Tulsi and Neem Extract Effects on Mosquito Larvae
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The effect of Neem and Tulsi on mosquito larvae. The project is aimed at finding natural pesticides or bio-pesticides that effectively control the mosquito population by killing mosquito larvae. They could be used as alternatives to the current pesticides used that have adverse effects on the environment and other species besides the target pest. Mosquitoes are hosts for a very large number of diseases and therefore keeping larvae under control and disallowing the emergence of adult mosquitoes is essential. Two plant extracts have been tested; Neem and Tulsi. The initial hypothesis was that both extracts would have some effect on the larval development and cause mortality in the larvae due to the properties of both plants. The secondary initial hypothesis was that Neem extract would be significantly more effective in killing the larvae than Tulsi at the same percentage. Concentrations of 1% to 10% of each extract were prepared and 10 trials each performed with 10 larvae were undertaken. The samples were checked on after 12 hours and once again after 24 hours and the number of larvae killed in each case was recorded. The results were conclusive and both extracts were very effective at killing larvae at 10% with Neem killing an average of 10 larvae out of 10 and Tulsi 9.6 Â± 0.52. However t-values showed that the initial hypothesis stating that Neem would be more effective at all concentrations was incorrect and only 2%, 9% and 10% were significantly more effective in 24 hours with t values of 3.13, 2.92 and 2.43 respectively (> table value of 2.10). This means that in 2%, 9% and 10% concentrations, Neem should be used instead of Tulsi as it is significantly better. This is all very exciting as environmentally safer pesticides can be developed from these two plant extracts.
Word Count: 299 words
Research question: What are the effects of medicinal plants like Ocimum sanctum (tulsi) and Azadirachta indica (neem) extract on the mortality of Culex larvae?
The conservative pesticides are augmented to combat complicated pest problems in mosquitoes. In human health during the past few decades these pesticides played an important role in the inhibition of diseases caused by mosquitoes either by inactivating or killing these mosquitoes. Later on when these pesticides are getting accumulated in the environment proved to be hazardous to the environment and mankind. Frequent application of synthetic organic insecticides resulted into pest resistance and outbreak. Most of the insecticidal compound falls within four main classes namely organochlorides, organophosphates, carbamates and pyrethroids. Out of these the major classes of insecticides, the insecticides used in present situation are organophosphates and carbamates. There are problems of pesticides resistance and negative impacts on non-target organisms including man and the environment. 
Environment problems due to the insecticides and pesticides such as destruction of beneficial organisms, non-target pest, some of the pesticide residues in the host plant resulting in stunted grow or killing other organisms have come to an end. This work is basically taken up by some of the herbal plants. These herbal plants possess some of the active ingredients which are eco-friendly but simultaneously have a severe effect on the mosquito populations. 
Medicinal plants are the most promising source and under extensive trails for their biological activity against various mosquito larvae. During the last 10 to 15 years, interest in botanical insecticides has increased to look for substitutions for synthetic insecticides with those based on naturally occurring substances. The use of botanicals in pest management is not only useful for suppression of pest population but also helps to maintain the sound ecological balance. 
BOTANICALS IN USE
The groups of plant possessing insecticidal substances are enormous. More than 2400 species of the plants in India possess insecticidal properties3. In the middle of the 17th century, most of the economically important natural plant compounds obtained from the plants like neem, tulsi, adathoda, chrysanthemum, turmeric, garlic, tridax etc. are used in commercial insect control. Despite the relative safety of the well-known botanical insecticides, most of these substances have their drawback hindering large-scale application. The chemicals obtained from these plants are unstable in the sunlight and are rapidly metabolized thus limiting their potency and application. 
Botanical insecticides break down readily in soil and are not stored in animal and plant tissue. Often their effects are not as long lasting as those of synthetic insecticides and some of these products may be very difficult to find. The plant parts used for extraction or assay were the leaves, roots, tubers, fruits, seeds, flowers, the whole plant, bark, sap, pods and wood. The most commonly utilized parts were the leaves, roots. The plant families Asteraceae, Fabaceae and Euphorbiaceae contain most of the insecticidal plant species reported.
