Abstract
Objective
To evaluate the larvicidal and pupicidal potential of the methanolic extracts from Moringa oleifera (M. oleifera) plant seeds against malarial vector Anopheles stephensi (A. stephensi) mosquitoes at different concentrations (20, 40, 60, 80 and 100 ppm).
Methods
M. oleifera was collected from the area of around Bharathiar University, Coimbatore. The dried plant materials were powdered by an electrical blender. From each sample, 100 g of the plant material were extracted with 300 mL of methanol for 8 h in a Soxhlet apparatus. The extracts were evaporated to dryness in rotary vacuum evaporator to yield 122 mg and 110 mg of dark greenish material (residue) from Arcang amara and Ocimum basilicum, respectively. One gram of the each plant residue was dissolved separately in 100 mL of acetone (stock solution) from which different concentrations, i.e., 20, 40, 60, 80 and 100 ppm were prepared.
Results
Larvicidal activity of M. oleifera exhibited in the first to fourth instar larvae of the A. stephensi, and the LC50 and LC90 values were 57.79 ppm and 125.93 ppm for the first instar, 63.90 ppm and 133.07 ppm for the second instar, 72.45 ppm and 139.82 ppm for the third instar, 78.93 ppm and 143.20 ppm for the fourth instar, respectively. During the pupal stage the methanolic extract of M. oleifera showed that the LC50 and LC90 values were 67.77 ppm and 141.00 ppm, respectively.
Conclusions
The present study indicates that the phytochemicals derived from M. oleifera seeds extracts are effective mosquito vector control agents and the plant extracts may be used for further integrated pest management programs.
Keywords: Moringa oleifera, Anopheles stephensi, Insecticide, Larvicide, Pupicide, Malaria, Phytochemical, Mosquito vector control, Plant extract, Larvicidal activity
1. Introduction
Vector-borne diseases, such as malaria, filariasis, dengue and hemorrhagic fever (DHF), are still major public health problems in the Southeast Asian countries because of their tropical or subtropical climate. Also owing to poor drainage system, especially during rainy seasons, the presence of many fish ponds, irrigation ditches and the rice fields provide abundant mosquito breeding places. Malaria and other vector-borne diseases contribute to the major disease burden in India.
Repeated use of synthetic insecticides for mosquito control has disrupted natural biological control systems and led to resurgences in mosquito populations. It has also resulted in the development of resistance[1], undesirable effects on non-target organisms, and fostered environmental and human health concern that initiates a search for alternative control measures[2]. Plants are considered as a rich source of bioactive chemicals and they may be an alternative source of mosquito control agents[3].
Plant products have been used by traditionally human communities in many parts of the world against the vectors and species of insects. The phytochemicals derived from plant sources can act as larvicides, insect growth regulators, repellents and ovipositional attractants and have deterrent activities observed by many researchers[4]. Repellents have an important place in protecting man from the bites in insect pests. An effective repellent will be useful in reducing man vector contact and in interrupting disease transmission. A repellent compound should be toxic, non-irritating and long lasting. Amides, imides, esters and other polyfunctional compounds are known to be good repellents[5]. Plants could be an alternative source for mosquito repellents because they constitute a potential source of bioactive chemicals and typically are free from harmful effects[6]. Because of this, much interest has been focused on plant extracts, or plant essential oils as potential mosquito repellent agents[7],[8] and studied the interactive effect of botanicals (Neem, Pongamia) and Leucas aspera, Bacillus sphaericus against the larvae of Culex quinquefasciatus.
Moringa oleifera (M. oleifera) is the most widely cultivated species of a monogeneric family-the Moringaceae, native to the sub-Himalayan tracts of India. This rapidly-growing tree (also known as the horseradish tree, drumstick tree, benzolive tree, kelor, marango, mlonge, moonga, mulangay, nebeday, saijhan, sajna or Ben oil tree), was utilized by the ancient Romans, Greeks and Egyptians, and it is now widely cultivated and has become naturalized in many locations in the tropics. All parts of the Moringa tree are edible and have long been consumed by humans. In the West, one of the best known uses of Moringa is to flocculate contaminants and purify drinking water with its powdered seeds[9]–[11]. This tree has in recent times been advocated as an outstanding indigenous source of highly digestible protein, Ca, Fe, Vitamin C, and carotenoids suitable for utilization in many of the so-called “developing” regions of the world where undernourishment is a major concern. In the present study an attempt was made to evaluate the toxicity of M. oleifera on malarial vector, Anopheles stephensi (An. stephensi).
