1 Introduction
Control of disease-bearing vectors relies heavily on the extensive and intensive use of chemical insecticides. These chemicals are to certain extent quite successful in controlling the diseases concerned. In view of some of the side effects of chemical agents used in vector control, environmental friendly approaches and the use of biological control agents have gained much importance. In the case of mosquitoes, phytoplankton deserves particular attention (Clements, 1992).

Some species of phytoplankton provide nutritious food for mosquito larvae, whereas others produce allelochemicals that are toxic to mosquitoes at different stages (Kiviranta and Abdel-Hameed, 1994; Gross, 2003; Legrand et al., 2003; Graneli and Hansen, 2006; Rey et al., 2009). It is common in nature for mosquito larvae to die before completing their development because they are poisoned by phytoplankton toxins or they starve to death while feeding on phytoplankton that are indigestible (Ahmad et al., 2001; Marten, 2007). Mosquito indigestible phytoplankton have good field characteristics as a biological control agent against mosquitoes because they are naturally present in the habitats of mosquito larvae and are able to multiply and persist in these habitats. Another major advantage of phytoplankton for mosquito control is the expectation that mosquitoes will not evolve resistance to their use (Ahmad et al., 2001). The objective of the present study is to investigate the effect of twelve species of marine phytoplankton on the survival and development of mosquito larvae of Culex quin -quefasciatus.
 
2 Materials and Methods
2.1 Isolation and maintenance of phytoplankton cultures
Twelve phytoplankton species including nine cyano bacteria (Chroococcus turgidus, Lyngbya confervoides, Nostoc commune, Oscillatoria fremyii, O. geminata, O. sancta, Phormidium corium, P. tenue and Spirulina major), two diatoms (Chaetoceros calcitrans and Skeletonema costatum) and the planktonic green alga (Nannochloropsis oceanica) were isolated from rocks, puddles and sea water of Arabian Sea coast (West coast of India).

The filamentous cyanobacteria were isolated by micr -opipette method, whereas unicellular cyanobacterium (Chroococcus turgidus), diatoms and planktonic green alga were isolated by agar plate method (Andersen, 2005). The cultures were microscopically examined for the assessment of growth and contamination. The successful axenic cultures were diluted and subcultured to 100 ml of culture media in 250 ml conical flasks. The cultures of diatoms and planktonic green alga were maintained in Walne’s medium at 20±2˚C, whereas cyanobacteria were cultured in f/2 medium at 28±2˚C and incubated under illumination of 1000 lux with 8 : 16 h light and dark regime.
 
2.2 Harvesting of cultures
The cultures were harvested at stationary growth phase by centrifugation at 3500 rpm for 10 min, whereas filamentous cyanobacteria cultures were recovered by filtration. The harvested cells were diluted with distilled water to obtain a concentration of OD at 620 nm = 0.6 (Ahmad et al., 2001). For filamentous cyanobacteria, the homogenized cultures to a similar cell density were used.
 
2.3 Evaluation of phytoplankton isolates as larvicides against mosquito larvae
Larvicidal activity of the phytoplankton suspensions against mosquito larvae was determined by transferring 200 ml suspension to a glass beaker containing 25 numbers of second-instar larvae of Culex quinquefasciatus. Three replicates were used each time and test was repeated two times. The control consisted of larvae in distilled water fed with 50:50 finely ground brewer’s yeast and egg albumin (Rey et al., 2009). The food was added to the containers at a rate of approximately 0.5 mg/larva/day.

The larval mortality was assessed after every 24 h and Lethal Time (LT) values were calculated using Probit analysis (Finney, 1971). The percentage of mortality for each test was calculated. Daily observations on larval and pupal mortality were continued through adult emergence or until termination of the test. Adult body size was determined by measuring the wing length (distance from axial incision to the apical margin, excluding fringe of scales) of each individual (Rey et al., 2009) and compared with the control by Analysis of Variance.

The larval digestibility was tested by transferring 25 larvae into the 100 ml of phytoplankton suspensions. The larvae were allowed to feed for one hour and removed from the suspension. The phytoplankton cells attached to larvae were washed with distilled water and placed in distilled water to allow for further digestion. The gut contents were then teased from the membrane into a vial containing sterile distilled water and observed under phase contrast microscope. Cell counts were carried out to determine the percentage digestion of the phytoplankton cells.

