Oregano (Plectranthus amboinicus) and Lemongrass (Cymbopogon) as Primary Ingredients for Liquid Mosquito (Culicidae) Larvicide

10 persons. Mosquitoes


Introduction
When you think of an organism who kill the most people a year, you think of a Lion, Sharks, and Snakes, but the animal who killed the most humans (except we humans ourselves) are tiny insects known as mosquito, mosquitoes kill at least 725,000 people every year whereas snakes kill an estimated 50,000 and sharks a mere 10 persons.Mosquitoes are one include dengue, which causes 50 to 100 million cases per year worldwide, yellow fever, which has a high mortality rate, or Japanese encephalitis, which causes more than 10,000 deaths per year, mostly in Asia.Not to forget Zika virus, with its recently described devastating and long-term neurological effects in babies born to infected mothers (Mosquitoes: World's Deadliest Animal, 2017).
Depending on situation, people are using mosquito repellents that work by blocking mosquito's sense of smell and prevent them on finding their target, or larvicides that can kill mosquito larvae and hatchlings before it matures and transform into a mosquito.However, some chemicals that are used in repellent and larvicides have a potential hazard for other organisms and the environment.The researchers would like to study the natural extract of the given herbal plant Oregano (Plectranthus amboinicus) and Lemongrass (Cymbopogon) that are commonly found in the backyard of homes if it can be a natural larvicide and in what concentration of it has the highest mortality rate on mosquito larvae.
According to the World Health Organization (2020), the WHO Global report on insecticide resistance in malaria vectors: 2010-2016 showed that resistance to the 4 commonly used insecticide classespyrethroids, organochlorines, carbamates, and organophosphatesis widespread in all major malaria vectors across the WHO regions of Africa, the Americas, South-East Asia, the Eastern Mediterranean and the Western Pacific.Over a period, mosquito will develop a resistance into the chemical we are using or the decreased susceptibility of a pest population to a pesticide, it is a big concern for the effect of insecticide along with its toxicity hazard to another organism and environment.
Extracts from plants may be alternative sources of mosquito egg and larval control agents, since they constitute a rich source of bioactive compounds that are biodegradable into non-toxic products and potentially suitable for use in control of mosquito larvae.
In fact, many researchers from A comparative Study of the Larvicidal Activity of Lemongrass (Cymbopogan citratus) from Different Methods of Extraction (2019), have reported on effectiveness of plant extracts or essential oils against mosquito larvae, such as Cymbopogan citratus (lemongrass).Lemongrass is extremely popular and used for medicinal, food and insect repellent products.The lemongrass oils are used in cosmetics, soaps, perfume, dyes, and odorizes along with thousands of other products.Lemongrasses are extremely safe and are listed on the EPA'S GRAS list (Generally Regarded as Safe), unlike other insecticides containing chemical compounds like DEET (N,NDiethlymetatoluamide) used as an active chemical ingredient in insect repellent.
Using mosquito larvicide is a better option to control the population of mosquitos than using repellent.Compared to repellent, Larvicide is applied to wet places where mosquitoes can lay their egg, killing its larvae (wriggles) before they mature into a mosquito reproduce, and become a vector for diseases.
The study aims to answer, will oregano and lemongrass solution be an effective mosquito larvicide?Specifically, it aims to answer the following questions: 1. Which of the following composition will have the highest mortality rate?1.1) 40% of 30 mL Oregano extract, 60% of 30 mL Lemongrass extract.
2. How much amount of the larvicide solution will it take before all larvae in the set-up container(s) dies? 3. How long will it take for the different larvicide solutions made of lemongrass and oregano to take effect?

