Larvicidal Activities of N-(2-Hydroxyl) Propyl-3-Trimethyl Ammonium Chitosan Chloride (HTCC) and Silver Nanoparticles against Two Mosquito Species, Aedes and Culex: A Comparative Study

Abstract

This paper mainly focuses on using biopolymer as a potential larvicide. This work initially starts with extracting chitosan from the shrimp waste by a chemical method and it was characterized further. Chitosan is a natural carbohydrate biopolymer derived by deacetylation (DA) of chitin that is nontoxic, biodegradable, and biocompatible. But usually chitosan will not completely dissolved in water at normal conditions. Therefore, to increase the solubility and reaction rate, quaternized derivative of chitosan, N-(2-hydroxyl) propyl-3-trimethyl ammonium chitosan chloride (HTCC), is obtained and characterized. It highly dissolves in water and has more positive charge (quaternary ammonium groups) compared with chitosan. Next the larvicidal activity of the obtained HTCC against two mosquito species, Aedes and Culex, is evaluated. Next part of the work continues with synthesizing silver nanoparticles from the plant Euphorbia Antiquorum and characterized by UV Spec, FTIR, and SEM; then the larvicidal activity is examined similar to HTCC and the results obtained are compared with each other; Though both, silver nanoparticles and HTCC, show similar larvicidal activity, this study suggests HTCC is a better larvicidal agent having in mind the drastic effects of silver nanoparticles on aquatic ecosystem; Nanoparticles in aquatic systems are responsible for agglomeration and aggregation, dissolution, redox reactions and transformation into new solid phases, whereas chitosan and chitosan derivatives are completely degradable, biocompatible, and nontoxic to plants, animals, or human.

Introduction

World health organization has declared mosquitoes as a public health pest throughout the world as they are responsible for the transmission of various dreadful disease-causing pathogens [1,2]. Though over 3,500 species of mosquitoes exist [3], only few of them are found to be awful, which act as vectors for a number of infectious diseases, belong to the genera Culex, Aedesand Anopheles.

Therefore, control over mosquito population is vital. It is more easier to control mosquitoes when they are in larval stages than controlling the dispersed adults [4] All the three stages of mosquito, i.e., egg, larvae, and pupa, need water for their growth and survival. Since a brood of larval mosquito can be killed while they concentrate in a pool of water. Using chemical pesticides such as DDT, organochlorines, organophosphates, methoprene, etc, were one of the methods followed in killing mosquitoes. Despite high cost, food safety concerns, and environmental hazardous [5,6], mosquitoes also develop genetic resistance [7,8] to these chemicals. Therefore, alternative vector control strategies, especially effective, environmental safe, biodegradable, low cost, and indigenous methods are extremely imperative [9-12]. Thereof, investigations and searching of natural and environmental friendly insecticidal substances are ongoing worldwide [13-15].

Biopolymers as Larvicidal Agent

Though silver nanoparticles are Eco synthesized, one of the effective methods widely used, they are researched to be responsible for biomagnification and heavy metal toxicity. Therefore, to overcome the issues of silver nanoparticle, biopolymer materials are used. One among the most applicable biopolymer is chitosan, extracted from the crustacean wastes, that has wellknown antibacterial and antifungal activities. More than that, after treatment they break down into monomers and at particular time completely disintegrates. Therefore, this work attempts to examine the larvicidal activity of N-(2-hydroxyl) propyl-3-trimethyl ammonium chitosan chloride (HTCC), synthesized from the chitosan extracted from shrimp shell wastes which is highly positively charged and easily soluble in water against two mosquito species Culex and Aedes. The obtained results are compared with the activity of silver nanoparticles synthesized from the plant Euphorbia antiquorum.

