Insecticidal and repellent activities of methanolic extract of Tribulus terrestris L. (Zygophyllaceae) against the malarial vector Anopheles arabiensis (Diptera: Culicidae)

Methanolic extracts of leaves and seeds from, Tribulus terrestris (Zygophyllaceae) was tested against 3 instar larvae and adults of mosquito, Anopheles arabiensis under laboratory condition. The seeds extract showed high insecticidal activity at all concentrations compared to the leaves extract and the Lc50 was 36.5 and 123.1ppm for seeds and leaves extract, respectively. All extracts exhibited remarkable effects on the fecundity, fertility and sterility index of adult females resulted from treated larvae, but the seeds extract was more effective than leaves extract. The repellent action of the present plant extracts varied depending on the plant parts and the dose of extract. The seeds extract was more effective in exhibiting the repellent action (100%) against the mosquito tested as compared with the leaves extract (79.5%) at the dose 1.0 and 2.0mg/cm, respectively. The seeds extract showed the same repellency percent (100%) of commercial formulation, N. N. diethyl toulamide (DEET) but at the lower dose (1.0mg/cm). The extracts in this study showed strong larvicidal, pupicidal, adulticidal, ovicidal, repellency and biting deterrence and these extracts may be used for further integrated mosquito management programs.


INTRODUCTION
Vector-borne diseases, such as malaria, dengue and hemorrhagic fever (DHF), are still major public health problems in the South of Kingdom Saudi Arabia countries because geographic location next to the Yemen malaria-endemic and their tropical or subtropical climate.High rainfall level, the prevalence of lakes and/or ponds levels, and the belated displacement system in many regions of Saudi Arabia, provided ideal environmental conditions for mosquito breeding and dense mosquito population .
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 (Liu et al., 2006), undesirable effects on non-target organisms, and fostered environmental and human health concern that initiates a search for alternative control measures (Hayes and Laws 1991).Plants are considered as a rich source of bioactive chemicals and they may be an alternative source of mosquito control agents (Georgewill et al. 2010).
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 (Babu and Murugan 1998).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 ( Kalyanasundaram 1982).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 (Isman 1995).The aim of the study was to determine the Insecticidal activity of T. terrestris methanolic extract against the malarial vector, An. arabiensis and to examine the repellent properties of this plant against mosquito bites.

Mosquito culture
Anopheles arabiensis larvae were collected from stagnant water area during December 2011 through dipping method from the natural sites located in a village called Sanba at a distance of 10 km from Jazan, Kingdom Saudi Arabia to establish the mosquito colony in our laboratory in the Department of Biology faculty of science, Jazan University.Larvae were kept in plastic and enamel trays containing tap water.They were maintained, and all the experiments were carried out, at 27±2ºC and 75-85% relative humidity under 12:12 light and dark cycles.Larvae were fed a diet of brewer's yeast, dog biscuits, and algae collected from ponds in a ratio of 3:1:1, respectively.Pupae were transferred from the trays to a cup containing tap water and were maintained in wooden cages (45×45×40 cm) where adults emerged.Adults were continuously provided with 10% sucrose solution in a jar with a cotton wick.On day five, the adults were given a blood meal from a pigeon placed in resting cages overnight for blood feeding by females.Glass petri dishes with 50 ml of tap water lined with filter paper was kept inside the cage for oviposition.They were maintained and reared in the laboratory as per the method of Rahuman et al. (2008).

Plant collection and preparation of plant extract
Fully matured T. terrestris ( Family: Zygophyllaceae) was collected during November 2011 from the Sabia city (Sabia Jazan desert road).The taxonomic identification is made by Dr. Wael Kassem, Ass.Prof., Biology Department, Faculty of Science, Jazan University, and by comparison with the published plant description in flora of Saudi Arabia (Migahed, 1987).A voucher specimen has been deposited in the herbarium of Biology Department, Faculty of Science, Jazan University.The leaves and seeds were washed and dried in the shade at room temperature (27-31 °C) for 10 days till they become brittle, then pulverized to powder in a hammer mill.100g of powder from each part was extracted three times separately with 300 ml of methanol at room temperature.After 24 h., the supernatants were decanted, filtrated through Whatman filter paper No. 5. and dried in a rotary evaporator at 40 ºC to obtain 12.9g and 9.1g of a semi solid leaves and seeds crude extract, respectively.The dry extracts were kept in deep freezer (-4ºC) till used for experiments.