Methods
Researchers used experimental research design to know if the extract from Oregano and Lemongrass is effective as a larvicidal solution.The elements that remained constant throughout the research or the control group are the water level wherein the larvae has hatched and experimented with, dropper, the mosquito larvae, and the amount of solution that was used in the experiment.Also, the independent variable is the ratio of the two (Oregano, and Lemongrass) extract.Meanwhile, the dependent variables are the Mortality rate and duration of effectivity.The materials that were used by researchers for the larvicide are blender, kettle, container, droppers, strainer, syringe, and plastic cup(s).While the ingredients used for the larvicide are Oregano and Lemongrass which are divided into three (3) different solutions.The three (3) different solutions are 40% of 30 mL Oregano extract and 60% of 30 mL Lemongrass extract, 50% of 30 mL Oregano extract and 50% of 30 mL Lemongrass extract, and a 40% of 30 mL Lemongrass extract and 60% of 30 mL Oregano extract.The researchers prepared the materials for getting the extract of oregano.First, is to wash your ingredients which are the Oregano and Lemongrass then let it dry for a minute.Second, put the oregano into the blender and grind it until it turns to liquid.After you liquefied the Oregano use a strainer to separate the small pieces of leaves to the extract and set asiIn getting the Lemongrass extract the experimenter will use different ways compared to getting the Oregano extract.First, is to slice the Lemongrass into small pieces and slightly crush it before putting in the kettle/casserole with five (5) cups of water then put it on fire for one (1) to (2) hours.Pour the extract on a clean container and let it cool.Prepare the paper cup that will serve as the container for the mosquito larvae.Use the dropper to safely separate the larvae from others and set aside.In every container there will be twenty (20) pieces of mosquito larvae.Next, prepare the first vessel that will contain 60% Lemongrass oil and 40% Oregano extract.In the second vessel mix 50% of Lemongrass oil and 50% of Oregano extract.For the third vessel blend 40% of Lemongrass oil and 60% of Oregano extract.Once

Result and Discussion
This chapter shows the investigation and observation of Lemongrass and Oregano as alternative larvicide solutions.A total of two (2) set-ups.In set-up one (1) there are four (4) containers including the two (2) control groups per set-up which is sixty (60) mL of water.The second set has four containers consisting of experimental and control groups.The corresponding analysis and interpretation of the data is also included in this chapter.The study of Chandra, Chowdhury, N., & Ghosh, A., (2012) stated that active toxic agents that are present in plants have been used since ancient times to control mosquito breakout.Non-toxic agents that are cheap and eco-friendly.Since there are synthetic chemicals in the commercials larvicide that can also damage other living organisms near it and the environment, people have been studying the use of natural active agents that can be found in plants.
The experiment consists of two (2) experimental groups (Experimental Group Set 1 & Experimental Group Set 2) both group sets will be conducted with three (3) set-ups (Set-up A, Set-up B, Set-up C) to get a precise and accurate result.The experiment also has a control group where the researchers will not apply any solution and will be the standard to which comparisons are made on this experiment.All of it contains the same amount of mosquito larvae (20) alive mosquito larvae and the same volume of water (60 ml), the only thing that is subjected to change is the ratio of concentration of the solution (Oregano and Lemongrass Extract).As shown on the graph in Set A container one (1) eighty-five percent (85%) mortality rate, container two (2) has zero percent (0%) mortality rate, and container three (3) has eighty percent (80%) mortality rate.Meanwhile, in set B container one (1) the mortality rate was zero percent (0%), container two (2) had zero percent (0%) mortality rate unlike container three (3) which has sixty percent (60%) mortality rate.The study of Chandra, G., Chowdhury, N., & Ghosh, A., (2012) stated that active toxic agents that are present in plants have been used since ancient times to control mosquito breakout.Non-toxic agents that are cheap and eco-friendly.Since there are synthetic chemicals in the commercials larvicide that can also damage other living organisms near it and the environment, people have been studying the use of natural active agents that can be found in plants.
The experiment consists of two (2) experimental groups (Experimental Group Set 1 & Experimental Group Set 2) both group sets will be conducted with three (3) set-ups (Set-up A, Set-up B, Set-up C) to get a precise and accurate result.The experiment also has a control group where the researchers will not apply any solution and will be the standard to which comparisons are made on this experiment.All of it contains the same amount of mosquito larvae (20) alive mosquito larvae and the same volume of water (60 ml), the only thing that is subjected to change is the ratio of concentration of the solution (Oregano and Lemongrass Extract).As shown on the graph in Set A container one (1) eighty-five percent (85%) mortality rate, container two (2) has zero percent (0%) mortality rate, and container three (3) has eighty percent (80%) mortality rate.Meanwhile, in set B container one (1) the mortality rate was zero percent (0%), container two (2) had zero percent (0%) mortality rate unlike container three (3) which has sixty percent (60%) mortality rate.
Looking back to the study of Shah G, Shri R, Panchal V et al. (2011), they stated that Lemongrass (Cymbopogon) has been used in the past year as insecticide/ pesticide and repellents against mosquitoes.According to the study, they used different kinds of mosquito species and produced the result that 95% of the species was repelled due to the strong odor that the Lemongrass has.As stated by Arumugam, G., Swamy, M. & Sinniah, U. ( 2016), Oregano has a distinct odor that can be used as pesticide/insecticides and repellents; aside from that it also has excessive number of bioactive compounds that are commonly the main components for antibacterial and anti-fungal effects.
In Experimental Group Set one (1) the result varied in every Set-up, in set-up A where the solution concentration is sixty percent (60%) Lemongrass and forty percent (40%) Oregano extract and has a total number of twenty (20) larvae, where seventeen (17) pieces of larvae or eighty-five percent (85%) of larvae died.In Set-up B where the concentration of the solution is forty percent (40%) Lemongrass and Sixty percent (60%) Oregano extract and has a total number of twenty (20) larvae, where there is no death recorded or zero (0%) of larvae died.
. In addition, at the first ten (10) to thirty (30) minutes the specimen stops moving and can make them mistakenly dead, but after one (1) to two (2) hours they will start moving again.Moreover, the researchers ensure that the specimen is regularly given a food and are placed in a cool place to make sure that there is no other cause of their death.
It shows that in set-up A that the solution concentration of the Lemongrass is sixty percent (60%) and (40%) Oregano extract were seventeen (17) out of twenty (20) larvae died and the remaining three (3) survived in three (3) days.
The experiment revealed that Oregano and Lemongrass larvicide solution is not enough to reach what this study aims for.Also, experiment results indicates that Oregano and Lemongrass solution is not enough to kill the larvae of mosquitoes.
Therefore, the researchers conclude that the oregano (Plectranthus amboinicus) and lemongrass (Cymbopogon) larvicide solution is not effective as an alternative mosquito (Culicidae) larvicide solution.
The following recommendations are presented.
Recommendations for future researchers.
1. To use a different species of mosquito because the researchers only used Aedes aegypti mosquito eggs in the experiment.

