Larvicidal , Ovicidal , and Repellent Activities of Marine Sponge Cliona celata ( Grant ) Extracts against Culex quinquefasciatus Say and Aedes aegypti L . ( Diptera : Culicidae )

Solvent extracts of marine sponge Cliona celata (Grant) were screened for larvicidal, ovicidal, and repellent properties against the filarial vector Culex quinquefasciatus Say and dengue vector Aedes aegypti L. Larvicidal and ovicidal activities of hexane, ethyl acetate, andmethanol extracts were tested in four different concentrations ranging as 62.5, 125, 250, and 500 ppm. Among the three solvent extracts of C. celata, methanol extract showed the highest larvicidal activity at 500 ppm against both mosquito species. The LC 50 and LC 90 values of C. celata methanol extract were recorded as 95.63 and 242.16 ppm against C. quinquefasciatus larvae and 158.40 and 780.16 ppm againstA. aegypti larvae, respectively. Ovicidal activity was high inmethanol extract, in which 100% ovicidal activity was recorded in C. quinquefasciatus, and 72% ovicidal activity was recorded in A. aegypti at 500 ppm. The hexane extract was found to be the most effective protectant against the adult mosquitoes of both species. The mean protection time recorded in hexane extract was up to 273 and 165min at 5mg/cm dosage against C. quinquefasciatus and A. aegypti, respectively. Considering these bioactivities, C. celata could be used to obtain some novel pesticidal molecules.


Introduction
Mosquitoes are the major arthropod vectors and the most dangerous human health pests.The war against mosquitoes using chemical pesticides has failed due to the resistance developed by mosquitoes [1,2].Chemical control of mosquitoes is also causing many unwanted effects on human health and nontarget animals.By understanding these side effects of chemicals, people are now showing interest towards biopesticides and botanical formulations, which are considered as eco-friendly.Nature is providing numerous bioactive products against vector mosquitoes in the form of plant products, marine products, microbial products, and other biological derivatives.Culex quinquefasciatus is a major vector of Wuchereria species causing lymphatic filariasis; it is widely distributed in tropical regions with around 120 million people getting infected and 44 million people under clinical manifestation [3].India alone contributes around 40% of global filariasis burden, and the estimated annual economic loss is about 720 crore Indian rupees [4].The mosquito Aedes aegypti is more widely distributed in tropical and subtropical regions of the world.It is a major vector of arboviruses causing chikungunya and dengue fever.Natural insecticides are generally pest specific, biodegradable, usually nonallergic to human as well as nontarget organisms [5], and they may possess novel compounds with a wide range of activities [6,7].In recent years, plant products and phytochemicals have been studied for the control of mosquitoes [8][9][10][11][12].Likewise, some investigators have reported that the secondary metabolites of marine sponges possessed insecticidal activities [13][14][15][16].
Sponges (Phylum: Porifera) are the oldest metazoan organisms and have been recognized as a rich source of biologically active compounds that are of potential interest to mankind [17,18] and are good alternatives for synthetic pesticides [19].They are a potential source of novel antimicrobial agents [20].Till date, nearly 8000 species of sponges have been described throughout the world, and 108 species of sponges have been identified in Gulf of Mannar, India [21].Till date, practically very few species of sponges have been studied for their mosquito larvicidal activity [22][23][24][25].However, studies on mosquitocidal properties of marine sponges from Indian waters are limited, and the larvicidal, ovicidal and repellent efficacy of the marine sponge Cliona celata (Porifera: Hadromerida: Clionidae) has not been studied previously.Hence, the present study was undertaken to screen the crude extracts of marine sponge C. celata from the Gulf of Mannar for their larvicidal, ovicidal, and repellent activities against two vector mosquitoes C. quinquefasciatus and A. aegypti.

Collection of Sponges.
Marine sponge C. celata was collected from the Gulf of Mannar (between 8 ∘ 47  to 9 ∘ 05  N Latitude and 78 ∘ 12  to 79 ∘ 07  E Longitude, India) at depths varying from 15 to 25 feet by scubadiving.Sponges were gently removed from the substratum and transferred to laboratory within 48 h; then, the specimen was identified and deposited (deposition number: MBRC/ZSI-S.225) in National Zoological Collection of Marine Bio Resource Centre, Zoological Survey of India (13 ∘ 04  N Latitude and 80 ∘ l7  E Longitude, India).