Recently several other plants viz. Neem, Adathoda, Chrysanthemum, Turmeric, Onion, Garlic, Ocimum, Ginger and some other plants have been reported as insecticidal plants which can be used in insecticide preparation. The leaf extracts of Tulsi (Ocimum basilicum, O. sanctum) and vetiver (Vetivera zizanoides) are useful in controlling leaf miners in potato, beans, eggplant, tomato, chilies, etc.
Among all the important insecticidal plants, Neem (Azadirachta indica) is the most promising source of bio-pesticide and its various formulations are extensively used for pest control.  Neem leaves, stems, seeds and oil have been used for pest control in sericulture.  The limonoids present in it and its products have made it a harmless to mankind while functioning as insecticide, bactericide, fungicide, pesticide etc. It is likely to provide a solution to many of pest and disease problem in sericulture.  Generally, extracts of plant leaves or seed are prepared and sprayed; otherwise, seeds are dried under sunshade, powdered finely and applied as dust.
More than 20 Neem based biopesticides are available in the market (Table 3). Neem gets biodegraded in a matter of weeks when exposed to sunlight or in soil. Neem products are highly photodegradable and normally degrade within a week. No problem of development of pest resistance and resurgence has been reported from neem products. Hence they have characteristics suitable for IPM strategy. Much of the information's are available on insecticidal properties of plants having some sort of toxic property against insects. The plant parts to be used should be removable: leaves, flowers or fruit and harvesting should not mean destruction of the plant.
Neem is perhaps the most well known of all herbs and has been used in ayurveda in India for centuries. The active ingredient in Azadirachta indica (neem) is azadirachtin. It a member of the Meliaceae family and is a botanical cousin of mahogany. Neem is very hard and virile. Neem is especially useful due to its climatic tolerance that allows it to be grown all over the world though it is usually found in tropical regions. It has insecticidal, antifeedant, growth regulating and development-modifying properties and has properties that make it attractive in insect control. It reduces fecundity and longevity as well as increased development time of immature insects. On insects it has been shown to act as a growth retardant and cause molting disorders, change of behavior and morphogenetic defects. Mosquito control is essential as they act as carriers for malaria, filariasis and a host of other diseases as well as being a nuisance. There is a high demand for a less environmentally harmful insecticide as the ones currently used have high neurotoxic effects. Recent studies have also demonstrated neem-induced effects on vitellogenesis and severe degeneration of follicle cells during oogenesis in mosquitoes. It also has several medical properties such as a cure blood morbidity, biliary afflictions, itching, skin ulcers, burning sensations and pthysis. It is also an effective cure against ringworm, eczema and scabies. In English the tree is known as margosa and attains a maximum height of 40 to 50 metres.
Tulsi is an aromatic plant in the Lamiaceae family. Tulsi has been used for thousands of years in Ayurveda due to its diverse healing properties. Tulsi's extracts are used in ayurvedic remedies for common colds, headaches, stomach disorders, inflammation, heart disease, various forms of poisoning, and malaria. Traditionally, tulsi is taken in many forms: as herbal tea, dried powder, fresh leaf, or mixed with ghee. Essential oil extracted from Karpoora Tulsi is mostly used for medicinal purposes and in herbal cosmetics, and is widely used in skin preparations due to its anti-bacterial activity. For centuries, the dried leaves of Tulsi have been mixed with stored grains to repel insects. 
These plants in harmonious integration with other safe methods of pest control like biological control can provide eco-friendly and economically viable solutions for pest problems in near future.
Plants producing the compounds having insect growth regulators (IGR), feeding deterrents, repellents and confusants activities are known by the farmer because most of the time they grow in the same general area. Some of these products may be these products act very quickly inhibiting insect feeding even though long term they do not cause insect death. Since most of these products have a stomach action and are rapidly decomposed they may be more selective to insect pests and less aggressive with natural enemies. Most of these compounds are not phytotoxic and have rapid action and low toxicity to mammals and plants. Resistance to these compounds is not developed as quickly as with synthetic insecticides.
