Repellent Effects of Codium flabellatum and Caulerpa taxifolia Seaweed Extracts on Aedes aegypti

Received 05 Jul, 2024

Accepted 21 Aug, 2024

Published 22 Aug, 2024

This study investigated the repellency activity of extracts from two green seaweeds, Codium flabellatum(C. flabellatum)and Caulerpa taxifolia(C. taxifolia),against the dengue vector Aedes aegypti. Extraction was conducted using ethanol as the solvent, with subsequent evaluation of repellency through the hand-introducing method. Various concentrations of seaweed extracts (20%, 30% and 40%) were prepared and tested. Results indicated moderate repellent effects across all samples, with the highest repellency observed in C. flabellatumat a 40% concentration, achieving 100% repellency at 6 hours. The LT₅₀ values (lethal time at which 50% of mosquitoes repel due to the applied sample on human skin) showed that C. flabellatumat 40% concentration exhibited the highest repellency (LT₅₀ value: 12 hours) against Aedes aegyptifollowed by Ctaxifolia(LT₅₀ value: 10 hours) at the same concentration. Conversely, a 20% concentration of seaweed extracts demonstrated the most negligible effectiveness, with an LT₅₀ value of 3 hours against Aedes aegypti. These findings suggest that seaweed possesses promising potential as an eco-friendly mosquito repellent and efficient biocontrol agent against mosquitoes.

INTRODUCTION

The proliferation of mosquitoes in our surroundings, thriving in breeding grounds such as stagnant water and punctured tire shops, has become a growing concern (Tariq & Zafar, 2000). Many synthetic repellents containing chemicals like N-N Diethyl-Meta-toluamide (DEET), Allethrin, and Dimethyl phthalate pose risks due to adverse effects on human health (Tabassum, 2016; Asadollahi et al., 2019). Sprays such as DDT (Dichloro diphenyl trichloroethane), once used to combat mosquitoes, are banned in various countries due to their detrimental impact on wildlife and the environment (Hira et al., 2017: Hira et al., 2018; Kabasenche & Skinner, 2014; da Costa et al., 2019).

Mosquitoes play a significant role in transmitting mosquito-borne diseases such as dengue, yellow fever virus, Zika, filariasis, schistosomiasis, and Japanese encephalitis (JE), causing illness and death worldwide (Gurunathan et al., 2016; Asadollahi et al., 2019). Dengue fever, in particular, has seen a significant rise, with approximately 100 countries now affected, compared to only nine before 1970 (Tan et al., 2019; Yu et al., 2015). The transmission of mosquito-borne diseases affects over 700 million people annually (Shammemrani K., 2018). Malaria alone claims the lives of 3 million people yearly, with one child succumbing every 30 seconds (Alarif et al., 2010).

Scientific studies have investigated the insecticidal activity of volatile constituents from various algae, with Actinotrichia fragilis volatile oil showing promising results (Qari & 2018; Shameel, 2008; Pasdaran et al., 2016). Metabolites obtained from Caulerpa racemoseare also alternative to synthetic insecticides (Alarif et al., 2010; Nagaraj et al., 2014).

The primary objective of this study is to develop a natural insecticidal product utilizing indigenous seaweeds (Arumugam et al., 2018; Chan & Matanjun, 2017), potentially manufacturing mosquito repellents. Mosquitoes cause nuisance through their bites and serve as significant vectors for severe human diseases (Amerasan et al., 2012; Alarif et al., 2010). Effective mosquito control strategies are crucial in combating mosquito-borne diseases (Anuradha et al., 2016). Using synthetic insecticides poses risks to non-target organisms, including beneficial insects like honey bees, highlighting the importance of eco-friendly alternatives (Ananth et al., 2017; Tariq & Qadri., 2001; Mahyoub, 2018). Natural mosquito repellents offer a safer, environmentally friendly solution to mitigate the adverse environmental and human health effects (Tan et al., 2019; Khattab et al., 2017). Given the current situation in Pakistan, where cases of Dengue fever are rampant, natural mosquito repellents could play a pivotal role in reducing or preventing such outbreaks and other diseases transmitted by mosquitoes (Ali et al., 2013; Yu et al., 2015). It is imperative to explore alternative approaches that are environmentally sustainable, cost-effective, and biodegradable (Ali et al., 2013; Yu et al., 2015). Therefore, the current research was planned to explore the potential of green seaweeds available in the Karachi coastal area (Pati et al., 2016) to control the impact of mosquitoes. This study provides a valuable natural seaweed extract as a natural mosquito repellent.

MATERIAL AND METHODS

Collection of Seaweeds
The study area encompasses a range of habitats, including small intertidal pools, rocky ledges, sublittoral zones, cobbles, shallow sand bottom flats and widely scattered boulders that exhibit a diversity of seaweeds.

Green algae specimens utilized in this investigation were collected from the Buleji coast in Karachi from February to November, excluding June, July and August (2020). These algae were collected by hand-picking and placed in plastic bags during low tides. The chosen seaweeds for this study were Codium flabellatum and Caulerpa taxifolia. Codium flabellatum was typically encountered in drift form, while Caulerpa taxifolia was firmly anchored to the substratum through its rhizoids.

