Abstract: A total of twenty marine algae were collected from the rocky intertidal and subtidal regions of the southwest coast of India and extracted in methanol. The extracts were evaluated for larvicidal activity against the second and third instar larvae of the human vector mosquito Culex quinquefasciatus and Aedes aegypti (Diptera: Culicidae). Analysis on the activity profile of the above marine algae indicated that the early stage larvae were very sensitive to seven seaweed extracts that had been tested. Among the seven marine algae, Lobophora variegata was highly potential, showing LD50 value of 70.38 and 79.43 μg mL-1 on the 2nd instar larvae of A. aegypti and C. quinquefasciatus respectively. The rank of larvicidal potency in highly active algae in the descending order is as follows: Lobophora variegata (Dictyotaceae)>Spatoglossum asperum (Dictyotaceae)>Stoechospermum marginatum (Dictyotaceae)>Sargassum wightii (Sargassaceae) >Acrosiphonia orientalis (Acrosiphoniaceae)>Centroceras clavulatum (Ceramiacea)>Padina tetrastromatica (Dictyotaceae). This is the first report that envisaged the mosquito larvicidal efficacy of L. variegata from the Indian coast. Therefore, this marine alga could be recognized as a potential resource of natural insecticide and can be developed to replace synthetic insecticides in future.
INTRODUCTION
Mosquitoes coming under the order Diptera, are ravaging humans and other animals for generations. There are nearly 2,500 mosquito species in the world but a mere fraction of them transmit an array of pathogens including viruses (e.g., arboviruses), protozoans (e.g., malaria) and nematode worms (e.g., lymphatic filariasis). According to Taubes (1997), annually more than 700 million people suffer from mosquito-borne diseases. The immense usage of many synthetic aerial, terrestrial and aquatic insecticides offer logistic problems on the environment (Chowdhury et al., 2008) and causes resurgence of different mosquito-borne diseases (Milam et al., 2000). Costs and complexity of mosquito control have increased strikingly since the passage of the Environmental Protection Act in 1969. Environmental managers practiced the bio-control agents such as fishes, birds, dragonflies and frogs but feedback on the effectiveness of these agents is only anecdotal and some mosquito species have developed high levels of resistance to microbial control agents (Rajkumar and Jebanesan, 2005). It is therefore high time to develop an alternative strategy in mosquito control management in order to circumvent existing resistance thus reducing the risk of new resistance.
For many centuries, phyto-products have been used traditionally by human communities to fend off vector and pest species of insects (Jang et al., 2002; Pavela, 2007). The application of phyto-products is considered to be one of the counter measures to control pests and vectors (Manilal et al., 2009b). The first observation pertaining to larvicidal activities of botanicals was reported by Campbell et al. (1933). In recent years, marine natural products from sponges, microbes and algae have been screened for larvicidal activity (Rao et al., 2008; Selvin and Lipton, 2004). Marine flora and fauna produce diverse novel bioactive metabolites unique to the environment (Manilal et al., 2009a, 2010a, 2011a,b). Over the last decade, numerous secondary metabolites with complex chemical structures and different bioactivity spectra have been characterized from marine algae and recognized as a virtually untapped reservoir of novel drug leads (Manilal et al., 2010c). Many of these metabolites have toxicological, pharmacological and ecological importance (Manilal et al., 2010b). Therefore, the marine derived algal products may serve as a suitable alternatives to synthetic insecticides in future as they are relatively safe, biodegradable and are easily available around the world. The objective of the current study was to evaluate the larvicidal potency of 20 marine algae collected from the southwest coast of India against second and third instar larvae of two urban mosquito species.