Larvicidal test
For the larvicidal bioassay, the WHO standard protocols (WHO, 1996) with slight modifications were followed.The different test concentrations were prepared by adding different range of the stock solution (1%) to 200 ml of water in a 500 ml capacity of glass beaker.Then, twenty of third instar larvae were put immediately into plastic cups contained 200 ml of test concentration of extract.Mortality was recorded daily and the dead larvae and pupae were removed until adult emergence.The experiments were replicated three times and the data were subjected to Probit analysis4.The control experiments were run parallel with each replicate.
Larval mortality percent was estimated using the following equation (Briggs, 1960): larval mortality % = A -B / A × 100 (where: A = number of tested larvae, B = number of tested pupa).Pupation rate was estimated using the following equation: Pupation % = A / B × 100 (where: A = number of pupae, B = number of tested larvae).The pupal mortality percent was estimated using the following equation: Pupal mortality % = A -B / A × 100 (where: A = number of produced pupae, B = number of observed adults).Adult emergence of males and females were counted and calculated using the following equation: Adult emergence % = A / B × 100 (where: A = number of emerged adults, B = number of tested pupae).

Reproductive potentiality of females: Fecundity:
The emerging adult females from the treated 3 rd instar larvae were collected and transferred to the wooden cages (20×20×20 cm) using an electric aspirator and fed with 10% sugar solution for three days.These treated adult females were accompanied with some normal adults males.The adult males and females were left for one day without sugar solution.At the 5 th day, starved females were allowed to take a blood meal from a pigeon and allowed to lay egg on clean water (oviposition traps) in the cages.The number of eggs was counted using a binocular and then mean value was counted.

Fertility:
The fertility egg or hatchability was calculated using the following equation: Egg hatchability % = A / B × 100 where: A = total number. of hatched eggs, B = total number of eggs laid.According to the formula of Toppozada et al. (1966), Sterility percentage = 100 -[a×b /A×B×100], Where: a = number of eggs laid / female in treatment, b = percentage of hatched eggs in treatment, A = number of eggs laid / female in control, B = percentage of hatched eggs in control

Repellency test
Standard cages (20×20×20cm) were used to test the repellent activity of plant extracts.Different weight from each extract was dissolved in 2ml (70% ethanol) in glass 4×4cm to prepare different concentrations.One ml from each concentration was directly applied onto 5×6cm of ventral surface of pigeon after removed feathers from the abdomen to evaluate the repellency against An.arabiensis, compared with commercial repellent Deet (N.N. diethyl toulamide) (Johnson Wax Egypt) as a positive control.After 10 minutes of treatment, the treated pigeons were placed in the cages containing at least 20 An. arabiensis starved females 5-7 d-old for 4h.Control tests were carried out alongside with the treatments using ethanol or water.Each test was repeated three times to get a mean value of repellent.
Repellency % was calculated according to Abbott, (1925).Repellency % = [% A -% B / 100 -% B] × 100 Where: A = percent of unfed females in treatment, B = percent of unfed females in control 6.Statistical analysis Statistical analysis of the data was carried out according to the method of lentner et al., (1982).Lc 50 was calculated using multiple linear regression (Finney, 1971).

