Mosquitoes are the root cause of many diseases, especially dengue, malaria, and Chikungunya. These are endemic diseases in Asia and Africa, which also affect severely on human health.1,2 Aedes aegypti is responsible for vector diseases.3,4 According to world health organization (WHO) postulated globally 400 million peoples are currently affected by dengue. Day today in India dengue fever is increased due to the water pollution, especially Delhi, a city in North India, is endemic for dengue infection with last reported in 2014.5
Mosquitoes spread dengue fever by way of biting. This kind of dengue virus is known as Flavi virus. It has four types they are DENV 1, DENV 2, DENV 3, DENV 4.6,7 Thsese four are some way or other have distinct Aedes aegypti transmit dengue in large number. This type of mosquitoes breeds only still waters. In general, it bites a human being at day time for 2-7 days in the infected person’s blood this virus is being circulated. Gradually mosquito transforms dengue from one person to another in the way of bitting infected persons.8
One of the method to control the growth of dengue causing mosquito by synthetic insecticides.9 This method have some disprove such as environmentally contaminated by air, water and soil. There is best way to replace by a synthetic method by green evolution. Current scenario has proved the plant extracts are alternative larvicides because their derivatives were used to kill the mosquitoes. Tephrosia purpurea is a flowering plant and its belongs to the Pea family, this plant containing many properties.10 This is used to kill the mosquitoes, which cause dengue. And also it has been recommended as a blood purifier.11 It cures diseases apart from dengue like bronchitis, bilious febrile attacks, liver obstructions, spleen and kidney.12,13 They are two important types of mosquitoes existing in the world, namely nuisance and vector mosquitoes. For the past twenty years, bacterial larvicides like Bacillus thuringiensis and Bascillus sphaericus are being used in order to control these two mosquitoes. Bacillus sphaericus, a soil bacterium that kills mosquito larvae in water. It is developed as commercial larvicide. But in several countries that is not used in outnumber.14-16
In this research, the methanolic leaf extract of T. purpurea and bacterial insecticide of B. sphaericus were tested against larval and pupal of Dengue vector for analyzing their inhibitory activity. The larvicidal and pupicidal activities were checked by the under laboratory as well as field conditions.This plant was selected based on their ethnobotanical literatures.17 Therefore this study provide first report mosquito larvicidal and pupicidal activity, combined effect of T. purpurea leaf extract B. Sphaericus against A. aegypti.
MATERIALS AND METHODS
Collection of plant and preparation of extract
T. purpurea plant was collected from in and around the A.V.V.M.Sri Pushpam College, Campus, Poondi, Thanjavur. The plant was identified at The Rapinat Herbarium, St. Joseph’s College, Trichy, India. T. purpurea leaves were washed with tap water and shade dried at room temperature. The dried plant materials (leaves) were powdered by an electrical blender. From the powder 500 g of the plant sample was extracted with 1.5 L of organic solvents of methanol for using a Soxhlet apparatus boiling point 60-80ºC for h.18 The extracts were filtered through a Buchner funnel with Whatman number 1 filter paper. The crude plant extracts were evaporated to dryness in rotary vacuum evaporator. 1g of the plant residue was dissolved in 100 mL of acetone (stock solution) considered as 1% stock solution. From this stock solution concentrations were prepared ranging from75, 150, 225, 300 and 375 ppm, respectively.
Collection of eggs and maintenance of larvae
The eggs of A.aegypti were collected from National Centre for Disease Control field station at Mettupalayam, Tamil Nadu, India, using an “O”-type brush. These eggs were brought to the laboratory and transferred to 18×13×4 cm enamel trays containing 500 mL of water for hatching. The mosquito larvae were pedigree dog biscuits and yeast at 3:1 ratio. The feeding was continued until the larvae transformed into the pupal stage.
Maintenance of pupae and adults
The pupae were collected from the culture trays and that are transferred into the to plastic containers (12×12 cm) containing 500 mL of water with the help of a dipper. The plastic jars were kept in a 90×90× 90 cm mosquito cage for adult emergence. Mosquito larvae were maintained at (27 ±2) ºC, 75% - 85% relative humidity, under a photoperiod of 14:10 (light/dark). A 10% sugar solution was provided for a period of 3 days before blood feeding.
