Screening of Different Extracts of Marine Macro Green Algae for Larvicidal Activity against Dengue Fever Mosquito, Aedes aegypti (Diptera: Culiadae)

. The present study larvicidal activities of hexane, chloroform, ethyl acetate, acetone and methanol extracts of Halimeda macroloba, Decsne, Caulerpa racemosa (Frosk) Weber-Van-Bosses and Ulva lactuca Lin, (Chlorophyceae) against Aedes aegypti. The marine macro green algae extracts were tested against early 4 th instar larvae of A. aegypti using WHO protocol and concentrations of 200, 400, 600,800 and 1000 ppm. The observed mortality was made 24 and 48 h after treatment, data was subjected to probit analysis to determine the lethal concentration (LC 50 and LC 90 ) of the treated larvae of the tested species. Among the tested extracts the maximum efficacy was observed in the ethyl acetate extracts. The ethyl acetate extracts of the seaweeds showed the presence of terpenoids, tannins and phenolic compounds stronger than the other extracts. The results revealed that all the extracts showed varied levels of larvicidal activity against A. aegypti tested. However, the ethyl acetate extract of C. racemosa showed remarkable larvicidal activity against A. aegypti (LC 50 =579.9 and LC 90 =1255.4 ppm values at 24 h and LC 50 =495.4 and LC 90 =1073.9 ppm at 48 h) followed by U. lactuca (LC 50 =588.1 and LC 90 =1290.7 ppm values at 24 h, and LC 50= 530.8, and LC 90 = 1160.0 ppm at 48 h), respectively. The lowest larval mortality was observed with hexane extract of H. macroloba against A. aegypti with values of LC 50 =1116.8 and LC 90 = 1824.5 ppm (after 24 h) and LC 50 =1059.9 and LC 90 =1768.3 ppm (after 24 h). The present studies indicate that the larvicidal activity and phytochemicals derived from the ethyl acetate extract of C. racemosa have the potential to be used as an ideal eco-friendly approach and effective mosquito vector control agent.


Introduction
Marine algae are one of the most important producers of biomass in the marine environment. They produce a wide variety of chemically active metabolites in their surroundings to protect themselves against other settling organisms [1]. So far, it was evidenced that marine algae are potential antimutagenic, anticoagulant and antitumor agent. Up to now, numerous studies have confirmed the anti-HSV activity of algae that increase the interest in algae as a source of antiviral compounds. Marine macro algae are an excellent source of vitamins such as A, Bl, B12, C, D, E, riboflavin, niacin, pantothanic acid and folic acid as well as minerals such as Ca, P, Na and K [2]. Chlorophyceae seaweeds, popularly known as green algae, are widely distributed in both inter-tidal and deep-water regions of the seas. More recent reports indicate that in many parts of the world marine algae are still used in folk medicine for the treatment of a variety of disease [3].
Vector-borne diseases are dengue, malaria, filariasis, dengue haemorrhagic fever schistosomiasis, leishmaniasis, chagas disease, yellow fever, lymphatic filariasis, African trypanosomiasis and onchocerciasis [4]. Aedes aegypti (Culicidae) occurs in Asia, Africa, Central and South America and transmits virus of Flavi virus genus, etiologic agents of human diseases like dengue and yellow fever [5]. On the other hand, there is no vaccine for dengue fever; vector control is the only method used to minimize the transmission of the virus. Dengue fever can be caused by four serotypes of the dengue arbovirus and clinically can happen in asymptomatic forms, classic dengue fever, hemorrhagic dengue fever and other more severe forms. Worldwide, 2.5 billion of people are in risk to acquire the disease and 50 million are infected every year, characterizing a pandemia [6].
Plant extracts and phytochemicals have potential as mosquito control agents because many of them are selective, may biodegrade to nontoxic products and may be applied to mosquito breeding sites in the same way as conventional insecticides [7]. Each year, an increasing number of novel marine metabolites are reported in the literature, indicating that the marine environments is likely to continue to be a prolific sources of more natural products for many years to come. Several studies have demonstrated that marine algae have biological activity such as larvicidal, antifeedant and anticoagulant [8].
Therefore, the present study was carried out to evaluate the larvicidal properties of marine macro green algae of different extracts against the dengue fever mosquito, A. aegypti.

Preparation of extracts
Algal species were handpicked from rocks and shells submerged under water during low tide. They were washed thoroughly with sea water to remove all unwanted impurities, epiphytes, animal casting and adhering sand particles etc., Morphologically distinct thallus of algae were placed separately in new polythene bags and were kept in an ice box containing slush ice and transported to the laboratory. Then, the samples were blot dried using sterile tissue paper. The seaweed materials dried in one week in room temperature. After getting from all the samples were grounds in to a fine powder. Five hundred grams of finely ground algal powder material were packed in Whatman filter paper. The powdered samples were extracted with different organic solvents in a Soxhlet apparatus for 72 hours with increasing polarity viz., hexane, chloroform, ethyl acetate, acetone and methanol. The extracts were concentrated to solvents free by evaporation in a rotary vacuum evaporator (Heidolph, Germany) at a temperature less than 40 °C. The crude extracts were obtained were kept at 4 °C for larvicidal assay.