RESULTS
Data given in table (1&2) indicated the insecticidal activity of methanolic extract of T. terrestris (leaves and seeds), respectively against the 3 rd instar larvae of An. arabiensis.Complete larval mortality percent (100%) occurred at the highest concentrations (250 and 150ppm), while the lowest mortality percent (18.3 and 23.3%) occurred at the lowest concentrations (50 and 10ppm) for leaves and seeds extract, respectively compared to 5.0% for the control.The lethal effect of leaves and seeds extract was extended to the pupal and adult stages, but the seeds extract was more effective than leaves extract.The highest pupal mortality was 100.0 and 45.0% at 100 and 200ppm for seeds and leaves extract, respectively, vs. 0.0% for the control.A remarkable reduction in the percentage of adult emergence from pupae produced by treated larvae with the seeds and leaves extract was observed.Also, the seeds extract was more effective than leaves extract, the adult emergence percent (0.0 and 55.0%) was occurred at the concentration 100 and 200ppm for seeds and leaves extract, respectively, vs. 0.0% for the control.
From the aforementioned results it is obvious that the toxicity values of the tested methanolic extract of different plant parts of T. terrestris based on Lc 50 values (Table 3) may be arranged in a descending order as follows: seeds > leaves.As summarized in Table (4&5), the seeds extract exerted a profound reducing effect on the female fecundity (0.00.0, 29.87.3 and 41.97.9 eggs/♀ at   The repellent activity of the present extracts against starved An. arabiensis females varied according to the plant parts and the doses used (Table 6).At doses 1.0 and 2.0 mg/cm 2 , the seeds and leaves extract induced a degree of repellency as calculated in 100.0 and 79.5%, respectively, compared to 100% repellency for Deet at a dose1.5 mg/cm 2 .

DISCUSSION
The plant tested in the present study are known to be eco -friendly and are not toxic to vertebrates.Moreover, it is clearly proved that crude or partially purified plant extracts are less expensive and highly efficacious for the control of mosquitoes rather than the purified compounds or extracts (Jang et al., 2002;Cavalcanti et al., 2004 andMaurya et al. 2009).The present study showed high bioactivity of the different extracts from T. terrestris which are grown widely in Saudi Arabia especially Jazan region.Such results may offer an opportunity for developing alternatives to rather expensive and environmentally hazardous organic insecticides.
The present study showed that, the toxicity of the tested plant extracts against 3 rd larval instar was varied according to plant part used and concentration of the extract.The larval mortality percent was increased by increasing extract concentration for all plant extracts tested.The toxicity of methanolic extracts based on Lc 50 was seeds > leaves.These results agree, to some extent, with the previously mentioned suggestions of (Maurya et al. 2009 andKovendan et al. 2012).Extracts from several other plant species were tested on different species of mosquitoes by many authors worldwide.The activity of the plant extracts on larval mortality of An. arabiensis, in the present study, were in agreement with the results obtained by Coria et al. (2008), Maurya et al. (2009), Govnidarajan (2010), Patil et al. (2010 and2011) and Kovendan et al. (2012).The evaluation of T. terrestris Linn (Zygophyllaceae) acetone extract for larvicidal activity against mosquito vector, An. stephensi was tested by Singh et al. (2008).The LC 50 values of leaves and seeds acetone extract were 124 and 72ppm, respectively.In the present study, Also, methanolic extract of leaves showed the same effect against An.arabiensis with Lc 50 123.1ppmbut the seeds extract seemed to be more effective than other extract with LC 50 values of 36.5ppm.
A remarkable decrease in the pupation percent was induced by all plant extracts in the present study.The pupation% decreased as the concentration of the plant extract increased.The present study showed that the toxic effect of methanolic extracts of leaves and seeds had been extended to the pupae.In addition, these extracts induced reductions of the adult emergence.The reduction was found as a concentration-dependent.These results are comparable to earlier results of Sharma et al. (2006) using petroleum ether extract of A. annua against An.stephensi and C. quinquefasciatus larvae, respectively, Wiesman and Chapagain (2006) using one fraction obtained from the silica gel column chromatography of the methanol extract against Ae.aegypti mosquito larvae, Gunasekaran (2009) using Neem Azal against Ae.Aegypti, Patil et al. (2011) Plumbago zeylanica (Plumbaginaceae) and Cestrum nocturnum (Solanaceae) plant extracts against Ae.aegypti.and Kovendan et al. (2012) using Carica papaya (Caricaceae) leaf extract against, Aedes aegypti.Gang et al. (2000) concluded that, some plants such as T. terresteis, Eupatorium fortunei and Datura stramouium have value for development as botanical insecticides against C. pipiens.
Results obtained in the present study indicated that the toxicity of plant extracts against the 3 rd instar larvae of An. arabiensis was extended to the adults causing mortality reached to 33.3 and 66.7% for leaves and seeds extract at the concentrations 200 and 50ppm, respectively.Similar results were obtained by Jeyabalan et al. (2003) using methanol extract of Pelargonium citrosa leaf against An.stephensi, Nathan et al. (2005) using the neem A. indica extract against An.Stephensi, Nathan et al. (2006) using methanolic extracts of leaves and seeds from the chinaberry tree Melia azedarach against An.stephensi and El-Sheikh et al. (2011) using leaf and stem extracts from Cestrum nocturnum (solanaceae) against Culex pipiens.
Almost all plant extracts tested against the 3 rd instar larvae of An. arabiensis significantly reduced the female fecundity and induced the sterility % of females.The fecundity and sterility percents depended on the plant part and concentration.
Moreover, a remarkable decrease in the hatchability % of eggs laid by females resulted from treated larvae especially with seeds extract was observed.The hatchability of eggs decreased as the conc. of the extract increased.These results are in consistent with those obtained by many authors using different plant extracts against different mosquito species (Jeyabalan et al., 2003;Nathan et al., 2006;Coria et al., 2008 andPavela, 2009).
Whatever the concentration, the plant extracts used in the present study exhibited some repellency activity against the starved female adults of An. arabiensis.The repellent action of the plant extracts tested was varied depending on the plant part used and the dose of the extract.The present study indicate that the seeds extract was more effective in exhibiting the repellent action against the mosquito tested as compared with the leaves extract and showed same repellency percent (100%) but at the lower dose (1.0mg/cm 2 ) as compared with a commercial formulation, N,Ndiethyl-m-methylbenzamide (1.5mg/cm 2 ).
Many plant extracts and essential oils manifest repellency activity against different mosquito species.The present results are in accordance with such results obtained by Sharma et al. (1995) using neem oils against mosquito bites of Anopheles spp., Culex spp.and Ae.spp., Govere et al. (2000) using extracts of fever tea (Lippia javanica) rose geranium (Pelargonium reniforme) and lemon grass (Cymbopogon excavatus) against An.arabiensis, Yang et al. (2004) using methanol extracts from 23 aromatic medicinal plant species against female blood-starved Ae. aegypti, Choochote et al. (2007) using repellent activity of selected essential oils from ten plant species against Ae.Aegypti, Mullai et al. (2008) using leaf extract of Citrullus vulgaris against the malarial vector, An. stephensi, Singh et al. (2008) Govnidarajan and Sivakumar (2011) tested the repellent activities of crude hexane, ethyl acetate, benzene, chloroform, and methanol extracts of leaf of Eclipta alba and Andrographis paniculata at three different concentrations of 1.0, 2.5, and 5.0 mg/cm 2 against important vector mosquitoe Ae. aegypti and they suggested that the leaf solvent plant extracts have the potential to be used as an ideal eco-friendly approach for the control of mosquitoes.In the present study the efficacy of seeds extract from T. terrestris at the dose (1.0 mg/cm 2 ) produced the highest protection (100%) during the entire research period of 4h post-treatment.