MATERIALS AND METHODS
The present study was carried out in Marine Bio-Prospecting Laboratory, Bharathidasan University, Trichy, India between April to December 2010. Algae belonging to Chlorophyta, Phaeophyta and Rhodophyta were collected from the intertidal and subtidal habitat of Kollam (08° 89' N and 76° 54' E) area (Table 1) during April to December 2010 when algal diversity remains greater. The study area, southwest coast of India exhibits small intertidal pools, large and shallow sand bottom flats, numerous irregularly distributed laterite rocks, cobbles and scattered granite boulders inhabited with a wide variety of seaweed floristic biomass (Sulekha and Panikkar, 2006; Mantri, 2006). Live and healthy plants were handpicked and washed thoroughly in running water to remove adhering detritus matters. Cleaned algal samples were wrung between blotting paper and shade dried until it becomes brittle. After completing the shade drying process, they were chopped into small pieces and dried in an oven at 40°C. The completely dried material was weighed and ground coarsely in a mechanical grinder. Algae were extracted in methanol as per Manilal et al. (2010b). The resultant extractive were collected in air–tight plastic vials and stored in the refrigerator at 4°C for further activity studies.
Bioassays: The assessment of larvicidal activity of various extracts was tested against the urban mosquitoes C. quinquefasciatus and A. aegypti using standard bioassay protocol. Eggs of respective mosquitoes were obtained from drainage systems of Tiruchirappalli city (Tamilnadu, South India). Eggs were reared under standard insectary conditions at ambient temperature (29±3°C), relative humidity 80±5%, 12:12 light: dark photoperiod and fed with ground shrimp feed daily. Larval development was monitored for seven days. The second and third stage larvae were collected at the tip of a pasture pipette and placed in cotton bud to remove excess water and transferred gently to the test vial (10 / vial) by tapping. The larval mortality was observed using various concentrations of seaweed extracts (100, 200, 300 and 400 μg mL-1) including positive (with 2% methanol) and negative controls (without vehicle).
| Table 1: | Extract yields of algae collected from the southwest coast of India |
The number of larvae surviving after 24 h of exposure was recorded. Wriggling movement after probing it with a needle identified deceased larvae. Toxicological effects were recorded in all trials in order to maximize consistency. All the experiments were repeated six times to validate the findings statistically (Wardlaw, 1985).
RESULTS
Overall objective of the present study was to compare the ability of organic extract of marine algae collected from the same habitat to inhibit the survival of mosquito larvae. Larvicidal assay was performed with the extract of twenty marine macro algae against the second and third instar larvae of C. quinquefasciatus and A. aegypti. The data for the amount of algal extract yielded are presented in Table 1. Of the 20 marine algae, yield of extract was highest (0.185 g dry-1 weight) for the Gracilaria corticata and lowest (0.034 g dry-1 weight) for the Enteromorpha intestinalis. The data also revealed the considerable variability in the concentration of the extract and larvicidal activity. Comparing the two species representing the different mosquito genera, C. quinquefasciatus was found to be more sensitive to seaweed extracts than A. aegypti. It was also found that the red and brown seaweed groups had high potency than green algae. Data obtained from the larvicidal activity of seaweed extracts at the end of 24 h suggested that the second instar larvae of both species were highly sensitive than the third instar. Of the 20 seaweed extracts tested, the dose level 200 μg mL-1 of seven species showed 100% mortality in both species of second instar larvae. These algal extracts showed high larvicidal activity, their LD50 after 24 h ranged between 70.38 and 97.41 μg mL-1 against second instar larvae of A. aegypti (Table 3) and 79.43 and 97.94 μg mL-1 against C. quinquefasciatus (Table 2). Among the algae tested, L. variegata was the highly potential and showed LD50 value of 70.38 and 95.52 μg mL-1, respectively against the second and the third instar larvae of A. aegypti.
| Table 2: | LD50 values of C. quinquefasciatus larvae against effective seaweed extracts |
| Values are Mean±SD (n = 6) | |
| Table 3: | LD50 value of A. aegypti larvae against effective seaweed extracts |
| Values are Mean±SD (n = 6) | |
The same algae produced a LD50 value 79.43 and 96.52 μg mL-1, respectively against C. quinquefasciatus. The second most effective seaweed was S. asperum which showed LD50 at 81.28 and 96.13 μg mL-1 against A. aegypti larvae and in C. quinquefasciatus, LD50 was 83.17 and 97.71 μg mL-1, respectively. However, a lower level of activity was observed in other species of algae and their LD50 value was above 150 μg mL-1 (data not shown). Although the trend of larval mortality within 24 h was consistent between the larval stages and the mosquito exposed to 100 μg mL-1 of L. variegata extracts showed exceptionally 80% mortality at the end of 24 h. In the highest dose level of L. variegata (200 μg mL-1), the 80% larvae (second and third instar) of both vectors showed behavioral changes. After 1 h of exposure, the larvae exhibited primary phase of behavioral changes including restive wriggling with rapid surface surge and loss of co-ordination while the secondary phase began after 6 h and larvae exhibit loss of wriggling activity. During the tertiary phase (12-24 h) the larvae showed loss of response to tapping and probing with needle and finally inclined to the bottom of the test vial followed by mortality.