Materials And Methods

Synthesis of Silver Nanoparticles

Ten grams of fresh plant was washed and chopped in to pieces, later it was boiled in 100 ml deionized water for 10 minutes. The extract was then cooled and filtered using Whatman No 1 filter paper. 10 ml of this plant extract was added 90 mL of 1Mm silver nitrate solution. The solution was allowed to react at room temperature, after 24 h color change was observed from pale yellow to brick red. The reduction mechanism of silver ions in to nanoparticles was confirmed through the UV-visible spectrophotometer analysis at 420 nm. It is centrifuged at 13000 rpm and the pellet was air dried and collected in an air-tight eppendorf tube. Thus-obtained silver nanoparticles were characterized using UV-Vis spectrometer and SEM.

Extraction and Characterization of Chitosan and HTCC from Shrimp Waste

Ten grams of shrimp shell powder is mixed with 4% NaOH for 21 h and washed several times until neutral pH and dried at 60°C for 3 hours. It was followed by demineralization using 2% HCl for 2 h at 70°C. It was washed several times and the left out thus obtained is chitin. Thus-obtained chitin is DE acetylated using 60% NaOH for 72 hours. After deacetylation samples are cooled at room temperature and washed several times using distilled water until neutral pH, the remaining is the chitosan that is oven-dried completely for further use. HTCC was synthesized according to a known method [16].

Selection and Culturing of Mosquito Species

Mosquito species Aedes aegypti and Culex were selected for this study. Entire analyses were carried out against laboratoryreared vector mosquitoes which are free of insecticidal and pathogenic exposure.

Larvicidal Bioassay of Silver Nanoparticles

Larvicidal activity of silver nanoparticles was performed using a standard protocol of WHO. Synthesized nanoparticles were diluted to 5.0, 4.0, 2.0, 1.0, and 0.5 mg/l using double distilled water. Ten larvae of each species is added to each test solution with beakers containing 200 ml of water, and control is a beaker with distilled water. The mortality was recorded after 24 and 48 h and the experiment was repeated for four times for average value. Percentage mortality of both experiments was recorded using Abott’s formula.

Larvicidal Bioassay of HTCC

Larvicidal bioassay of HTCC was carried out according to WHO standard procedures with slight modifications. Different concentrations ranging from 200 to 1000 ppm of HTCC were redissolved in 200 ml of tap water of 250 ml beakers and 10 larvae per concentration were used for all the experiments. A control was 200 ml of water. The number of dead larvae at the end of 24 and 48 h was recorded.

Results

Characterization of Silver Nanoparticles

Color conformation: Change of the solution color from pale yellow to brick color after 48 hours initially confirms the formation of silver nanoparticles, as shown in Figure 1.

Figure 1: Formation of silver nanoparticles by color changing from pale yellow to brick red after 48 h. Figure 2.

UV-VIS Spectrometer

Figure 2 shows the absorption spectra of AgNPs formed in the reaction media that have absorption maxima at 450 nm due to surface plasmon resonance of AgNPs confirmed the formation of silver nanoparticles.

Figure 2: UV Spectroscopy of silver nanoparticles synthetized from Euphorbia antiquorum.

SEM

Figure 3 shows the SEM of silver nanoparticles synthesized from Euphorbia Antiquorum. The silver nanoparticles were spherical in shape with particle size range from 5 to 40 nm and they are aggregated to each other.

Figure 3: SEM Image of Silver nanoparticles synthetized from Euphorbia antiquorum.

Characterization of Chitosan and HTCC

Table 1 shows color, yield percentage, ash content, molecular weight, degree of deacetylation, solubility, water and oil absorbing percentage, pH and viscosity of the extracted and obtained chitosan and HTCC.

Table 1: Physical Properties of Chitosan and HTCC extracted from shrimp shell.

FTIR of Chitosan and HTCC

Figure 4 shows FTIR peak of chitosan. There is a broad band at 3438 cm^-1 that is because of –NH² groups, -OH groups, and intermolecular hydrogen bonds overlap each other. The peak at 2991 cm^-1 represents the CH stretch [17]. Peak at 1639 cm^-1 shows the presence of carbonyl group, which is due to incomplete deacetylation of chitin to chitosan. A significant peak is also observed at 1560 cm^-1 indicates N-H bending of the primary amine.

