Evaluation of cypermethrin and Acorus calamus L. extract against 4 instar larvae and adults of Aedes albopictus (Skuse) and Aedes aegypti (L.)

The current study was conducted to evaluate bioassay of both Acorus calamus Linn. crude hexane extract and Cypermethrin 80SC against the 4 instar larvae and adults of Aedes albopictus and Aedes aegypti with LC50 and LC90 values. LC50 and LC90 values of Cypermethrin on the 4 instar larvae of A. albopictus were 0.1470 and 0.6120 ppm respectively while on the A. aegypti were 0.0047 and 0.0108 ppm respectively as compared to the Acorus calamus extract on the 4 instar larvae of A. albopictus were 4.1360 and 22.2766 ppm respectively while on the A. aegypti were 5.4420 and 12.4938 ppm respectively. Both Cypermethrin and Acorus calamus extract showed a significant difference (P<0.05). LC50 and LC90 values of Cypermethrin on adults of A. albopictus were 3.5277 and 5.2844 ppm respectively while on A. aegypti were 0.8220 and 3.4267 ppm respectively and a significant difference (P<0.05) was to be found as compared to that of A. calamus extract on the adults of A. albopictus were 52.8842 and 544.1275 ppm respectively while on the A. aegypti were 18.5270 and 344.8478 ppm respectively and no significant difference (P<0.05) was to be found.


Introduction
The primary vector of dengue in Southeast Asian countries including Pakistan is Aedes aegypti (L.) while Aedes albopictus (Skuse) is attributed as secondary vector for spreading dengue fever [1].During the last few decades dengue virus infection has become a serious health problem.Annually 55 million people affects due to dengue fever and approximately two fifth of the total population is expected to be at risk of dengue across the world [2].Because of thousands of deaths and every year millions of dengue fever cases, it is known as one of the major public health concern to the urban, suburban and rural tropical areas [3].Fatal diseases are potential threat to children causing by the dengue viruses [4].About 2.8% deaths occur every year among children in all over the world due to dengue epidemics [5].Ae. aegypti were found in brackish water (BW), an environment which limits the osmotic gradient [6]. A. albopictus and A. aegypti are the known suspected vectors of dengue in Lahore, Pakistan [7].DHF (Dengue Hemorrhage Fever) in Pakistan was first reported in Karachi in 1994 [8].In 2011, more than 21000 positive cases and above 300 deaths, Lahore have witnessed a severe epidemic of dengue.In Pakistan, the dengue is an alarming situation.Vector control is the only option for having no vaccine and proper antiviral drug to control the disease [9].It is necessary to know the biology and ecology of vectors to establish a strategic control for mosquitoes [10].In over populated areas female mosquitoes in a few numbers are enough to cause an outbreak [11].In all over the world, mosquitoes are known to be insect which affects the health of humans and domestic animals.About 3520 species of mosquitoes which are belonging to 50 different genera are to be found in all over the world, but the most important genera are known to be Anopheles, Culex and Aedes which transmit diseases [12].The intensive migration and urbanization of people results in extended infestation of Aedes mosquito, which is known to be principal vector of dengue infections [13].
For the last 2-3 decades, different types of chemical insecticides like organochlorides, organophosphates and carbamates have been used successfully for the control of mosquito vectors.In all over the world insecticide pyrethroid is used for the control of mosquitoes most successfully [14].The annual use of insecticides from 2000 to 2012, against dengue vectors was 396 tons of organophosphates and 160 tons of pyrethroids [2].Cypermethrin, a pyrethroid affects the nervous system which causes paralysis in insects [15].Cypermethrin ultimately causing the death of insects such as mosquitoes when acting on sodium gated channel target site [16].In future, the most promising mosquito control programs will rely on plant derived chemicals [17].Thus, the researches for new environmentally safe and target specific insecticide are being conducted.To find new modes of action and also to develop active agents which will be based on natural products, efforts are being made to isolate, screen and develop phytochemicals having pesticidal activity [18].The traditional approach to initiate dengue control programmes once the dengue virus is introduced into a population and dengue cases received to hospitals makes it difficult to control, evident from the cases all over the world [19].The economically feasible natural, biodegradable compounds are the inevitable measures for the control of mosquito vector that will replace the expensive synthetic insecticides.In Multan, from the last few years, the dengue cases are reported from urban, suburban and tropical areas as well.Thus, in current study, focusing Multan, the efficacy of plant extracted Acorus calamus Linn (Fam.Araceae) will be evaluated in comparison to the synthetic pyrethroid type-II cypermethrin 80SC against A. albopictus and A. aegypti in the laboratory.