Mosquito larvicidal assay
The eggs of A. aegypti were received from the Field Station, Centre for Research in Medical Entomology (ICMR-Government of India), Viruthachalam. The egg rafts of A. aegypti were kept in the tray containing tap water (culture medium) at a laboratory condition (29 ± 3 °C , 75 to 85 % RH, 14:10 light and dark photo period). After 24 h of incubation, the eggs were observed for hatch out in to first instar larvae. Appropriate amount of nutrient (sterilized fed with Brewer's yeast/ dog biscuit 1:3 ratio). The 4 th instar larvae were used in the study.
The larvicidal activity was observed as per the standard procedures recommended [10]. The seaweeds extracts were dissolved in 2 ml dimethyl sulfoxide (DMSO) and prepared into different International Letters of Natural Sciences Vol. 62 concentrations viz., 200, 400, 600, 800 and 1000 ppm were prepared with distilled water. Twenty larvae (in a 100 ml beaker) of early fourth instar stage were used for larvicidal assay and five replicates were maintained for each concentration. During this experiment, no food was offered to the larvae. The larval mortality was calculated after 24 and 48 hours of the exposure period. All moribund mosquito larvae were considered as dead. The larval mortality was also checked for water and DMSO individually.

Statistical Analysis
The results are expressed as the mean  SD. All statistical analyses were performed using SPSS version 16.0 statistical software (SPSS Inc., Chicago, IL, USA). The average larval mortality data were subjected to probit analysis calculating LC 50 , LC 90 and other statistics, 95% confidence limits and chi-square values were calculated.

Phytochemical analysis
The hexane, chloroform, ethyl acetate, acetone and methanol extracts were analyzed for phytochemicals, terpenoids, tannins, cardiac glycosides, steroids, alkaloids, phenolic compounds and coumarins ( Table 1). The ethyl acetate extracts of H. macroloba, C. racemosa and U. lactuca showed the presence of phytochemicals, terpenoids, tannins and phenolic compounds strongly than the other extracts. Among the phytochemicals, cardiac glycosides were present in all the extracts except acetone and methanol. Steroids were present in all the extracts of C. racemosa, H. macroloba and U. lactuca expect actone and methanol extracts of H. macroloba. Alkaloids and coumarins were absent in all the extracts of H. macroloba, C. racemosa and U. lactuca.

Larvicidal activity
Current strategies based on the elimination of breeding sites and applications of chemical insecticides for larval and adult mosquito control have resulted in development of resistance without eliminating the constant risk of dengue epidemics [11]. Thus new approaches are urgently needed. Interest on possible use of environment friendly natural products such as extracts of plants or plant parts increased for vector control. Plant derived products have received increased attention from scientists and more than 2000 plant species are already known to have insecticide properties [12]. Algae synthesize a number of chemically diversified secondary metabolites. Among them, some of the compounds are recognized as insecticides. The control of adult mosquito is an unsuccessful strategy, as the adult stage occurs beside human inhabitation and they can easily overcome remedial measures [13].
Padina minor showed significantly greater larvicidal activity compared to D. linearis at LC 50 . Greater larvicidal activity of Padina than Dictyota was also observed in P. tetrastromatica and D. dichotoma from southwest Coast of India [17] .   Table 3. Mortality effects of 48 hours A. aegypti exposed to different concentration of crude extracts of selected Marine macro algae.  [19].
In this study, the ethyl acetate extract of C. racemosa green alga the highest larvicidal activity may be due to the presence of phytochemicals, terpenoids, tannins and phenolic compounds. Many International Letters of Natural Sciences Vol. 62 of the secondary metabolites produced by plants for its protection against microorganisms and predator insects are natural source for the discovery of new plants based products to combat A. aegypti. Many researchers have been focused on natural products for controlling Aedes mosquito as insecticide and larvicide with varied results [20]. The biological activities of this marine plant extracts might be due to various compounds, including phenolics, terpenoids, flavonoids, saponins and alkaloids existing in plant. These compounds may jointly or independently contribute to produce larvicidal activity against mosquitoes [17].

Conclusion
The results of the present study suggest the larvicidal activity of hexane, chloroform, ethyl actate, acetone and methanol extracts of H. macroloba, C. racemosa and U. lactuca against A. aegypti. Among the ethyl acetate crude extract of C. racemosa could be used for mosquito control. The C. racemosa ethyl acetate extract can be well utilized for preparing biocide or insecticide formulation, effects on non-target organisms and field evaluation are necessary. In that way, the results of the present study offer a possible way for further investigations to find out the active molecule needed to elucidate this activity against a wide range of all stages of mosquito species.