Blood feeding of adult A.aegypti
The adult female mosquitoes were allowed to feed on the blood of a rabbit (a rabbit per day, exposed on the dorsal side) for 2 days, to ensure adequate blood feeding for 5 days. After blood feeding, enamel trays with water from the culture trays were placed in the cage as oviposition substrates.
B. sphaericus was obtained from K.K Biotech, Lab Service, Chennai, Tamil Nadu, India. The organism was grown in a liquid medium containing (in g per liter of distilled water): FeSO4·7H2O,0.01; MnSO4, 0.1; MgSO4·7H2O, 0.2; CaCl2, 0.08; K2HPO4, 0.025; yeast extract, 2; peptone, 4; and D-glucose, 1 and casein, 5. Solutions of yeast extract, peptone casein, D-glucose, K2HPO4 and CaCl2 were separately prepared, sterilized, and added before inoculation. The pH of the medium was adjusted to 7.1 before sterilization. The required quantity of B. sphaericus was thoroughly mixed with distilled water and prepared at various concentrations ranging from 10, 20, 40, 60 and 80 ppm, respectively.
Larval/pupal toxicity test
Laboratory colonies of mosquito larvae/pupae were used for the larvicidal/pupicidal activity. Twenty-five numbers of first to fourth instars larvae and pupae were introduce into 500 mL glass beaker containing 249 mL of de-chlorinated water and 1 mL of desired concentrations of plant extract and B. sphaericus were added. Larval food was given for the test larvae. At each tested concentration two to five trials were made and each trial consisted of five replicates. The control was setup by mixing 1 mL of acetone with 249 mL of dechlorinated water. The larvae and pupae were exposed to dechlorinated water without acetone served as control. The control mortalities were corrected by using Abbott’s formula.19
The LC50 and LC90 were calculated from toxicity data by using probit analysis.20
For the field trial, the quantity of plant extract residues and B. sphaericus (Bs) required (based on laboratory LC50 and LC90 values) quantity for each treatment was determined by calculating the total surface area of drinking water bodies in each habitat. The required quantities of T. purpurea and B. sphaericus were mixed thoroughly with water in a bucket with constant agitation. Teepol was used as emulsifying agent (0.05%). Field applications of the T. purpurea leaf extracts and B. sphaericus were done with the help of a knapsack sprayer (Sujatha Products, India, Private Limited, 2010) and uniformly on the surface of the drinking water bodies in each habitat. Dipper sampling and counting of larvae monitored the larval density before 24, 48 and 72 h after the treatment. A separate sample was taken to determine the composition of each larval habitat. Six trails were conducted for T. purpurea of the plant extracts and B. sphaericus alone and combined the treatment. The percentage of reduction was calculated by the following formula:
Where C is the total number of mosquitoes in control, T is the total number of mosquitoes in treatment.
All data’s were subjected to analysis of variance; the means were separated using Duncan’s multiple range tests by Alder and Rossler.21 The average larval mortality data were subjected to probit analysis for calculating, LC50, LC90 and other statistics at 95% confidence limits of upper confidence limit (UCL) and lower confidence limit (LCL) and chi-square values calculated using the (Statistical software package). The values were expressed as mean ± standard deviation of five replicates. Results with P < 0.05 were considered to be statistically significant.
Table 1 shows the phytochemicals present in the methanolic leaf extract of T.purpurea contains alkaloids, carboxylic acid, coumarins, flavanoids, quinines, proteins and resins.Larval and pupal mortality of A.aegypti after the treatment of methanolic leaf extract of T.purpurea was observed. Table 2 provides the results of larval and pupal mortality of A.aegypti (I and II instars) at different concentrations (75 to 375 ppm). Forty two point four percent mortality was noted at I instar larvae by the treatment of T.purpurea at 75 ppm, whereas it has been increased to 83.4 at 375 ppm of T.purpurea leaf extract treatment. Similar trend has been noted for all the instars of A.aegypti at different concentrations of T.purpurea treatment. The lethal concentrations (LC50 and LC90) were represented as LC50 value of Ist instar was 139.24 ppm, IInd instar was 176.24 ppm, IIIrd instar was 580.34 ppm and IVth instar was 256.27 respectively. Whereas LC90 value of Ist instar 480.72, IInd instar 541.21, IIIrd instar 580.34 and IVth instar 672.20 respectively. The pupa value of LC50 and LC90 is 326.29 and 762.80 ppm respectively.