Figure 4: FTIR peak of Chitosan Synthesized from Shrimp waste.

Figure 5 shows FTIR of HTCC with a new peak at 1470 cm^-1 represents the C-H bending of trimethylammonium group of HTCC. It should also be noted that the peak at 1560 cm^-1 in chitosan is disappeared in HTCC due to the change of primary amine in chitosan to secondary amine in HTCC. The peak at 3430 cm^-1 confirms the N-H stretching of a secondary amine and this peak proves the synthesis of HTCC by forming N-H group.

Figure 5: FTIR peak of HTCC synthesized from Chitosan.

From the experiments carried out, Aedes was found to be resistive than Culex to both silver nanoparticles and HTCC and the rate of mortality was dose and time dependent.

Table 2 shows the larvicidal activity of silver nanoparticles on Aedes and Culex at 24 and 48 h, respectively. It can be seen that percentage of mortality increases with increasing concentration. In Culex, at least concentration, 0.5 ppm, the mortality was 10%; whereas at highest concentration, 5 ppm, mortality was 100%. In case of Aedes, it was 6% and 100% at 0.5 and 5pmm, respectively. Therefore, at all concentrations, Culex is more susceptible than Aedes.

Table 2: Percentage Mortality of Silver Nanoparticles on Culex and Aedes at 24 h.

Table 3 shows the percentage mortality of HTCC against Aedes and Culex. As before, Culex is more susceptible than Aedes and mortality increased with increasing concentration. Culex showed 12% mortality at low concentration and 100% at highest concentration. Aedes showed 10% mortality at low concentration and 100% at highest concentration, respectively. Both Silver nanoparticles and HTCC showed 50% mortality at 2 ppm and 600 ppm, respectively.

Table 3: Percentage Mortality of HTCC on Culex and Aedes at 24 h.

Table 4 shows lethal concentration (LC50 and LC90) values of the silver nanoparticles and HTCC on Culex quinquefasciatus and Aedes aegypti. The LC50 values of silver nanoparticles and HTCC on Culex quinquefasciatus were 1.179 and 2.212, respectively; 2.392 and 2.097, respectively, for Aedes aegypti. The LC90 values are 4.555 and 4.521 for Culex and 5.129 and 4.428 for Ades treated with Silver nanoparticles and HTCC, respectively, and the chi-square values are significant at p.

Table 4: Lethal concentration (LC₅₀ and LC₉₀) values of the silver nanoparticles and HTCC on Culex quinquefasciatus and Aedes aegypti.

Discussion

Larvicidal activity of silver nanoparticles was already reported by literature [12-16] Besides the larvicidal activity, negative impact of silver nanoparticles on the aquatic life after treatment is more concerned. The most important processes affecting the fate of nanoparticles in aquatic systems are agglomeration and aggregation, dissolution, redox reactions and transformation into new solid phases [18-22] though used in very less quantity. The use of nanomaterials and their potential environmental and human health risks [23] are of increasing concern and social debate [16,24] and have been the subject of many government reports.

There are several reports evidenced the drastic effects of nanoparticles on aquatic environment. Julia Fabrega, et al. reported that concentrations of Ag NPs, as low as, just a few ng/L, can affect prokaryotes, invertebrates, and fish and also they also studied the mechanisms of toxicity. researched Ag ion toxicity in vivo in freshwater fish species with LC10 values as low as 0.8 μg/ L had effect on certain freshwater fish species [25].