2. Materials and methods
2.1. Plant collection and preparation of plant extract
The plant M. oleifera was collected from the area around Bharathiar University, Coimbatore. The dried plant materials were powdered by an electrical blender. From each sample, 100 g of the plant materials were extracted with 300 mL of methanol for 8 h in a Soxhlet apparatus. The plant extracts were evaporated to dryness in rotary vacuum evaporator to yield 122 mg and 110 mg of dark greenish material (residue) from Arcang amara and Ociumum basilicum, respectively. One gram of each plant residue was dissolved separately in 100 mL of acetone (stock solution) from which different concentrations, i.e., 20, 40, 60, 80 and 100 ppm were prepared.
2.2. Test for larvicidal activity[12]
An. stephensi was used to test the the larvicidal and pupicidal activity of M. oleifera. It was maintained at (27±2) °C, (75%-85%) RH, under 14 L: 10D photoperiod cycles. The larvae were fed with dog biscuits and yeast at 3:1 ratio. Twenty-five I, II, III and IV instar larvae and pupae of An. stephensi were kept in 500 mL glass beaker containing 249 mL of dechlorinated water and 1.0 mL of desired plant extract concentration. Three replicates for each concentration were set up. A control was set up with 1.0 mL of acetone in 249 mL of dechlorinated water. The control mortality was corrected by Abbott's formula[13] and LC50, LC90, regression equation, and 95% confidence limit of lower confidence limit (LCL) and upper confidence limit (UCL) were calculated by using probit analysis[14].
2.3. Pupicidal activity
A laboratory colony of mosquito pupae was used for pupicidal activity. Ten freshly emerged pupae were introduced into each testing cup (sterilized plastic drinking cup of 150 mL capacity), which contained 100 mL of dechlorinated tap water. A measured volume of stock solution was added to obtain the desired concentrations. Experiments were carried out with a series of five-seven concentrations, 20%, 40%, 60%, 80%, and 100%, respectively, each with 5 replicates and a final total number of 100 pupae for each concentration. The LC50 and LC90 were determined by a probit analysis program[14]. Control mortality was accounted by the formula of Abbott's[13].
2.4. Repellent activity
Repellent activity of plant compounds was tested with human volunteers. For the repellent activity of plant extracts percentage protection in relation to dose method was adopted[7],[12]. Three to four days old blood starved female adult mosquitoes (100) were kept in a net cage. The arms of the tested person were cleaned with isopropanol. After air-drying the arm only 25 cm2 of the dorsal side of the skin on each arm was exposed, the remaining area being covered by rubber gloves.
The plant extract was dissolved in isopropanol and the alcohol served as control. The plant extract at 0.5, 1.0 and 2.0 mg/cm2 concentrations was applied. The control and treated arms were introduced simultaneously into the cage. The number of bites was counted over 5 min every 60 min, from 20:00 to 6:00. The experiment was conducted five times. The percentage protection was calculated by using the following formula.
 |
T = the number of mosquitoes collected from treated areas.
2.5. Smoke toxicity test
M. olifera seed extract was used for smoke toxicity assay. The mosquito coils were prepared following the method of Saini et al[15] with minor modification by using 4 g of coconut shell, charcoal powder as burning material. All the three was thoroughly mixed with distilled water to form a semisolid paste. Mosquito coils (0.6 cm thickness) were prepared manually and shade dried. The control coils were prepared without the plant ingredient.
The experiments were conducted in a glass chamber of 140 cm × 120 cm × 60 cm. A window of 60 cm × 30 cm was situated at mid bottom of one side of the chamber. Hundred of three or four days' old blood starved adult female mosquitoes, fed with sucrose solution, were released into the chamber. A belly shaven pigeon was kept tied inside the cage in immobilized condition. The experimental chamber was tightly closed. The experiment was repeated five times on separate days, including control mosquitoes of the same age groups. The data were pooled and average values were subsequently used for calculations. Controls were maintained in two sets. One set was run with coil lacking the active ingredient of plant powder (control I), the other was a commercial coil (Mortein coil) which was used for positive control to compare the effectiveness of plant coils. After the experiment over fed and unfed (active and dead) mosquitoes were counted.