Preparation of Crude Extracts.
The Marine sponge C. celata was thoroughly washed with distilled water to remove the sand particles and cut into small pieces before shade drying for 48 h.Then the sponges were homogenized and extracted sequentially with hexane, ethyl acetate, and methanol.Initially the powder of the C. celata (500 g) was soaked in 1 litre hexane for 72 h and filtered through filter paper.The residue was dried and then extracted sequentially with ethyl acetate and methanol solvents after 72 h of soaking in each solvent separately.The extracts were condensed separately under reduced pressure by using vacuum evaporator, and the solvent free crude extracts were collected in glass vials and stored in 4 ∘ C until use.Stock solutions (10% in acetone) of all the three solvent extracts were prepared and then subjected to bioassay screening.

Test Insect.
Eggs, larvae, and adults of C. quinquefasciatus and A. aegypti were obtained from the stock culture maintained at Entomology Research Institute, which were free of exposure to pathogens, insecticides, or repellents.Laboratory rearing was done at a temperature of 27 ± 2 ∘ C, 75-85% relative humidity, and a photoperiod of 11 ± 0.5 h.

Larvicidal Bioassay.
Larvicidal activity was evaluated by following the methods of WHO [26] with slight modifications.Twenty numbers of early fourth instar larvae of C. quinquefasciatus and A. aegypti were introduced into the test containers.The extracts taken in four concentrations were 500, 250, 125, and 62.5 ppm.Normal control and solvent control (Acetone in water) were maintained separately.Mortality rate were registered after 24 h exposure period.The moribund and dead larvae were collected, and larval mortality was calculated for each concentration.The bioassays were performed at a room temperature of 27 ± 1 ∘ C with five replicates for each concentration.Mortality was converted into percent mortality (a), and corrected mortality was calculated using Abbot's formula (b) [27].
(a) Percentage of mortality is as follows: no. of dead larvae no. of larvae introduced × 100.
(b) Corrected percentage of mortality is as follows: where  is the number of larvae, T is the number of larvae survived in treated, and C is the number of larvae survived in control.
The corrected percentage mortality value of each concentration was considered to estimate LC 50 and LC 90 values using SPSS Probit analysis statistical pack, version 11.5.

Ovicidal Bioassay.
Ovicidal activity was evaluated by following the method of Elango et al. [28] with slight modification.Twenty five freshly laid eggs of C. quinquefasciatus and A. aegypti were treated separately with the C. celata extracts at 62.5, 125, 250, and 500 ppm concentrations.Normal control was kept separately.Acetone in water was served as solvent control.Each treatment was replicated five times.The ovicidal activity was assessed up to 120 h posttreatment and thereafter control; treated eggs were observed under the microscope and photographed using stereo zooming microscope (Wild M7S TYP 308700, Switzerland).The nonhatched eggs with unopened opercula were counted in each treatment, and the percent mortality was calculated using the following formula and analysed in Graph Pad Prism version LC 50 lethal concentration that kills 50% of the exposed larvae, LC 90 lethal concentration that kills 90% of the exposed larvae, LL lower limit (95% confidence limit), and UL upper limit (95% confidence limit).
*  ≤ 0.05, level of significance of chi-square values.was obtained.The experiments were replicated five times in separate cages, and in each replicate different volunteer was used to nullify any effect of skin differences on repellence.The protection time of each extract was calculated using previously established methods [29,30].

Statistical Analysis.
The results were presented as mean ± SD.Statistical analyses of all the data obtained in larvicidal activity were evaluated using Probit analysis (SPSS Program; Version 11.5).The differences were considered as significant at  ≤ 0.05.

Larvicidal Activity and Lethal Doses.
All the three extracts of the sponge C. celata showed larvicidal activity.The larvicidal activity varied between the solvent extracts and the mosquito species.1).In normal control and solvent control, all the larvae were active and exhibited normal movement.But in the treated larvae, restless movement was observed.After 1 h, tremor and convulsion were observed in all the treated larvae and dead larvae settled down as reported earlier [31].