3 Results
Among the 12 phytoplankton isolates, mortality of the Culex quinquefasciatus larvae was observed in the suspensions of cyanobacteria species namely, Nostoc commune, Phormidium corium, P. tenue and planktonic green alga Nannochloropsis oceanica. No mortality of larvae was observed up to 3 days except in Nannochloropsis oceanica suspension which showed 32% of larval mortality. After 7 days, the percentage mortality of larvae fed with Nostoc commune, Phormidium corium, P. tenue and Nannochloropsis oceanica was found to be 44%, 48%, 48% and 100%, respectively (Table 1).

The development of larvae fed with phytoplankton cells was delayed compared to the control (larvae fed with normal food) with respect to the first pupation period. But larvae fed with P. tenue did not show significant difference with control. The days taken for the 50% of adult emergence was comparatively high than the control, whereas growth and adult emergence was faster in larvae fed with diatom Chaetoceros calcitrans as that of control. The adults emerging from the treatments were further analyzed for their body size by measuring the wing length. The wing length of the adults emerged from the larvae fed with phytoplankton cells were shorter than control and those of adults emerged from Chroococcus turgidus, Oscillatoria geminata, Chaetoceros calcitrans and Skeletonema costatum treatment were similar in size to that of control. From the statistical analysis, very few significant treatment effects on development times and mosquito size in the individual trials were observed. The larvicidal property of the isolates was determined by calculating the Lethal Time (LT50 and LT90). More than 50% of mortality was seen only in the larvae fed with N. oceanica. The LT50 and LT90 were found to be 4.98 and 6.14 days. The percentage of undigested N. oceanica cells was found to be 91.23% (Table 2).

4 Discussions
Phytoplankton are consumed by mosquito larvae (Marten, 2007). But some species of phytoplankton such as Oscillatoria agardhii, Microcystis aeruginosa, Anabaena solitaria, A. circinalis, Akashiwo sanguine and Chlorella ellipsoidea have lethal effects in the development and survival of mosquito larvae (Kiviranta and Abdel-Hameed, 1994; Saario et al., 1994; Harada et al., 2000). The effects of phytoplankton could be toxic to aquatic stages of mosquitoes, local reduction or elimination of mosquito populations by their indigestibility or modification of the reproductive cycles (Tuno et al., 2006; Rey et al., 2009).

In the present study, the planktonic green alga, Nannochloropsis oceanica was found to be the most effective larvicide against the test mosquito larvae, with most dying with their guts full of microalgae cells. The larvae showed no growth and died within few days during the second or third instar stage of development. The larvae which reached the fourth instar stage were usually in an emaciated condition. The 100% mortality of larvae fed with N. oceanica cells was observed. Ahmad et al. (2004) reported the similar observation when Aedes aegypti larvae treated with Chlorella vulgaris culture. They also observed the shrunken appearance of treated larvae and concluded that microalgae may induce some morphological abnormalities in the mosquito larvae. In the remaining culture suspension, no significant larvicidal effect was seen. Rashed and El-Ayouty (1992) investigated that Chlorella vulgaris has some mosquito regulating effects and it is not a sufficient food source for larval development when tested against Culex pipiens.

The digestibility of microalgae cells by Culex quinquefasciatus showed that Nannochloropsis oceanica cells were found to be resistant to digestion (92.23%). This suggested that death of the larvae at second/third instar stage of development might be due to the indigestibility of microalgae cells. The poor digestibility of N. oceanica may be caused by the resistance of the thick and rigid cell wall to disruption by digestive processes (Becker, 2007; Marshall et al., 2010). Ahmad et al. (2004) reported that digestibility of the microalgae in larval food is determined by the resistant properties of their outer wall and duration of exposure in the gut. Many researchers found that cell wall of Chlorophytes consist of sporopollenin, a carotenoid polymer impervious to all mosquito larval digestive enzymes (Atkinson et al., 1972; Ahmad et al., 2004).