CONCLUSION
In general, it could be concluded that seeds and leaves methanolic extracts of T. terrestris used in the present study act as repellent or antifeedant, ovicidal, larvicidal, pupalcidal, adulticidal and possess emergence inhibiting against the mosquito vector, An. arabiensis.Furthermore, the results of the present study may contribute to a reduction in the application of synthetic insecticides, which in turn increases the opportunity for natural control of various medically important pests by botanical pesticides.Further studies on the tested plants including mode of action, synergism with the biocides under field condition are needed.
using acetone leaves extract of T. terrestris against An.stephensi, El-Sheikh et al. (2009) using sixteen ethanolic and petroleum ether extracts of 4 indigenous plants as repellent in the field against wild mosquitoes, Elango et al. (2011) using methanol extract of Andrographis paniculata against An.Subpictus and Prabhu et al. (2011) using Moringa oleifera methanolic extracts against An.stephensi.

Table 4 :
Effect of methanolic extract Tribulus terrestris (leaves) on fecundity, fertility and sterility index of female Anopheles arabiensis.

Table 5 :
Effect of methanolic extract Tribulus terrestris (seeds) on fecundity, fertility and sterility index of female Anopheles arabiensis.

Table 6 :
Repellency effect of methanolic extract of Tribulus terrestris on Anopheles arabiensis.