Efficacy of Plant Extracts Against Subterranean Termites i.e., Microtermes obesi and Odontotermes lokanandi (Blattodea:Termitidae)

Leaf and seed crude extracts at three concentrations (high, medium and low) of Euphorbia helioscopia L., Cannabis sativa L., and Calotropis procera (Ait.) were tested against workers and soldiers (4-5th instar) of Microtermes obesi Holmgren and Odontotermes lokanandi Chatarjee and Thakur (Blattodea: Termitidae). Results revealed that all extracts showed moderate toxic effect. 100% mortalities were found in M. obesi and O. lokanandi on day 11 and 7 respectively. Our results showed that Mortalities in both species were concentration depended. Maximum mortalities were observed in high concentration, followed by medium and low. Our results also indicated that M. obesi was more resistant than O. lokanandi.


Introduction
Subterranean termites are highly destructive polyphagous insect pests [1], which largely damage house hold materials. They damaged goods, plants and agricultural crops such as sugarcane, millet, barley and paddy [2]. It is estimated that billions of dollars are spent annually to control termites worldwide [3]. In the past, the control of termites has been totally based on chemicals, especially synthetic insecticides such as persistent organo-chlorine (OC) and organophosphate (OP) insecticides [4,5]. The maximum residual effects as well as the development of insecticide resistance in target pests along with adverse effects on human health and concerns for environmental deterioration are some of disadvantages that hinder widespread use of pesticides [6].
Replacement of synthetic insecticides with bio-pesticides is a universal acceptable and practical approach worldwide [7]. Plant extracts offer a vast, virtually untapped reservoir of chemical compounds with many potential uses. One of these uses is in agriculture to manage pests with less risk than with synthetic compounds that are toxicologically and environmentally undesirable. Various experiments using plant extracts in human and animal health protection, agriculture and household pest management have been particularly promising [8,9]. The plant extracts with complex mixtures of such compounds have been investigated for their insecticidal, repellent, and anti-feedant properties [10][11][12][13]. The deleterious effects of photochemical or crude plant extracts on insects are manifested in several ways, including suppression of calling behaviour [14], growth retardation [15], toxicity [16], oviposition deterrence [17], feeding inhibition [18] and reduction of fecundity and fertility [19].
The present study was focused to find out the insecticidal potential of Euphorbia helioscopia L., Cannabis sativa L., and Calotropis procera (Aiton) against Microtermes obesi and Odontotermes lokanandi.

Collection of Experimental Termites
The experimental termites were collected from an infested termites building situated in Rawal Town, Islamabad by trapping technique used by [36] and these termites were acclimatized in Entomological laboratory of National Agriculture Research Center, Islamabad. These termites were identified by using the taxonomic keys designed by Chaudhry et al.

Extracts preparation
Three fresh and healthy plants of Euphorbia heliocsopia L. ( Sun spurge), Calotropis procera (Ait.) (Ak) and Cannabis sativa L. (Bhang) were collected from Islamabad. These plants were brought in the Entomological Laboratory of National Agriculture Research Center, Islamabad. Aqueous extracts of leaves and seeds of each plant were prepared in three levels i.e., 50, 33 and 25% (high, medium and low) by using the methodology of [37] with some modifications.

Bioassay
Force-feeding tests were conducted following the procedure adopted by [38]. Petri dishes having (dia. 5.5 cm) were used as experimental units. These were sterilized in the oven at 200°C. Circular filter papers were cut and the bottom of each sterilized glass Petri dish was provided with two of them and the lid of each Petri dish with one. Each filter paper in the bottom was soaked with 0.2 mL of the respective extracts concentrations to the extent that it was fully absorbed. Soaking was carried out with the help of a syringe. For each concentration a new syringe was used. Distilled water was used for control. Each treatment was replicated three times. Then populations of 50 termites (45 workers and 05 soldiers) of 4 th -5 th instar (as determined by size) were added to each Petri dish. The Petri dishes were placed in the desiccators having 92% relative humidity. These desiccators were kept in laboratory at temperature (27 ± 30°C) and relative humidity (60 ± 5%). Daily observations were taken and the dead individuals in each Petri dish were removed through forceps.

Statistical Analysis
The data was converted to percentage by using the following formula: Percent Mortality = Total number of dead termites after treatment x 100/ Total number of termites before treatment Then the percent mortality was corrected by using Abbots formula [39]. The experiment was designed as a completely randomized experiment. Statistical computing was performed by using Co-Stat. Means were separated by using Least Significant Difference (LSD) at P<0.05
The analysis revealed that the percent mean mortality recorded at medium and low concentrations was found non-significant (P>0.05), but significantly differed from high aqueous concentration; whereas percent mean mortalities in M. obesi by using seed extracts at high, medium and low concentrations of E. helioscopia were 100.00 ± 0.00, 100.00 ± 0.00 and 94.17 ± 3.06, respectively, which was statistically similar (P>0.05).

Microtermes obesi
Results (Table 3) indicated that percent mean mortalities in M. obesi at high, medium and low aqueous concentrations of leaf extracts of Cannabis sativa were 100.00 ± 0.00, 98.01 ± 0.10 and 95.00 ± 0.98, respectively in 11th day. Statistically the percent mean mortality at high concentration was found non-significant (P<0.05) from percent mean mortality at medium, but significantly higher (P<0.05) from observation recorded at low concentration; while 100% mortality was recorded at high concentration of seed extracts of Cannabis sativa in 11th day, which is statistically non-significantly different (P>0.05) from percent mean mortality recorded at medium concentration and significantly higher (P<0.05) from percent mean mortality recorded at low concentration.

