Larvicidal activity of marine algae, Sargassum swartzii and Chondria dasyphylla, against malaria vector Anopheles stephensi.
Malaria is still a major endemic disease in foci located in south and southeast of Iran. The annual malaria cases have been reported from 66,075 to 6,211 during 1995-2009, indicating the sharp decline of disease. It is unstable with two seasonal peaks mainly in spring and autumn. These areas include the provinces of Sistan and Baluchistan, Hormozgan and Kerman (1). In this part of the country, six anopheline mosquitoes including An. culicifacies, An. stephensi, An. dthali, An. fluviatilis, An. superpictus, and An. pulcherrimus (Diptera: Culicidae) are known to be the malaria vectors and An. sacharovi and An. maculipennis are considered as malaria vectors in northern part of the country (2-7).
Chemical control methods have been applied against either the immature or the adult of malaria vectors. Applying chemical parricides is the most important part of such program. Mosquito control, using chemical larvicides has been performed during the fight against malaria in Iran and still considered as an important part of vector control. Chemical larvicides are now considered as toxic material to fish and other non-target organisms as well as the environment. They are also responsible for increase of insecticide resistance in arthropods. The extract of whole leaf and essential oil of certain plants have been investigated, and showed toxic effect against some public health pests (8-10). Several species of marine algae from coastlines of Iranian islands and Hormozgan province have been reported (11). Marine algae produce different secondary metabolites with a wide range of biological activities (12). Many studies have been achieved on the screening of biological effects of marine organisms and many active compounds were isolated and characterized (13). Red algae from genus Chondria are known as a producer of cyclic polysulfides, terpenoids, amino acids and amines. Domoic acid derivatives with larvicidal and lowering blood pressure activity have been identified in Chondria armata (14). Secondary metabolites with cytotoxic and antitumor activity have been extracted and identified in Sargassum species (15-16). This study was aimed to determine the larvicidal activity of different extracts of S. swartzii and C. dasyphylla, collected from coastlines of the Persian Gulf, southern Iran, against main malaria vector An. stephensi.
MATERIAL & METHODS
Brown algae, Sargassum swartzii C. Agardh (Sargassaceae), Chondria dasyphylla (Woodward) C. Agardh (Rodomelacea), were collected from Asaluye-Niband marine protected area of the Persian Gulf in February 2008. The algae were identified by Dr J. Sohrabipour at the Agriculture and Natural Resource Research Center of Hormozgan (herbarium numbers are 20,424, 20,426 respectively) and the voucher specimens were deposited at this center.
Extraction of marine algae
The algae were air-dried in the shade at room temperature and were smashed to make a powder with a mortar and pestle. Each sample of 200 g was extracted with MeOH-[H.sub.2]O (70:30) (5x200 ml) at room temperature. The combined extracts were evaporated under vacuum. The residues were subjected to Silica gel (230) mesh and diluted successively with n-Hexane, CH[Cl.sub.3], EtOAc and Methanol. Removal of the solvents resulted in the production of n-Hexane, CH[Cl.sub.3], EtOAc and MeOH-[H.sub.2]O fractions.
Different extracts of S. swartzii and C. dasyphylla were evaluated against late III and early IV instar larvae of An. stephensi. The mosquitoes were collected from malarious areas of Iran, and then were maintained at the insectary of School of Public Health & National Institute of Health Research, Tehran. The reared susceptible larvae to different insecticides were exposed to different concentrations of the S. swartzii and C. dasyphylla extracts which were prepared in methanol. The minimum concentration was 2.5 mg/l and the maximum was 40 mg/l. These concentrations gain the appropriate mortality to plot the regression line. Mortality was determined after 24 h exposure period. All the tests were conducted at 30 [+ or -] 1[degrees]C and 60 [+ or -] 5% relative humidity, and 10 : 14, dark : light periods respectively in the laboratory conditions (17,18). For each concentration, at least 4 replicates of (25) individuals were used (19).
The mortality data were subjected to probit analysis using Finney studies (20). From the regression line between logarithmic dose and probit mortality all the parameters including [LC.sub.50] and 95% confidence interval, [LC.sub.90] and 95% confidence interval were determined (21). The regression line was plotted using Microsoft Excel.
Mortality data of An. stephensi exposed to different extracts of two algae, S. swartzii and C. dasyphylla are shown in Table 1. The EtOAc fraction of both S. swartzii and C. dasyphylla were found to be more effective than the other fractions and total extract. Other fractions didn't show significant larvicidal effect against An. stephensi. For EtOAc fractions the chi-square values were significant at p <0.05 level (22). [LC.sub.50] and [LC.sub.90] values for S. swartzii were 11.7584 and 53.472 ppm respectively, and values for C. dasyphylla were 10.625 and 56.394 ppm, respectively (Table 2). The probit regression line is plotted in Fig. 1. From this probit regression line different parameters about efficacy of product against malaria vector can be calculated.