Therefore, completely nontoxic biocomponent is indeed for treating mosquito larvae which reside in water bodies. Chitosan is a polysaccharide which forms the basis of the main constituent of the outer skeleton of insects and crustaceans like shrimp, crabs, and lobster [26]. There are several studies evidenced the antimicrobial activity of chitosan [27,28] and also reported that chitosan has effective antifungal activity which inhibits spore germination, germ tube elongation, and radial growth. Few works also evidenced antiprotozoal activity of chitosan [15,29]. The killing mechanism is due to the interaction mediated by the electrostatic forces between the protonated NH⁺³ groups and the negative residues [30], presumably by competing with Ca²⁺ for electronegative sites on the membrane surface of the microbes.

To improve further the solubility of chitosan in water, various derivates of chitosan were prepared. Among the various chitosan derivatives, the derivatives with quaternary ammonium groups have shown higher efficient activity against microbes compared to those of chitosan [31,32]. This may be due to enhanced positive charged quaternary amine group, known to targeted on the negatively charged cytoplasmic membrane of microbes, altering membrane properties and impeding nutrients entering the cells [33,34]. Therefore, same mechanism may occur in killing mechanism of mosquito larvae [35-40].

From the results it can be concluded that HTCC has equivalent and better larvicidal activity when compared with silver nanoparticles [41-46], which shows 100% mortality 1000 ppm in both Aedes and Culex. And the activity can also further enha nced by synthetizing HTCC nanoparticles [47-50].

Conclusion

This study compares the larvicidal activity of three silver nanoparticles and HTCC. Despite equal efficacy, this study suggests using HTCC as a larvicidal agent is advisable and more beneficial; because chitosan is completely degradable, biocompatible, and nontoxic to plants, animals, or human. This study can be further extended in future by synthesizing HTCC nanoparticles and examining its larvicidal activity. This work also aims to explore the parasitic activity of HTCC in future studies.

Acknowledgement

Author would like to thank Armats biotec for providing the lab facilities to carry out the research and also would like to thank Department of Environmental Technology, Central Leather Research Institute, Guindy for proving the instrumentation facilities for this research.

