3 died)	15+	15+

0.1	R+	S+	R-	S-
11/30	11	5	15+	15+
12/2	4 (7died)	All died	15+	15+

The table below show the number of flies that were present, died or reproduced in different concentrations of hemp seed oil in the presence of mantis sounds. 

0.2	R+	S+	R-	S-
11/16	6	2	4	5
11/18	3 lost	Died replaced with 3	4	5
11/23	3	3	4	5
11/30	10(more reproduced)	9 new flies put in	15+	15+
12/2	20+	All died	15+	15+

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