The protection given by the smoke from plant samples against the biting of adult mosquito was calculated in terms of percentage of unfed mosquitoes due to treatment.
 |
2.6. Field trial
For the field trial study, mosquito breeding sites were at the endemic districts of Tamil Nadu. The field trials were conducted by using required concentration of plant extracts and bacterial pesticide in different breeding habitat, such as overhead tank, cement tank and cement container. Selection of the localities was decided on the basis of the breeding potential and operational convenience. Field application of the plant extracts and bacterial pesticides was done with the help of a knapsack sprayer (or) hand sprayer. Biopesticide was sprayed uniformly on the surface of the water in each habitat. The mean larval density was calculated on the basis of 5 dips per each habitat. Prior to the experiment the surface area of the breeding habitat was measured along with the pre-spray density of larvae. 24 h after the treatment the post-spray density of larvae was recorded. Successive observations were made at an interval of one day. The percentage reduction was calculated by the following formula[7].
% reduction=100 - (C1 / T1 × T2 / C2)
Where, C1 and T1 are pre-treatment density and T2 and C2 are the post-treatment density of larvae per dip in the control and treated habitats, respectively.
2.7. Statistical analysis
The mortality observed (ppm) was corrected using Abbott's formula during the observation of the larvicidal potentiality of the plant extracts. Statistical analysis of the experimental data was performed using the computer software SPSS 14 version and MS EXCEL 2003 to find the LC50, regression equations (Y = mortality; X = concentrations) and regression coefficient values.
3. Results
The results of larvicidal and pupicidal activity of M. oleifera were presented in Figure 1. The plant extract exhibited larvicidal activities on different instars (I, II, III and IV) and pupa of An. stephensi. The LC50 and LC90 values of M. oleifera for I instar larvae were 57.79 ppm and 125.93 ppm, II instar 63.90 ppm and 133.07 ppm, III instar 72.45 ppm and 139.82 ppm, IV instar 78.93 ppm and 143.20 ppm, respectively. The LC50 and LC90 values of M. oleifera for pupa were 67.77 ppm and 141.00 ppm. The regression equation values of M. oleifera for I instar larvae were Y= -1.087 01 + 0.018 81X, II instar Y= 0-1.184 07 + 0.018 53X, III instar Y= -1.378 29 + 0.019 02X and IV instar Y= -1.574 0 + 0.019 94X, respectively. The regression equation values of pupae were Y= -1.186 04 + 0.017 50X. The LC50 and LC90 values of pupae were 6.792%, 5.449% and 16.925%, 15.474%. Among the different larval stages, the I instar larvae were more susceptible than the other instar larvae. The plant extract showed considerable larval and pupal mortality. The Chi-square values were significant at P<0.05 level (Table 1).
Figure 1. Larval and pupal toxicity effect of M. oleifera on malarial vector, An. stephensi Liston.
*Significance at 0.05% level (P>0.05%).
Table 1. LC50 and LC90 of larval and pupal toxicity effect of M. oleifera on malarial vector, An. stephensi Liston.
| Larval and pupal stage | LC50 and LC90 (ppm) | Regression equation | 95% Confidence limit |
Chi-square value (χ2) | |
| LCL LC50 (LC90) | UCL LC50 (LC90) | ||||
| I | 57.79 (125.93) | Y=-1.087 01 + 0.018 81X | 51.38 (112.49) | 64.02 (146.47) | 1.51* |
| II | 63.90 (133.07) | Y=-1.184 07 + 0.018 53X | 57.64 (118.45) | 70.54 (155.67) | 1.60* |
| III | 72.45 (139.82) | Y=-1.378 29 + 0.019 02X | 66.28 (124.51) | 79.72 (163.45) | 0.24* |
| IV | 78.93 (143.20) | Y= -1.574 0 + 0.019 94X | 72.67 (127.88) | 86.72 (166.64) | 2.27* |
| Pupa | 67.77 (141.00) | Y= -1.186 04 + 0.017 50X | 61.19 (124.42) | 75.14 (167.43) | 3.06* |
*Significance at P < 0.05 level.
Table 2 showed the repellent activity of M. oleifera against An. stenphensi and it was does dependent. Repellency was increased after the does increased. For example 90.41% repellency was noted at 100% concentration and 23.28% repellency was reduced after the treatment of 20% concentration. The repellent activity was carried out in the evening from 5.00 -10.00 pm. The repellency was low at 20% concentration whereas it has been increased at 100% concentration. An average production was at 60% concentration which could make 58.90% production against An. stephensi.