Most of these products are not truly insecticides since many are merely insect deterrents and their effect is slow. They are rapidly degraded by UV light so that their residual action is short and breakdown is rapid, requiring more precise timing of and/or more frequent application. Not all plant insecticides are less toxic to other animals than the synthetic ones. They are not necessarily available and sometimes cost is more. Most of them have no established residue tolerances and there is lack of test data and sometimes lack of state registration of some materials. There are no legal registrations establishing their use. Not all recommendations followed by growers have been scientifically verified. The above statements are altered if you find anything that can be added or deleted you can do it.
Insects adapt themselves to aquatic habitats inspite of their terrestrial origin. The aquatic and semi-aquatic insects are remarkable for their diversity of forms, reflecting adaptation to a wide variety of niches such as salt water pools, saline ponds, hot springs, high mountain lakes, large rivers, temporary and permanent ponds.
Mosquitoes are known as vectors of the pathogens causing human diseases. They belong to the order Diptera; the true flies. Like all flies they have two wings but unlike other flies they have scales and the females have sucking proboscis. There are over 2500 species of mosquitoes. Culex mosquitoes are known to be painful and persistent biters and are a nuisance.
The reason why I chose this topic is because the pesticides that are currently used in pest control contain chemicals that have diverse side effects on the environment and organisms other than the target pest. I decided to use natural substances found in nature and see if they would work as a form of pest control instead. I have used two extracts; Neem and Tulsi, in order to see which one was more effective in killing these mosquitoes. Neem, Tulsi and eucalyptus have been used as they are prevalent in the tropical and sub-tropical countries of Africa and Asia with a large mosquito population and there have been significant prospects of being used as an insecticide on a commercial scale due to their useful properties.
In early literature mentioned above it was noted that the mosquitoes inhibit both temporary and fresh water stagnant bodies and they are found in abundance causing nuisance to the human population. The aim of the present study was undertaken on the following aspects
- To determine the number of Culex larvae killed in 12 hours and 24 hours using different plant extracts like Neem and Tulsi in crude form.
- To determine the number of Culex larvae killed in 12 hours and 24 hours by Neem and Tulsi under various concentrations (1%-10%).
- To determine the statistical analysis t - tests were performed.
Materials and Method
Materials / Equipments
250 ml beaker, Mortar and pestle, Pipette, Stirrer, Water, Fishing net (for catching larvae), Dropper, Watch glass, Petri dishes.
For the present study, the larvae of Culex mosquitoes were used throughout the investigation. These larvae were chosen because they were most abundant in the water bodies which are very active fast wriggling movement. The Culex larvae were collected from stagnant water bodies at Varthur Lake in Bangalore (fig.1) using a hand net. They were transported in plastic buckets containing clean water to the lab. Larvae were categorized based on their size as large and small. The large sized Culex larvae were about 0.7 cm and the small sized Culex larvae measured about 0.2cm in length. For the present study I have used only large sized larvae (3rd and 4th instars stage).
Preparation of Stock solution:
Azadirachta indica: [Neem]
I collected neem leaves from my school campus. Only young leaves were collected and dried in the absence of sunlight in the shady region. The complete method of preparing 10% stock solution of this plant extract is mentioned below. 10 grams of dried young leaves were then grinded using mortar and pestle along with methanol and dried. Then add 100 ml of distilled water to make out 10% stock solution. Then the 10% neem stock solution was decanted after centrifugation. Now from this 10% stock solution different concentration solutions in percentage were prepared [ranging from 1% - 10%] i.e., 10ml of 10% stock solution in 90ml of distilled water gives 1%, 20ml of 10% stock solution in 80ml of distilled water will give 2%, 30ml of 10% stock solution in 70ml of distilled water will give 3%, 40ml of 10% stock solution in 60ml of distilled water will give 4%, 50ml of 10% stock solution in 50ml of distilled water will give 5%, 60ml of 10% stock solution in 40ml of distilled water will give 6%, 70ml of 10% stock solution in 30ml of distilled water will give 7%, 80ml of 10% stock solution in 20ml of distilled water will give 8%, 90ml of 10% stock solution in 10ml of distilled water will give 9%, and for 10% the stock solution itself was used.