The different concentrations of (10 to 80 ppm) B. sphaericus to treat the larval mortality of A.aegypti (Table 2). The treatment of Ist instar larvae by B. sphaericus at 10 ppm, 31% was recorded, whereas it has been increased to 68.2% at 80 ppm of B. sphaericus treatment. Eighteen percentage of mortality was observed at pupal by the treatment of B. sphaericus at 10 ppm and it has been increased to 45% at 80 ppm. Likewise all the different concentrations have been noted. The LC50 and LC90 values were represented as Ist instar was 46.16, 141.68 ppm followed by IInd instar 56.23, 172.46 ppm, IIIrd instar 69.82, 184.21 ppm and IVth 80.81, 193.31 ppm respectively. The pupa value of LC50 and LC90 was 96.12 and 218.16ppm respectively Table 3.
The combined larval mortality after treatment of T.purpurea and B. sphaericus for all the larval instars was presented in Table 4. The concentration at 75+40 combined T.purpurea and B. sphaericus treatment for IVth instar mortality was 65.6% recorded. The LC50 and LC90 values are represented as Ist instar was 80.08: 108.39ppm followed by IInd instar 82.21; 118.21 ppm, IIIrd instar 88.0; 136.75ppm and IVth instar 92.21;149.02ppm respectively. The pupa value of LC50 and LC90 was 98.16; 153.24 ppm respectively. The X2 values are significant at P < 0.05level. The 95% confidence limits at P < 0.05 level. The 95% confidence limits at LC50, LC90 (LCL-UCC) values were calculated. Larval and Pupal mortality was observed after 24 h exposure. In observation of control mortality was absent.
In drinking water body systems totally 409 A. aegypti larvae have been noted. T. purpurea extract was treated against A .aegypti the larval density was reduced by 17.8%, 40% and 87% at 24, 48 and 72 h respectively Table 5.
As well as the reduction of A.aegypti larval densities after treatment with B. sphaericus were 9.5%, 31.5% and 79.46% respectively. Combined effect of T .purpurea and B. sphaericus were 44.74%, 79.95% and 100% at 24, 48 and 72h respectively Table 6.
Plants based organic products are preferred for the control of insect vectors of human diseases. The effectiveness of secondary metabolites such as alkaloids, isoflavonoids, saponine and steroids has potential mosquito larvicides.22-24 According to Pedro et al25 phytocompounds such as flavonoids, alkaloids and saponins are responsible for insecticidal activity. Kotkar et al26 reported that flavonoids isolated from Annona squamosa are effective as insecticides against Callosobruchus chinensis. In the recent study, the methanolic leaf extract of T.purpurea contain good amount of flavonoids and alkaloids were recorded Table 1.
From 19th century plant extract treated for the control of vector mosquitoes has been reported.27 While subsequently plants for identifying the potential activity not only against the larval stages but also include all other stage of mosquitoes.28 Taken from plants are successful and vital source of several elements which are used in distinct ways.29
Larvicidal and pupicidal actions of methanol leaf extract E .hirta against the malaria inducing vector, A. stephensi was shown the LC50 values in initial larva instars to till fourth larval instars respectively 172.65, 217.5, 269.37 and 332.39 ppm. The results obtained show that this plant material exhibited significant activity and could be considered as potential natural larvicidal agent.16 Similarly Maheshkumar et al30 reported that the efficacy of Solanum xanthocarpum leaf extract in the larval and puppal of A. stephensi with LC50 value of initial instars to fourth instars respectively 155.29, 198.32, 271.12, 377.44 and 448.41 ppm. Likewise, LC90 value of first to fourth instars larvae and pupae 687.14, 913.10, 1011.89, 1058.85 and 1141.65 ppm, respectively.