References

1. W.H.O. Lymphatic Filariasis. The disease and its control. WHO Report, Geneva.1992.
2. W.H.O. Report of the WHO informal consultation on the evaluation and testing of insecticides. CTD/ WHOPES/IC/96. 1. Control of Tropical Diseases Division. Geneva, WHO.1996.
3. Jaeger and Edmund C. A Source-Book of Biological Names and Terms. Springfield. Ill: Thomas. 1959;3:398-617.
4. El Hag, et al. Toxic and growth retarding effects of three plant extracts Culexpipiens larvae (Diptera : Culicidae). Phytother Res, 1999;13:388-392.
5. Yang Y.C, Lee S.G, Lee H.K, Kim M.K, Lee S.H and Lee H.S. A piperidine amide extracted from Piper longum L. fruit shows activity against Aedesaegypti mosquito larvae. J. Agric. Food Chem, 2002;50:3765-3767.
6. Junwei Z, et al. Adult repellency and larvicidal activity of five plant essential oils against mosquitoes. J. Amer. Mosq. Cont. Assoc., 2006;3:515-522.
7. Hartzell A and Wilcoxon F. A survey of plant products for insecticidal properties. Contrib Boyce Thompson Institute, 1941;12:127.
8. Jacobson M and Crosby D. G. Naturally Occurring Insecticides. Marcel Dekker Inc, 1971;585.
9. Amer A and Mehlhorn H, Repellency effect of fortyone essential oils against Aedes, Anopheles and Culex mosquitoes, Malaria Bulletin: A Compendium of Current Literature. Parasitology Research, 2006;230-234.
10. Chowdhury N, Ghosh A and Chandra G, Mosquito larvicidal activities of Solanum villosum berry extract against the dengue vector Stegomyiaaegypti. BMC Complementary Altern. 2008;8:10.
11. Piyarat S.W.K, Freed M and Roy S. Biologically active plant extract for the control of mosquito larvae. Mosq. News, 1974;34:398.
12. Kalyanasundaram M. Larvicidal and synergistic activity of plant extracts for mosquito control. Indian J. Med. Res, 1985;82:19-23.
13. Kuo P.M, et al. Insecticidal activity of essential oil from ChamaecyparisformosensisMatsum. Hlzforschung, 2007;61:595-599.
14. Balandrin M, et al. Natural plant chemicals: Sources of industrial and medicinal materials. Science, 1985;228:1154-1160.
15. Shin W. S, Kil J.C, and Park GM. Antiprotozoal activity of deacetylated chitosan oligosaccharide (dp 2-8) on Trichomonas vaginalis. J. Microbiol. Biotechnol, 2006;16:1984-1989.
16. RS/RAE,Nanosciences and nanotechnologies: opportunities and uncertainties.2004.
17. Andujar P, et al. Respiratory effects of manufactured nanoparticles. Rev. Mal. Respir, 2009;26:625-637.
18. Handy R.D, et al. The ecotoxicology and chemistry of manufactured nanoparticles. Ecotoxicology, 2008;17:287–314.
19. Ghosh A, Chowdhury N and Chandra G. Laboratory evaluation of a phytosteroid compound of mature leaves of day jasmine (Solanaceae: Solanales) against larvae of Culexquinquefasciatus (Diptera: Culicidae) and nontarget organisms. Parasitol Res, 2008;103:271-277.
20. Assis O. B. G,The fungistatic action of chitosan toppings of fresh-cut apples, Evaluation by image analysis. Iran J Parasitol, 2008;7:92-96.
21. Helfenstein M, et al. Effects of combustion-derived ultrafine particles and manufactured nanoparticles on heart cells in vitro. Toxicology, 2008;253:70-78.
22. Nowack B and Bucheli T.D. Occurrence, behavior and effects of nanoparticles in the environment. Environmental Pollution, 2007;150:5-22.
23. Andujar P, et al. Respiratory effects of manufactured nanoparticles. Rev. Mal. Respir, 2009;26:625-637.
24. B Feder. Technology's future: A look at the dark side. The New York Times.2006,
25. Birge W and Zuiderveen J. The comparative toxicity of silver to aquatic biota. Proceedings, 3rd Argentum International Conference on the Transport, Fate, and Effects of Silver in the Environment, Washington, DC.1995.
26. Kumar A. B. V, et al.  Characterization of chito-oligosaccharides prepared by chitosanolysis with the aid of papain and Pronase, and their bactericidal action against Bacillus cereus Biochem. J., 2005;391:167-175.  
27. Chen C. S, Liau W. Y and Tsai G. J.Antibacterial effects of N-sulfonated and N-sulfobenzoyl chitosan and application to oyster preservation J. Food Prot,1998;61:1124-1128.
28. Hadwiger L. A, et al. Chitosan both activated genes in plants and inhibits RNA synthesis in fungi, in: "Chitin in nature and technology". Muzzarelli, R. A. A., Jeuniaux, C. & Gooday, G. W. (Eds.), Plenum, New York. 1981.