Table 2. Repellent activity of M. oleifera (methanol extract) on malarial vector An. stephensi Liston.
| Repellent activity observation | Number of mosquito fed |
|||||
| Control | Concentration of extract (%) |
|||||
| 20 | 40 | 60 | 80 | 100 | ||
| 5.00-6.00 | 24.0±1.2a | 17.1±2.0a | 15.0±1.2a | 10.3±0.9a | 6.0±0.9a | 2.0±0.9a |
| 6.00-7.00 | 19.8±1.2b | 16.0±1.2b | 13.4±1.2b | 9.4±1.2ab | 5.0±2.0ab | 2.1±0.5b |
| 7.00-8.00 | 13.1±0.9c | 10.0±0c | 6.0±0.9c | 5.0±1.2b | 3.0±1.2c | 1.3±0c |
| 8.00-9.00 | 10.0±0.9bc | 8.3±0.9bc | 4.1±2.0d | 3.0±2.0c | 2.2±1.2d | 1.1±0.5bc |
| 9.00-10.00 | 7.0±1.2d | 5.0±1.2d | 3.3±0.8e | 3.3±0.9c | 1.3±0.5e | 1.0±0.8d |
| Fed mosquitoes | 73 | 56 | 41 | 30 | 17 | 7 |
| Unfed mosquitoes | 27 | 44 | 59 | 70 | 83 | 93 |
| Percentage of protection | 23.28 | 43.83 | 58.90 | 76.71 | 90.41 | |
Table 3 provided the results of smoke toxicity effect of M. oleifera on biting activity of An. stephensi. Two grams of plant ingredients from M. oleifera plant were used for smoke toxicity. The control was maintained without plant ingredients. It acts as negative control. The commercially available (Mortein) mosquito coil was used as positive control.
Table 3. Smoke toxicity effect of M. oleifera leaf, seed and oil on An. stephensi.
| M. oleifera | No. of mosquitoes tested | Fed mosquitoes | Unfed mosquitoes | Total | % Unfed over control I | |
| Leaf | 100 | 24ab | 31b | 45b | 76b | 58ab |
| Seed | 100 | 25b | 40a | 35ab | 75ab | 64b |
| Control I | 100 | 82a | 18c | 0c | 18c | - |
| Control II | 100 | 14c | 26ab | 60a | 86a | 68a |
Within column means followed by the same letter(s) are not significantly different at 5% level by DMRT; Control 1 = Negative control - blank without plant material; Control 2 = Positive control - Mortein coil.
One hundred of 4 -3 days starved An. stephensi mosquitoes were used. After the treatment of the plant, the fed and unfed mosquitoes were counted. There were 24 fed and 76 unfed mosquitoes after the treatment of M. oleifera leaf. In the treatment of M. oleifera seed, fed 25 and unfed 75 were counted. Among the two parts tested against biting of adult, there was more increased mortality after the smoke emerged from the coil made up of seed than other parts. The production given by the smoke from the M. oleifera leaves and seeds was respectively 58% and 64%. Comparisons with other plant showed that its efficacy was very high, but the combined effect of each plant showed good smoke toxicitic effect on An. stephensi.
Table 4 showd the field trial after using M. oleifera seed extracts against malarial vector, An. stephensi. The field study was conducted in mosquito breeding site, such as overhead water tank and water storage places. Field trial was conducted by using the M. oleifera seed extract against malarial vector, An. stephensi (overhead tank) sprayed by using knapsack sprayer. Bioefficacy of plant extract was noted based on the lethal concentration of plants. The LC90 value was double for M. oleifera sprayed individually at different breeding sites of malarial vector. The percentage of larval reduction was noticed in 24 h, 48 h and 72 h at the breeding sites. After treatment with M. oleifera extract, the larval reduction was 73.9%, 84.8% and 94.2% at 24 h, 48 h and 72 h, respectively on malarial vector An. stephhensi.