In the present study, T.purpurea leaf extract against first to fourth instars larvae and pupae of A.aegypti has been studied in the laboratory condition. The lethal concentrations (LC50/LC90) of T. purpurea were 139.24, 176.24, 219.28, 256.27, and 326.29ppm respectively. The LC90 values of 480.72, 541.21, 580.34, 672.20, and 762.80 respectively.
Aloe vera methanolic leaf extracts and the insecticide of bacteria B.sphaericus for the mosquitocidal property was screened as potential species of mosquito vectors.31 This is a peculiar biodegradable method for inhibiting the vector programs. In the proposed study, the B. sphaericus at various concentrations were carried out against the various larval instars of dengue vector, A.aegypti. Likewise 85% mortality was observed using bacterial pesticides of Spinosad against the malarial vector, A. stephensi. Based on the above mentioned mortality rate indicate that the extract can be used as bio-pesticides. The LC50 values of second, third and fourth instars larvae A. stephensi were 0.27%, 0.28% and 0.30% observed respectively.32-35 According to Kamalakannan and Murugan36 reported that control the mosquito larval and pupal population in the lab and field trials using ten microbial products. The lethal concentrations (LC50 and LC90) at 0.25 and 0.5 at 24 h for A.aegypti suggest that highest larval motility in the lab. Kalfon et al 198437 reported that B. sphaericus which is highly toxic to dipteran larvae has opened the possibility of its use as a potential biolarvicide in mosquito eradication programe. In Amazonia strains of B. sphaericus against treated for the larvicidal activity in different concentration levels.38 In the present study also were supports with Subramanian et al31
|Phytochemical constituents||T. purpurea|
|Sample No.||Before treatment||T. Purpuria||B. Spheerious|
|24 hrs||48hrs||72 hrs||24 hrs||48 hrs||72 hrs|
|Sample No.||Before treatment||After treatment|
|24 h||48 h||72 h|
Various concentrations like 10, 20, 40, 60 and 80 ppm were used in laboratory bioassays for A. stephensi.16 Similarly, various concentrations of B. sphaericus were treated for C. quinquefasciatus after 24h it was observed. The percentage of mortalities were different for the different instars of C.quinquefasciatus, B. sphaericus against the first to fourth larvae and pupae was 0.051%, 0.057%, 0.062%, 0.066% and 0.073% respectively. The outcome of the result gives that B. sphaericus which has rich and powerful properties to control mosquito. And also it acts as eco-friendly. The previous report of Kovendan is that B.sphaericus is used to control A.stephensis, malarial vector if it is isolated from soil sample. But the recent outcome shows that B.sphaericus reduced the activity of larvicidal . Previously, Kovenden et al39 reported that B. sphaericus isolated from soil sample and used to control the malarial vector A. stephensi. In the present results showed that B. sphaericus dealing the decrease of larvicidal activity.
Field trials was conducted by Sharma et al a and b40,41 at Sundergarh district of Odisha, they are used the drug deltamethrin in the form of tablet for controlling malaria. The effective results were observed from field as well as laboratory conditions against A. culicifacies and A. stephensi. Similarly stiles et al42 reported that both cement and mud surfaces, found 100% motility was observed residual activity of deltamethrin against A. gambiae. On the other hand emulsified neem oil formulation was observed against A. stephensi larvae in tank and pits. Hundred percent of reductions were observed by V.K Dua et al.43 However in the present study combined activity of plant extract of T. purpurea and B. sphaericus in the field were 44.74%, 79.95% and 100% at 24 hrs, 48 hrs and 72 hrs respectively. This results show that T. purpurea and B. sphaericus can control the dengue vector, A. aegepti. Likewise Panneerselvam et al16 were conducted combined activity of plant extracts of E .hirta and B. sphaericus in the field were 44.23%, 81.64% and 100% at 24 hrs, 48 hrs and 72 hrs respectively.
The outcome of the present research obviously shows that both T.purpurea and B. sphaericus have astonished mosquito properties against A.aegypti. This is the first study on the joined mosquito larvicidal and pupicidal acttivities. The research confirms that T. purpurea and B. sphaericus are two vital biological agents that could control the dengue incidence. Besides, there is a scope and agte way for further research on the activities of larvicidal and pupicidal.