DISCUSSION
Many marine plants synthesize a variety of chemically diverse secondary metabolites in response to selection pressures from herbivores and microorganisms and some of these compounds are recognized as insecticides. The management of adult mosquito stocks using adulticides is not a successful strategy, as the adult stage occurs beside human inhabitation and they can simply overcome remedial measures (Service, 1983, 1992). Mosquito control management including periodic larviciding (WHO, 1975; Becker et al., 2003) is very helpful in favorable conditions. Larval stage of mosquitoes are exclusively aquatic, systematic treatment of plant based larvicides in their breeding habitat is a successful and safer way to interrupt larval stages of vectors rather than the infective adult stage (Thangam and Kathiresan, 1991).
Mosquitoes are the vector for large number of human pathogens than any other group of arthropods (El-Hag et al., 1999; Cepleanu, 1993). The uncontrollable breeding of mosquitoes is posing a serious threat to the humanity. The mosquito borne diseases not only cause high levels of morbidity and mortality but also cause great economic loss and social chaos in the developing countries including costs of health care and negative impacts on tourism and real estate values. India alone carries around 40% of global filariasis burden and the estimated annual economic loss is about 720 crores (Hotez et al., 2004). Recent figures from the World Health Organization (WHO) evidenced that malaria accounts for at least 500 million infections and 3 million deaths annually. The prevalence of dengue fever has increased over the last 50 years and over 2 billion people are under risk in more than 100 countries. In 2007, incurable crippling disease Chikun Gunya, spread primarily by A. aegypti species has resurfaced in Kerala, a southern state of India and claimed more than hundred lives.
Many plant derived natural compounds was tested for mosquito control (Oladimeji et al., 2007, 2008; Vinayagam et al., 2008; Chakkaravarthy et al., 2011; Almehmadi, 2011). Ishibashi et al. (1993) reported the extracts of Aglaia elliptifolia showed insecticidal activity. Polyhalogenated monoterpenes, aplysiaterpenoid A and telfairine isolated from Plocamium sp. exhibited insecticidal activity against the mosquito larvae (Watanabe et al., 1989). Early studies envisaged that the Indian marine plant extracts possessed potential larvicidal activity (Thangam et al., 1993; Rao et al., 1995). Furthermore, studies with secondary metabolites isolated from algae showed significant larvicidal activity (Selvin and Lipton, 2004; Alarif et al., 2010).
Of the algae screened, L. variegata was effective against, both mosquito species in relatively lower concentration. There is no previous report on the mosquito larvicidal activity of L. variegata from the Indian coast.
The organic extract of algae, D. dichotoma, H. musciformis and U. fasciata found to be larvicidal in the previous reports were less active in the present study. This may be due to the fact that the bioactivity of same taxa can vary with geographical zone and could depend on ecological parameters such as irradiance and nutrients (Marti et al., 2004).
In conclusion, overall data of this study illustrate that the marine alga, L. variegata is a potential candidate for the eco-congenial larvicide development programme as an alternate to chemical insecticides that are being currently used in mosquito vector control programs. Our investigations leads to the following suggestions: (1) L. variegata can be cultivated in coastal areas (2) the plant bioactives can be extracted by cost effective methods with high yield (3) larvicidal activity was potent and safe to non target cohabitants (4) synergistic activity will defend the development of resistance in vectors. Till date, no seaweed bioactives have been operationally used for mosquito control. Therefore, systemic bio-efficacy evaluation in field condition is further warranted.
ACKNOWLEDGMENT
Manilal, A is grateful to the Council of Scientific and Industrial Research (CSIR), New Delhi, India, for the award of Senior Research Fellowship.