Improve the solution by adding more ingredients
that can help to add to the effectiveness of the solution.
3. Explore using the solution on several types of insects especially for pests to test if the solution will also be effective aside mosquitoes. 5. Recommendations for people who will use the solution.
6. Using the solution may not kill all the mosquito larvae because the researchers only used Aedes aegypti in the experiment.
7. The effectiveness will only be effective if the solution will cover the mosquito larvae.
8. The continuous feeding of larvae may weaken the effectiveness of the solution, so it is recommended to use more.

done preparing the three ( 3 )
types of solution the experimenter can now proceed with the testing part of the experiment.The researchers used the percent mortality rate by which the researchers can obtained by dividing the number of dead larvae by the sample size of larvae and multiply by 100 percent.% Mortality Rate = (M ÷ n) x 100 Wherein: n= sample size of the larvae M= number of the dead larvae

Fig 1 :
Fig 1: Mortality Rate of three (3) different containers of both set A and set B.

Fig 2 :
Fig 2: Amount of solution taken before the larvae die in different containers of set A and set B.In Set-up C where the concentration of the solution is fifty percent (50%) Lemongrass and fifty percent (50%) Oregano extract and has a total of twenty (20) larvae, where 16 or eighty percent (80%) of larvae died.In the 2nd Set of Experimental Group where the experiment will be repeated the results are: In Set-up A where the solution concentration is sixty percent (60%) Lemongrass and forty percent (40%) Oregano extract and has a total number of twenty (20) larvae, all of them remain alive or zero percent (0%) of larvae died.In Set-up B where the concentration of the solution is forty percent (40%) Lemongrass and Sixty percent (60%) Oregano extract and has a total number of twenty (20) larvae, all of them remain alive or zero percent (0%) of larvae died.In Set-up C where the concentration of the solution is fifty percent (50%) Lemongrass and fifty percent (50%) Oregano extract and has a total of twenty (20) larvae, where 17 or eighty-five percent (85%) of larvae died and three (3) remain alive.According to the article of New Tech Bio's article Lemongrass is widely as medicinal plant, and insect repellent; and as stated in the study of Shah G, Shri

Fig 3 :
Fig 3: Duration of Different Solution to take effects

4 .
Do multiple trials to test the effectiveness of the solution.

Table 1 :
Research Design Table