Ocimum sanctum: [Tulsi]
I collected Tulsi leaves from my school campus. Only young leaves were collected and dried in the absence of sunlight in the shady region. The complete method of preparing 10% stock solution and the different concentration of this plant extracts in percentage was done by same method as mentioned above in Neem.
Method / Procedure
Mortality of larvae using crude plant extracts:
- The large sized Culex larvae were taken in the petridishes.
- 5 ml of crude plant extracts of neem and tulsi were taken in the different test tubes.
- 10 larvae were introduced in each test tube at the same time.
- Ten trails were carried out for the two different plant extract.
- The number of larvae killed was recorded for 1hr, 12hr and 24 hr.
- Larvae were feed with dog biscuit powder.
- Comparative analysis was carried out graphically between the two plant extracts at different time period.
Mortality of larvae using different concentration of plant extracts:
The large sized Culex larvae were collected and separated in the petri dishes.
5 ml of 1% Neem extract was pipetted in the test tube using a graduated pipette.
10 Culex larvae were introduced and the time was noted.
Ten trials were carried out at the same time for different concentration.
The larval mortality was recorded for 12 hours and 24 hours.
The same procedure was followed for different concentrations [2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% & 10%] of the Neem extract.
During this experiment the larvae was feed with dog biscuits in powdered form.
The above mentioned procedure was also carried out using Tulsi plant extract.
A comparative study of larval mortality was made by using these two plants extract on the Culex mosquito larvae.
The percentage of mortality (which will give us an indication of the effectiveness of the extract) will be calculated using the following formula.
Percentage of mortality = X 100
Dependant variable- Number of larvae killed
Independent variable-Different concentration of neem extract and tulsi extract, different time period [1hr, 12 hrs, 24 hrs]
Controlled variable-Number of larvae, Volume of water, Temperature, food
Null Hypothesis (Ho): Neem and Tulsi have no significant the mortality of the Culex larvae.
Positive hypothesis (H1): Neem and Tulsi have a significant effect on the mortality of the Culex larvae.
Null Hypothesis (Ho): There is no significant difference in the rate of mortality between Neem and Tulsi plant extracts.
Positive Hypothesis (H1): There is a significant difference in the rate of mortality between Neem and Tulsi plant extracts.
5.1 Mortality of larvae using crude plant extracts
The Culex larvae were exposed to different plant extracts in crude form to study the mortality rate. The number of Culex larvae killed was recorded after 1 hour, 12 hours and 24 hours separately (Table 1).
In one hour the mean number of larvae killed by Neem was 4.9Â±0.74 and for Tulsi it was 3.5Â±1.08. As time progresses the difference in effectiveness becomes less and the two extracts are approximately equal. The crude extract of Neem was more effective than Tulsi in one hour duration. At 12 hours the mean number of mosquitoes killed were 9.2Â±1.14 and 8Â±1.05 respectively. At 24 hours the Neem and Tulsi extracts showed almost the same level of effectiveness with mean values of 10 and 9.7Â±0.48 respectively.
The crude form of Neem extract was significantly more effective than Tulsi in killing the Culex larvae in 1 hour and 12 hours. However in 24 hours there is no significant difference in the effectiveness of Neem and Tulsi plant extracts.
5.2 Mortality of larvae using different concentration of plant extracts:
The mortality of the larvae using different concentrations of Neem extract was recorded. At 1% concentration the mean number of larvae killed was noted to be 2.4 Â± 0.52 in 12 hours and 3.5 Â± 0.53 in 24 hours. At 10% concentration the highest mortality rate was recorded as 9.6 Â± 0.52 and 10 for 12 hours and 24 hours respectively. As the concentration of the Neem extract increased, the mortality of the larvae also steadily increased (Table 2).