Table 4. Field evaluation of the M. oleifera (methanolic extract) seed extract on malarial vector, An. stephensi.
| No | Larval density |
|||
| Before treatment | After treatment |
|||
| 24 h | 48 h | 72 h | ||
| 1 | 80.0±4.0a | 31.0±0.8a | 20.0±1.6a | 9.3±0.9a |
| 2 | 68.0±1.6b | 26.3±1.2b | 13.0±0.8b | 5.6±0.4b |
| 3 | 57.6±2.0ab | 17.3±0.9ab | 10.3±0.4c | 4.3±0.4bc |
| 4 | 44.0±2.1c | 12.0±2.1c | 5.3±0.4bc | 5.3±0.9c |
| 5 | 32.3±2.0bc | 8.3±1.2bc | 3.0±0.8d | 1.6±0.4d |
| 6 | 26.6±2.4d | 3.6±1.2d | 2.3±0.8e | 0.3±0.4e |
| Total | 285 | 86 | 60 | 29 |
| Average | 47.5 | 14.3 | 10 | 4.8 |
| Reduction | - | 69.8% | 78.9% | 89.8% |
Within column means followed by the same letter(s) are significancely at 0.05% level by DMRT.
4. Discussion
Many researches have been conducted on plant derived chemicals which are non-toxic to man and domestic animals and serve as useful basis for the development of safer and more selective mosquito insecticides[16]. As compared with other herbal extracts, M. oleifera seed extract also acts as larvicidal and pupicidal agent and studies have been reported on water-extracted M. oleifera seeds (WEMOS) against Aedes aegypti larvae, and methanol-extracted M. oleifera roots against Culex quinquefasciatus and Aedes Albopictus. Results obtained after the treatment of M. oleifera against An. stephensi were encouraging. The obtained larval and pupal mortality may be due to the active chemical compounds present in M. oleifera. Quercetin and kaempferol are flavonoids, compounds of phenolic hydroxyl groups of M. oleifera with antioxidant action of potential therapeutic uses[17].
Since An. stephensi breeds in drinking water tank many of plant extracts are subject to risk factors in mosquito control. The plant extracts which are highly toxic against the An. stephensi are also toxic to human beings. In the present study M. oleifera seed extract shows good effect on An. stephensi and it is also non-toxic to human beings. Many previous studies proved that the extract of M. oleifera is a water purifying agent. M. oleifera seeds can be used as a natural coagulant (primary coagulant) in household water treatment as well as in the community water treatment systems[18]. Hence, it can be considered that the seed extract of M. oleifera is not only a mosquitocidal agent, but also a water treatment agent. The present study also revealed that the seed extracts of M. oleifera have a promising larvicidal efficacy. Plants are rich sources of bioactive organic chemicals and offer an advantage over synthetic pesticides as the plants are less toxic, less prone to development of resistance, and easily biodegradable. The seed extract of M. oleifera will play an important role in the control of mosquitoes.
Repellents are used as personal protection methods against biting arthropods with the major aim of avoiding nuisance[19]. Insect repellents are considered useful alternatives where other control measures are neither practical nor possible. Repellents properly utilized are an inexpensive means of reducing or preventing a wide range of vector[20].
Many plant extracts and essential oils manifest repellent activity against different mosquito species[16]. The biological activity of the plant extracts might be due to a variety of compounds in Solanum tribolium plant, including phenolics, terpinoids and alkaloids. These compounds may jointly or independently contribute to causing oviposition deterrent and skin repellent activity against An. stephensi[21].
The present findings have important implications in the practical control of mosquito larvae in the polluted aquatic ecosystem. The plants studied are available in large quantities. These extracts are easy to handle, inexpensive and safe natural products for mosquito control[5]. The extracts of murungai (Local Tamil Name) seed can also be used for water purification[22]. In view of residue problems in the environment and the development of insect resistance to synthetic insecticides like DDT and other chlorinated hydrocarbons, the recent trend is to explore plants to obtain extracts that are safe for non target animals and do not pose any residue problem but are still able to suppress pest populations. Though several compounds of plant origin have been reported as larvicides[23]–[32], there is a wide scope for the discovery of more effective plant products[15]. Further research undoubtedly will lead to improved formulations with enhanced activity which may eventually become environmentally acceptable and replace objectionable conventional insecticides for mosquito control. It may be concluded that the nature possesses numerous medicinal plants, which may be useful for control of vector borne diseases.
Acknowledgments
Authors are thankful to Prof. G. Vanithakumari, Head of the Department, Department of Zoology, Bharathiar University for giving facilities and encouragement during the study period.
Footnotes
Conflict of interest statement: We declare that we have no conflict of interest.
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