The larvae fed with phytoplankton isolates such as, Chroococcus turgidus, Lyngbya confervoides, Nostoc commune, Oscillatoria fremyii, O. geminata, O. sancta, Phormidium corium, P. tenue, Spirulina major and Skeletonema costatum were able to reach adult but growth was slow. The wing length of adult emerged from larvae fed with all cyanobacteria isolates except Chroococcus turgidus and Oscillatoria geminata comparatively shorter than the control but significant variation was not found. Rashed and El-Ayouty (1991) reported that some green algae produce substances that inhibit larval development and delay the development of the surviving larvae to the adult stage. The delay in the development of mosquito larvae fed with Chlorococcum sp. and Scenedesmus quadricauda was observed by Ahmad et al. (2004). Feeding of Aedes aegypti on Anabaena circinalis and Oscillatoria agardhii causes lesions in the midgut epithelial cells of the larvae (Abdel-Hameed and Kiviranta, 1993; Saario et al., 1994).

Conversely, larvae fed on Chaetoceros calcitrans showed enhanced development and no mortality was seen. All larvae developed normally to the adult stage and growth was rapid than the control. Similar observation was also made by Ahmad et al. (2001) in larvae of Aedes aegypti fed with Ankistrodesmus convolutus.

Rey et al. (2009) studied the effect of phytoplankton such as Skeletonema costatum, Chlorella pyrenoidosa, C. vulgaris, Scrippsiella sp., Nitzschia kuetzingiana, N. palea, Akashiwo sanguinea, Entomoneis cf. delicatula, Thalassiosira weissflogii, Melosira lineata, Microcystis aeruginosa, Pandorina morum, Prorocentrum micans and Scenedesmus quadricauda on development and survival of the mosquito larvae Aedes aegypti using log-growth phase and senescent-phase cultures. They observed that larvae exposed to Microcystis aeruginosa producing microcystin toxin had significantly longer development times than the controls or those grown with non-toxic strain and larvae exposed to the dinoflagellate Akashiwo sanguine showed significantly higher mortality than the controls. At cell lyses, during the senescent phase, many compounds are released in culture medium and some of these compounds may play a nutritive role for the larvae. But toxic compounds may also be released at lyses. Production and release of exudates by algal cells are also influenced by the physiological status of the cells (Subbarao, 2006; Amsler, 2008).

The mosquito larvicidal activity of transgenic cyanobacteria species was studied by many researchers. The constitutive expression of high mosquito larvicidal activity by transgenic Anabaena sp. strain PCC 7120 with combinations of two δ-endotoxin genes (cryIVA and cryIVD) and regulatory gene p20 of Bacillus thuringiensis subsp. israelensis against Aedes aegypti was reported by Xiaoqiang et al. (1997). The cyanobacterium, Agmenellum quadruplicatum PR-6 transformed with cryIVD behind its own strong phycocyanin promoter, P_cpcB, produced inclusion bodies and was mosquitocidal, but the onset of toxicity with Culex pipiens larvae was delayed (Murphy and Stevens, 1992). Using tandem promoters for expression of cryIVB in Synechococcus sp. strain PCC 7942 increased the mosquitocidal activity, but the activity remained relatively low against Culex restuans (Soltes-Rak et al., 1993).

5 Conclusions
The present study demonstrates ecological importance of marine phytoplankton in the control of mosquito Culex quinquefasciatus. The study showed that some species may serve as nutritious food for mosquito larvae, whereas others are harmful. Among the twelve species, Nannochloropsis oceanica (planktonic green alga) showed effective larvicidal activity. The death of the larvae might be due to the poor digestibility of Nannochloropsis cells. In future, such phytoplankton can be used for the development of natural pesticides against larvae of disease transmitting mosquitoes.
 
Author’s contributions
SVR designed and carried out the experiments. MR participated in the design of the experiment and also helped to draft the manuscript. All authors read and approved the final manuscript.

Acknowledgements
The authors are thankful to the Ministry of Earth Sciences, Government of India, New Delhi for the financial assistance and Dr. C. Krishnaiah, Co-ordinator, OASTC, Mangalore University for his help during the study period.

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