29. Patil C.D, et al. Larvicidal activity of silver nanoparticles synthesized using Pergulariadaemia plant latex against Aedesaegypti and Anopheles stephensi and nontarget fish Poecilliareticulata. Parasitol Res,2012;111:555-562.
30. Rejane C, et al   A review of the antimicrobial activity of chitosan Polímeros,2009;19.
31. Ignatova M, Manolova N and Rashkov I. Novel antibacterial bers of quaternizedchitosan and poly(vinyl pyrrolidone) prepared by electrospinning. European Polymer Journal, 2007;43:1112-1122.
32. Ignatova M, et al. Electrospunnanobre mats with antibacterial properties from quaternised chitosan and poly (vinyl alcohol). Carbohydrate Research, 2006;341:2098-2107.
33. Klaine S.J, et al.  Nanomaterials in the environment: Behavior, fate, bioavailability, and effects. Environmental Toxicology and Chemistry, 2008;27:1825-1851.
34. Rabea E.I, et al. Chitosan asantimicrobial agent: applications and mode of action. Biomacromolecules, 2003;4:1457-1465.
35. El Hag, et al. Toxic and growth retarding effects of three plant extracts Culexpipiens larvae (Diptera : Culicidae). Phytother Res, 1999;13:388-392.
36. Janes N and Playle R.C. Modeling silver-binding to gills of rainbow trout (Onchorrynchus mykiss). Environ. Toxicol. Chem, 1995;14:1847-1858.
37. Julia Fabrega, Samuel N. Luoma, Charles R., Tyler, Tamara S, Galloway, Jamie R, Lead
38. Kaliyan V, Marimuthu G and Hoti S.L.Evaluation of plant-mediated synthesized silver nanoparticles against vector mosquitoes. Parasitology Research,2014;4567-4577.
39. Li Y, et al. Physicochemical characterization and antibacterial property of chitosan acetates. Carbohydrate Polymers,2007;67:227.
40. Muthukumaran U, Govindarajan M and Rajeswary M. Mosquito larvicidal potential of silver nanoparticles synthesized using Chomeliaasiatica (Rubiaceae) against Anopheles stephensi, Aedesaegypti, and Culexquinquefasciatus (Diptera: Culicidae).Parasitol Res, 2015;114:989-999.
41. Navarro E, et al. Environmental behavior and ecotoxicity of engineered nanoparticles to algae, plants, and fungi. Ecotoxicology, 2008;17:372-386.
42. Priyadarshini K.A, et al. Biolarvicidal and pupicidal potential of silver nanoparticles synthesized using Euphorbia hirta against Anopheles stephensi Liston (Diptera: Culicidae).Parasitol Res, 2012;111:997-1006.
43. Salunkhe R.B,  Larvicidal potential of silver nanoparticles synthesized using fungus Cochlioboluslunatus against Aedesaegypti (Linnaeus, 1762) and Anopheles stephensi Liston (Diptera; Culicidae). Parasitol Res, 2011;109:823-831.
44. Shanmugasundaram T and Balagurunathan R,Mosquito larvicidal activity of silver nanoparticles synthesised using actinobacterium, Streptomyces sp. M25 against Anopheles subpictus,Culexquinquefasciatusand Aedesaegypti.Journal of Parasitic Diseases,2015;677-684.
45. Subarani S, Sabhanayakam S andKamaraj C. Studies on the impact of biosynthesized silver nanoparticles (AgNPs) in relation to malaria and filariasis vector control against Anopheles stephensi Liston and Culexquinquefasciatus Say (Diptera: Culicidae).Parasitol Res, 2013;112:487-499.
46. Tavassoli M, et al. Novel in Vitro Efficiency of Chitosan Biomolecule against Trichomonas gallinae. Iranian J Parasitol,2012;7:92-96.
47. Veerakumar K, Govindarajan M and Rajeswary M.Green synthesis of silver nanoparticles using Sidaacuta (Malvaceae) leaf extract against Culexquinquefasciatus, Anopheles stephensi, and Aedesaegypti (Diptera: Culicidae).Parasitol Res, 2013;112:4073-4085.
48. Veerakumar K, et at. Mosquito larvicidal properties of silver nanoparticles synthesized using Heliotropiumindicum (Boraginaceae) against Aedesaegypti, Anopheles stephensi, and Culexquinquefasciatus (Diptera: Culicidae).Parasitol Res, 2014;113:2363-2373.
49. Wood C.M., Hogstrand C., Galvez F., and Munger R.S., The physiology of waterborne silver toxicity in freshwater rainbow trout (Oncorhynchus mykiss) 1. The effects of ionic Ag+. Aquat. Toxicol, 1996;35-93.
50. Zhou B, et al. An in vitro biotic ligand model (BLM) for silver binding to cultured gill epithelia of freshwater rainbow trout (Oncorhynchus mykiss). Toxicol. Appl. Pharmacol, 2005;202:25.