Ovicidal Activity.
The ovicidal activities of C. celata extracts on C. quinquefasciatus and A. aegypti eggs are given in Figures 1 and 2. Methanol extract was found to be highly lethal to the eggs of both species.In C. quinquefasciatus, methanol extract presented 100% ovicidal activity at 500 ppm concentration after 120 h posttreatment period.The lowest concentration (62.5 ppm) of methanol extract caused 25.6% egg mortality in C. quinquefasciatus.A. aegypti eggs were found to be more tolerant to all the three extracts of C. celata at all concentrations.At the highest concentration (500 ppm) of methanol extract, 72% ovicidal activity was noticed in A. aegypti after 120 h posttreatment period.In both species, the ovicidal effect of all the three extracts was directly proportional to the concentration.In methanol extract treated C. quinquefasciatus eggs, most of them did not hatch and some were hatched in an abnormal way at 500 ppm and the larvae died before completion of eclosion (Figure 3(b)).The methanol extract-treated eggs of A. aegypti were shrunken and most did not hatch at 500 ppm concentration (Figure 4(b)).In normal control and solvent control, hatchability of eggs of both species was normal (Figures 3(a) and 4(a)).

Repellent Activity.
The complete protection times for all the three extracts of C. celata against both the species of mosquitoes were recorded by standard skin repellence experiments, and the results are given in Tables 2 and 3.The repellence was directly proportional to the dose and protection time (min) for each extract showed variations between two species.In general hexane extract gave maximum protection time against both mosquito species compared to ethyl acetate and methanol extracts.Furthermore, it was noted that all the three extracts were found to be more effective against C. quinquefasciatus than A. aegypti mosquitoes.Hexane extract gave a maximum protection time of 273 min against C. quinquefasciatus at 5 mg/cm 2 dosage (Table 2).The same treatment recorded the highest protection time of 165 min against A. aegypti at 5 mg/cm 2 dosage (Table 3).These results were comparable with negative control (N-N Diethyl benzamide 12%, w/w), which showed maximum of 323 and 182 min protection at 5 mg/cm 2 dosage against C. quinquefasciatus and A. aegypti mosquitoes, respectively.

Discussion
Searching for eco-friendly pesticide molecules among natural sources has become an important research in these days, and it is promoted for mosquito control than chemical insecticides [32].Nature is providing innumerable bioactive molecules for the well-being of mankind.Plant kingdom and marine organisms provide majority of the beneficial biomolecules or products that are used by people in their daily life.Marine sponges are known to produce toxins and other compounds to repel and deter predators [33,34].These compounds of marine sponges are reported to be of the bioactive compounds which can be used in the treatment of many diseases [35][36][37][38][39].
In recent years, researchers are concentrating on marine organisms to study their biological activities; especially, marine sponges (Porifera) have attracted significant attention from various scientific disciplines [40].The literature also indicates that the marine products possess maximum percentage of bioactive substances with novel biological properties than the molecules originating from terrestrial origin [24,41].Recently, marine sponge extracts and their compounds have been screened for antimicrobial [42,43], antiplasmodicidal [44], antifilarial [45,46], and antihelminthic activity [47].Marine sponge extracts have also been screened against agricultural pests.Edrada et al. [48] have reported an insecticidal and growth regulating compound from the philippine marine sponge Xestospongia ashmorica against Spodoptera littoralis, an important polyphagous pest.Supriyono et al. [49] have reported that two guanidine alkaloids, namely, hymenialdisine and debromohymenialdisine from the tropical marine sponge Axinella carteri exhibited insecticidal activity with LD 50 value of 88 and 125 ppm, respectively, against the neonate larvae of S. littoralis in feeding bioassay experiments.
In this regard, very few researchers have studied the mosquitocidal potential of marine sponge extracts.Venkateswara Rao et al. [23] have screened the methanoldichloromethane (1 : 1) extracts of 18 different sponges collected from Palk bay and Gulf of Mannar waters against A. aegypti and houseflies (Musca domestica).They found that Psammaplysilla purpurea (LC 50 = 25.9 ppm) and Haliclona  cribricutis (LC 50 = 31.46ppm) were more effective against A. aegypti mosquito larvae.In our study, we observed that the methanol extract of C. celata was the most effective in larvicide, and the LC 50 of methanol extract against C. quinquefasciatus was found to be higher (95.63 ppm) than A. aegypti (158.40 ppm) values.An interesting finding in this study was that the low polar solvent extract; that is, hexane extract showed repellent activity and the high polar solvent (methanol) extract showed larvicidal and ovicidal activities.This result was comparable with the earlier report of Sonia and Lipton [25] who have screened the methanol extracts of five marine sponges, namely, Acanthella elongata, Echinodictyum gorgonoides, Axinella donnani, Callyspongia subarmigera, and Callyspongia diffusa for larvicidal activity against Culex sp.They found that Acanthella elongata extract was the most effective with the LC 50 value of 0.066 mg/mL than other extracts.
In the present study, all the three solvent extract treatments were not equally effective against C. quinquefasciatus and A. aegypti larvae, eggs, and adults.C. quinquefasciatus life stages were found to be more susceptible to the treatments than A. aegypti.A similar result was reported by Martínez et al. [22]; they have screened the ethanol extracts of five marine sponges, namely, Amphimedon compressa, Topsentia ophiraphidites, Svenzea zeai, Ircinia campana, and Agelas sventres against fourth instar larvae of A. aegypti and C. quinquefasciatus.They found that Ircinia campana extract was the most effective against the larvae of two mosquitoes and the activity was higher in A. aegypti than C. quinquefasciatus.
Earlier, Rey et al. [50] and David et al. [51] proved that the plant extracts primarily affect the midgut epithelium, and this is based on the concentration, duration of the treatment, and mosquito species used.Further, Daniel Reegan et al. [52] proved that the orange peel extract primarily damages the midgut epithelial columnar cells, gastric caeca (GC), and brush border cells (BB) in the treated larvae of A. stephensi.Acetylcholinesterase (AChE) is the molecular target for mosquito repellents [53].Possibly, these extracts of marine sponge C. celata may have impact on midgut cells of the treated larvae and on AChE.