Secondary metabolites with broad range of activities have been found in marine algae. To evaluate the larvicidal effect of the algae from the Persian Gulf against An. stephensi, the samples were extracted with methanol (70%) and fractions were obtained by using various polar and non-polar solvents.
[FIGURE 1 OMITTED]
In a previous study on antiplasmodial and antimicrobial activities of South African marine algal extracts, the dichloromethane fraction of Sargassum heterophyllum showed the most antiplasmodial effect with [IC.sub.50] value of 2.8 [micro]g/ml against chloroquine sensitive strain of Plasmodium falciparum (D10) (23).
Exposure of An. stephensi larvae to sub-lethal doses of neem extracts in the laboratory prolonged larval development, reduced pupal weight, high oviposition deterrence and high mortality (24). Some researchers have shown ethanol extract of aerial parts of Tagetes minuta had larvicidal effects with [LC.sub.50] value about 2.5 mg/l (25). Also for Conyza albida, [LC.sub.50] value of 2 mg/l and for Artmisisa afra, [LC.sub.50] of 5 mg/l has been determined (26). In another report for Maytenus senegalensis, [LC.sub.50] value was about 3.9 mg/l and for Harrisonia abyssinica [LC.sub.50] 4.7 mg/l have been reported (27).
In conclusion, larvicidal effects of EtoAc fractions of S. swartzii and C. dasyphylla could be related to semi-polar compounds existing in both algae. The extracts from these plants may be useful for improvement of new natural insecticides, however, further investigations are needed to identify and purify the effective components and their mechanisms of actions of these algae.
This study is a part of Pharm. D. thesis funded and supported by the Tehran University of Medical Sciences
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Mahnaz Khanavi , Pouyan Bagheri Toulabi , Mohammad Reza Abai , Nargess Sadati , Farzaneh Hadjiakhoondi , Abbas Hadjiakhoondi  & Hassan Vatandoost 
 Department of Pharmacognosy and Medicinal Plant Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran;  Department of Medical Entomology & Vector Control, School of Public Health & National Institute of Health Research, Tehran University of Medical Sciences, Tehran, Iran
Correspondence to: Dr Hassan Vatandoost, Department of Medical Entomology & Vector Control, School of Public Health & National Institute of Health Research, Tehran University of Medical Sciences, P.O. Box 6446-14155, Tehran, Iran.
E-mail: email@example.com; firstname.lastname@example.org
Received: 13 May 2011
Accepted in revised form: 13 December 2011
Table 1. Comparison of larvicidal effect of different extracts of Sargassum swartzii and Chondria dasyphylla on An. stephensi larvae Concentration Total Total Mortality Samples (ppm) tested dead (%) Control 70% (methanol) 50 0 0 Sargassum swartzii Total extract 40 50 4 8 Chloroform 40 50 0 0 Ethyl acetate 40 52 50 96.1 MeOH 40 49 3 6.1 Chondria dasyphylla Total extract 40 50 3 6 Chloroform 40 49 0 0 Ethyl acetate 40 51 47 92.1 MeOH 40 50 2 4 Note: The larvae were exposed to a 40 ppm concentration of different extracts of Sargassum swartzii and Chondria dasyphylla which were prepared in methanol. Mortality was determined after 24 h exposure period. For each extract, at least 2 replicates were used. Table 2. Probit regression line parameters of extract of S. swartzii and C. dasyphylla against larvae of An. stephensi Intercept (a) Slope (b [+ or -] S.E.) [LC.sub.50] in [LC.sub.90] in ppm (95% C.L.) ppm (95% C.L.) S. swartzii -2.0854 1.9484 [+ or -] 0.446 4.9945 41.2960 (11.75-33.78) (53.47-75.35) C. dasyphylla -1.8145 1.7680 [+ or -] 0.385 4.5996 42.2081 (10.62-12.46) (56.39-83.64) Intercept (a) [e.sup.2] [e.sup.2] p-value (heterogeneity) table (df) S. swartzii -2.0854 23.515 7.81 (3) 0.05 C. dasyphylla -1.8145 18.334 7.81 (3) 0.05
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|Author:||Khanavi, Mahnaz; Toulabi, Pouyan Bagheri; Abai, Mohammad Reza; Sadati, Nargess; Hadjiakhoondi, Farza|
|Publication:||Journal of Vector Borne Diseases|
|Article Type:||Clinical report|
|Date:||Dec 1, 2011|
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