The mortality of the mosquitoes for Tulsi extract was recorded in the same way as Neem. At 1% concentration the mean number of larvae killed was noted to be 1.8 ± 0.63 in 12 hours and 3 ± 0.67 in 24 hours. At 10% concentration the highest mortality rate was recorded as 8.7 ± 0.67 and 9.6 ± 0.52 for 12 hours and 24 hours respectively. As the concentration of the Tulsi extract increased, the mortality of the larvae also steadily increased (Table 3).
A t-test was performed for the values obtained for the comparison of the crude extracts. The t-test values for crude Neem and Tulsi extracts are effective if seeing if Neem is significantly more effective than Tulsi in a certain time period. The calculated t value for 1 hour is 3.38. This is higher than the table t value of 2.10. Hence in 1 hour Neem is significantly more effective in killing larvae. In 12 hours the calculated t value is 2.45 and therefore we can draw the same conclusion. However in 24 hours the calculated t value in 1.96 and thus there is no significant difference in the effectiveness of Neem and Tulsi. By this point it is evident that, as far as Hypothesis 1 is concerned, that both Neem and Tulsi are effective in killing mosquito larvae. With regard to the crude extract we can say that in 1 and 12 hour periods, Neem is more significantly more effective but not in 24 hours (Hypothesis 2).
T-tests were also performed to compare the significant difference in mortality of Neem and Tulsi extracts at different concentrations. Separate t-tests were performed for 12 hours and 24 hours.
As seen in the tables above the t-values are significant in 12 hours for 1%, 2% and 3%. They are significant as at these concentrations the calculated t values are greater than the table t value of 2.10. It is also observed in 9% and 10% concentrations in 12 hours. In 24 hours significance is observed in 2%, 9% and 10%. What these means is that for these concentrations in their respective time periods, Neem is significantly more effective than Tulsi in killing larvae.
The main effect that Azadirachta indica has on larvae is the growth regulatory effect. It is because of this property that Neem acts as an excellent natural insecticide. Exposure of culex larvae to sub lethal doses prolongs the larval development and causes reduced pupal weight and oviposition. Neem works by intervening at several stages of the insect's life. The ingredients present in Neem are approximately the same shape and structure of vital hormones for the insects. The larvae absorb these Neem compounds as if they were real hormones which blocks their endocrine systems. This leaves the insects so sonfused in brain and body that they cease to reproduce and thus the population plummets.
Tulsi extract exhibits high mortality, especially during the molting process. The molting of larvae takes place under the influence of the ventral nerve cord neurosecretory cells. These cells release the tanning hormone. The extract may have an inhibiting effect on such cells. They may also act on epidermal cells that produce enzymes necessary for the circular oxidation process.
Both plant extracts affect reproduction and inhibit the emergence of adult mosquitoes from their larvae. They also decrease the feeding time for larvae and cause less food to be ingested. This has been noted to cause a fall in the carbohydrate levels of the larvae. A study noted that the extracts cause the amount of DNA and RNA in the larvae to fall thus suggesting that the extracts may affect nucleic acid synthesis. The feeding in the larvae may have decreased due to indigestion caused by the plant extracts as they inhibit metabolic processes. 
Both Ocimum sanctum and Azadirachta indica are available easily and in large quantities. They do not require professional handling, are inexpensive and safe which makes them very valuable as pesticides. Additionally Tulsi has properties that enable it to disinfect water. The extracts could help replace harmful pesticides used such as DDT as they are safe for non target animals and do not pose residue problems but are still effective in killing larvae and suppressing the adult mosquito population. In conclusion further effort should be taken to produce bio-pesticides from Neem or Tulsi as well as research to find other plants products that may be more ideal. UV rays may affect the Neem and Tulsi extracts thus making them ineffective so research should be done in order to find plant products that do not degrade in the presence of sunlight thus making them completely versatile.
The findings have important implications in the practical control of mosquito larvae, especially, in a polluted aquatic environment.
Ocimum sanctum and Azadirachta indica are the two plants out of which the extracts were prepared.
Solutions of concentrations varying from 1% to 10% were prepared from the stock solution for each plant.
Each concentration was tested on 10 larvae. 10 trials were undertaken and the results were noted.
Both plants were deemed effective as far as larval mortality in concerned. Neem extract had a slightly higher mortality rate.
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