Conclusion
In Conclusion, the present study reports for the first time repellent, larvicidal, and ovicidal activities of marine sponge C. celata against C. quinquefasciatus and A. aegypti.The screening results suggest that the hexane and methanol extracts of C. celata are promising in mosquito control and repellent.This is an eco-friendly alternative to chemical insecticide which is promising in mosquito control.

Figure 1 :
Figure 1: Percent ovicidal activity of marine sponge C. celata extracts against the eggs of C. quinquefasciatus.

Figure 3 :
Figure 3: Hatched eggs of C. quinquefasciatus in control (a).The larvae of C. quinquefasciatus died in the egg case before completion of eclosion in methanol extract treated C. celata after 120 h posttreatment (b).

Figure 4 :
Figure 4: Hatched eggs of A. aegypti in control (a).Dead and shrunken eggs of A. aegypti in methanol extract treated C. celata after 120 h posttreatment (b).

Table 1 :
Lethal concentration (in ppm) of C. celata extracts against the larvae of C.
left undisturbed.The left arm served as control.N-N Diethyl benzamide (12%, w/w) was used as negative control.The mosquito bites were observed for three full minutes of every fifteen minutes.Protection time was recorded as the time that elapsed between extract application and the observation period immediately preceding that in which a confirmed bite * Culex quinquefasciatus and A. aegypti.

Table 1
shows the results on effective lethal concentration (LC 50 and LC 90 ) values of hexane, ethyl acetate, and methanol extracts of C. celata after 24 h treatment period.It was clear from the results that the methanol extract recorded the maximum larvicidal activity in both C. quinquefasciatus and A. aegypti.The LC 50 and LC 90Figure 2: Percent ovicidal activity of marine sponge C. celata extracts against the eggs of A. aegypti.values of methanol extract recorded in C. quinquefasciatus were 95.63 and 242.16 ppm, respectively (Table1).A. aegypti was less susceptible than C. quinquefasciatus, and the LC 50 and LC 90 values of methanol extract for A. aegypti were 158.40 and 780.16 ppm, respectively.Significant chi-square values were recorded in all the extracts (Table

Table 2 :
Complete protection time of three solvent extracts of C. celata against C. quinquefasciatus.

Table 3 :
Complete protection time of three solvent extracts of C. celata against A. aegypti.