Odontotermes lokanandi
Maximum (100 ± 0.00) percent mean mortalities in O. lokanandi were recorded at high concentration of leaf extract of Cannabis sativa in 7 th day, which was found similar (P>0.05) to percent mean mortalities (94.21 ± 3.22) recorded at medium concentration and significantly different (P<0.05) from percent mean mortality (81.58 ± 2.30) noted at lower concentration (

Microtermes obesi
Results (Table 5) showed that percent mean mortalities in M. obesi were 100.00 ± 0.00, 100.00 ± 0.00 and 95.80 ± 1.03 at high, medium and low concentrations of leaf aqueous extracts of Calotropis procera in day 11 of the trial. The analysis revealed that percent mean mortality at high and medium concentrations was found non-significant (P>0.05), but significantly differed (P<0.05) from percent mean mortality found at low concentration (

Odontontermes lokanandi
The results (Table 6) on the effects of leaf aqueous extracts of Calotropis procera when offered to workers and soldiers of Odontotermes lokanand for seven days in the form of soaked filter paper, percent mean mortality was 100.00 ± 0.00, 93.71 ± 1.83 and 87.43 ± 2.03 at high, medium and low concentrations respectively. The analysis showed that the percent mean mortality recorded at high concentration was found significantly different (P<0.05) from percent mean mortality noted at medium and low concentrations; while by force fed them on aqueous seed extracts of Calotropis procera, percent mean mortality was 100.00 ± 0.00, 100.00 ± 0.00 and 91.16 ± 1.15 at high, medium and low concentrations respectively. Results revealed that mortality recorded at high and medium concentrations was found similar, but significantly high (P<0.05) from mortalities found in low concentration.

Discussion
Different concentrations of leaf and seed extracts of Euphorbia helioscopia were tested against Microtermes obesi and O. lokanandi for eleven and seven days, respectively. Our results showed that percent mean mortality of both species were directly proportion to the concentrations of treatments. Maximum mortalities in both species were observed at higher concentration. Toxicity ranged in M. obesi 2.72 ± 0.68 to 100 ± 0.00 and 1.37 ± 0.69 to 100.00 ± 0.00 by using aqueous leaf and seed extracts of E. helioscopia, respectively; while toxicity ranged in O. lokanandi by using leaf and seed extracts of E. helioscopia 6.55 ± 0.30 to 100 ± 0.00 and 2.75 ± 1.37 to 100 ± 0.00, respectively. Our results showed that O. lokanandi was more sensitive that M. obesi. Essential oils and plant extracts are still an important natural resource of pesticides/ insecticides [40,41] or larvicides [42][43][44] or insect repellents [45][46][47]. The neem insecticide formulation and Margosan-O are observed toxic against the C. formosanus [48,49].
Park and Shin [23] report that garlic oil caus 100% mortality of Japanese termite, Reticulitermes spertus Kolbe after 24 h of treatment.
Verena and Hertel [50] also indicate that some plant extracts are used for termites control. Several higher plants have been tested to be effective against insect pests and diseases of various crops in the field as well as in store [51]. Our study indicated that extracts of the selected tropical herbal plants possess some insecticidal properties against M. obesi, but several variations occurred, based on the concentration of the extracts as these influenced the efficacy or biocidal activities of the plant materials. Euphorbia helioscopia is common weed almost every where in Islamabad. Being very chief source further studies are needed for the isolation of the factor (alkaloids) in the said plant.
Toxicity in M. obesi ranged from 3.39 ± 0.65 to 100.00 ± 0.00 and 7.36 ± 1.74 to 100.00 ± 0.00 by using leaf and seed extracts of Cannabis sativa respectively; while 2.80 ± 0.68 to 100.00 ± 0.00 and 2.13 ± 1.23 to 100.00 ± 0.00 when O. lokanandi were force fed on leaf and seed extracts of C. sativa, respectively. The results showed that aqueous extract of C. sativa contains insecticidal activities and percent mean mortality of both species were directly proportion to the concentrations of treatments. Our results also showed that seed extracts were more toxic that leaf extract of C. sativa. McPartlandC [52] indicates that C. sativa L. is used as a pest repellent. Seed extracts of Polygonum hydropiper L. and Cannbis sativa L. against Heterotermes indicola and Coptotermes heimi are effective more than leaf extracts in both species [53]. Thomas et al. [54] studies that Cannabis sativa caus 100% mosquito larvae mortality. Parihar and Singh [55] report that the aqueous extracts of Cannabis sativa are most effective against larval mortality of Heliothis armigera. Hiremath and Ahn [56] conclude that Cannabis sativa is effective against pest of rice, the paddy brown plant hopper (Nilaparvata lugens). The efficacy of Capparis deciduas and its combinatorial mixtures against Indian white termite Odontotermes obesus was studied and the results indicate that all the treatments had successfully controlled the ascending and descending movements of the termites and prohibited the tunnel formation by the workers [57]. Jalees et al. [58] determine the insecticidal properties of Cannabis sativa against the larvae of Anophles stephensi, Culex quinquefasciatus and Aedes aegypti in the laboratory.
Similarly, percent mean mortalities by using aqueous leaf and seed extracts of Calotropis procera ranged 2.73 ± 0.67 to 100.00 ± 0.00 and 3.42 ± 0.67 to 100.00 ± 0.00 in M. obesi respectively. However, percent mean mortality in O. lokanandi by forced feeding on leaf and seed extracts of Calotropis procera ranged 3.43 ± 1.35 to 100.00 ± 0.00 and