Preparation of extraction
Acorus calamus was grinded into smaller size to enhance extraction yield of the rhizome and Soxhlet apparatus was used with hexane for at least 22 hours for extraction.Under vacuum pressure the filtrate was evaporated for dryness.Against the 4 th instar larvae and adults of Ae. albopictus and A. aegypti the crude hexane extract of A. calamus was bioassay.Insecticide Cypermethrin 80SC was purchased from agricultural market of Multan city.

Bioassay against adults of Aedes albopictus and Aedes aegypyi
The indoor and outdoor surveillance of larvae of mosquitoes collected from various localities of Shah Rukn-e-Alam Town Multan, (Punjab) Pakistan.According to WHO [20] standard procedure the bioassay was conducted after some modifications.Laboratory colonies 2-6 days old 20 adult mosquitoes of A. albopictus and A. aegypti were exposed to filter paper (16X17cm) impregnated with different concentrations of A. calamus extract and Cypermethrin 80SC in the range of 0.3220ppm to 6.0ppm for testing against aegypti and 1.0ppm to 8.0ppm for A. albopictus for 2 hours.For Cypermethrin and control impregnated papers each concentration was diluted in 0.1% acetone; by diluting the crude extract with Tween 20 and 0.1% hexane the stock solution for A. calamus extract was prepared.For each 1, 3, 5, 10, 20, 30 and 60 minutes, the knockdown was recorded.With the help of clean filter papers all the mosquitoes were then transferred to holding tubes.The mosquitoes were fed in 10% sucrose solution with cotton pads soaked.Each experiment was done in duplicate.After 24 hours, the mortality rate of the adult mosquitoes was recorded.This experiment was repeated three times with the help of Probit Analysis program [21] and SPSS Software the experiment was analyzed.

Bioassay against 4 th instar larvae of A. Albopictus and A. aegypti
According to WHO [22] the bioassay was conducted after some modification.1000ml glass beakers which contained 250ml of prepared extract of A. calamus and Cypermethrin 80SC in different concentrations were prepared to expose 20 A. albopictus and A. aegypti larvae.In 0.1% hexane the A. calamus and control were diluted, while in 0.1% acetone cypermethrin 80SC and control were diluted.The mortality of the larvae was done in duplicate and repeated three times.The percentage mortalities were corrected by Abbott's formula [23] if the control mortality was between 5% and 20%.% test mortality -% control mortality X 100 100 -% control mortality By Probit Analysis [22] and SPSS software LC50 and LC90 values were determined.

Results
The results indicated that the LC50 and LC90 values of Cypermethrin on 4 th instar larvae of A. albopictus were 0.1470 and 0.6120 pp while LC50 and LC90 values of A. aegypti larvae of 4 th instar were 0.0047 and 0.0180 ppm respectively.A significant difference (P<0.05) was to be found on the effect of Cypermethrin to both A. albopictus and A. aegypti.Similarly, the values of LC50 and LC90 of A. calamus extract on 4 th instar larvae of A. albopictus were 4.1360 and 22.2766 ppm and the values of LC50 and LC90 with A. aegypti were 5.4420 and 12.4938 ppm respectively.So, a significant difference (P<0.05) was also to be found on the effect of A. calamus extract on both A. albopictus and A. aegypti larvae.Cypermethrin showed a significant difference (P<0.05) on larvicidal effect to A. calamus extract to A. albopictus and A. aegypti (Table 1).The results also indicated that the LC50 and LC90 values of Cypermethrin on A. albopictus adults of 3.5277 and 5.2844 ppm while LC50 and LC90 values with A. aegypti 0.8220 and 3.4267 ppm respectively.A significant difference (P<0.05) was to be found on the effect of Cypermethrin to A. albopictus and A. aegypti adults.Similarly, the values of LC50 and LC90 of A. calamus extract were 52.8842 and 54.1275 ppm on A. albopictus while the values of LC50 and LC90 on A. aegypti were 18.5270 and 344.8478 ppm respectively.However, there was no significant difference (P>0.05) on the effect of A. albopictus and A. aegypti to be effective as on adulticide as compared to that of Cypermethrin (Table 2).