Extraction:
After the collection, algae were washed thrice with tap water to remove the adhering salts, epiphytes and other detritus material (Afzal Rizvi & Shameel, 2005; Anuradha et al., 2016). Then, the shade is dried in a cage for a couple of weeks until it is completely dried. The algae were kept in air-tight polythene bags for further use. Some specimens, along with the holdfast, were also preserved in the form of herbarium. From the stock dried, Codium flabellatum was taken and soaked in ethanol in a ratio of 1:3. Similarly, Caulerpa taxifolia was also soaked in ethanol in a ratio of 1:3; both seaweeds were soaked for about 3 weeks/21 days. The solvents were evaporated from the crude extract by rotary evaporator at 50˚C. The crude ethanol extract was kept in air-tight glass vials and stored in a refrigerator for further use (Abbassy et al., 2014). The flow chart showed the extraction processes of seaweeds with ethanol (Fig.1).

Fig. 1: Flow chart of Extraction

Testing:
Mosquitoes Culturing:
Mosquitoes were cultured in the laboratory, and a total of 100 male and 100 female mosquito pupae were placed in a bowl filled with tap water (Ashrafi et al., 1966; Ramar et al., 2014). This bowl was positioned within a cage measuring 30×24×24 inches. Over the course of several days, adult mosquitoes emerged from the pupae. Throughout their developmental stages, from egg to larvae, pupae, and finally to adulthood, the mosquitoes were closely monitored to ensure they remained free of viral contamination. Upon emergence, the adult mosquitoes were initially provided with a blood meal, followed by a 5% sugar solution absorbed into a piece of cotton within a petri dish.

Repellency Test:
The Repellency study used the Hand Introducing (HI) method (Tariq & Qadri, 2001). Approximately (200) mosquitoes were kept in a cage (30×24×24”); these mosquitoes remained blood-starved for 3-4 days before the sample test. On the day of the assay, the test individual had no interaction with lotion, perfumes or fragrant soaps. The crude extract of seaweed was dissolved in ethanol to make 3 different concentrations viz. 20%, 30% and 40%. On the dorsal side of the right hand, an aliquot (0.5ml) of the test solution was applied to the particular area, which is 3cm×3cm.

In contrast, the remaining part of the treated hand was covered by wearing double gloves. The treated hand was exposed to the cage, and the number of bites and visits were counted for 5 minutes after every hour. Each day, one sample was applied. Swollen spots have been observed because of the biting of mosquitoes. The initial reading was considered a 0-hour reading. No skin irritation from the seaweed extract has been found (Sundar et al., 2013). After the testing of the sample, a 5% sugar solution was placed in a cage for those mosquitoes which could not suck blood due to the strong fumes of the samples. So, these mosquitoes were fed on that solution.

The repellency was calculated using the formula:

$$\text{Average biting}\mathrm{\%}=\frac{\text{total no. of biting in 5 minutes}}{\text{total no.of visits in 5 minutes}}x100$$

Repellency % = Control biting (100 %) – Average biting % in 5 minutes

Different concentrations were prepared using the formula:

$$\mathrm{Concentration\; in\; \%}=\frac{\text{wt. in grams}}{\text{solvent in ml}}x100$$

20 % = 1/5 × 100
30 % = 1/3.3 × 100
40 % = 1/2.5 × 100

RESULTS AND DISCUSSION

The current investigation assessed the mosquito-repellent activity of ethanol extracts (Hane & Raj 2018) from two seaweeds, C. flabellatum and C. taxifolia, against Aedes aegypti (Ahmad et al. 2016). In this study, crude extracts of C. flabellatum and Ctaxifolia dissolved in ethanol were prepared to make stock solutions and applied to a 3cm×3cm area of the right hand. These extracts are protected against mosquito bites without causing allergic reactions or irritation to the test subjects (Kumaresan et al., 2015). Additionally, the effectiveness of the repellent activity depends on the strength of the seaweed extract. The most substantial repellent effect against Aedes aegypti mosquitoes was observed with C. flabellatum and C. taxifolia at a concentration of 40% concentration, whereas C. flabellatum and C. taxifoliaat a concentration of 20 % exhibited the weakest repellency against the same mosquitoes. The findings revealed that both C. flabellatum and C. taxifoliademonstrated a minimal repellency of about 50% for 3 hours at a concentration of 20% (Table. 1). Conversely, at a concentration of 30%, both seaweed extract (Raj et al., 2018) displayed a moderate, ranging from 80% to 90% for 3 to 4 hours (Table. 2). Notably, the strongest repellency, reaching approximately 100%, was observed for 5-6 hours with both C. flabellatumand Ctaxifoliaat a concentration of 40% (Table. 3).

Table 1:

Repellency at 20% concentration

Duration of Repellency in hours	Seaweed Extract
	Codium flabellatum		Caulerpa taxifolia
	Biting rate in %	Repellency rate in %	Biting rate in %	Repellency rate in %
0	01	99	00	100
1	16	84	14	86
2	30	70	35	65
3	50	50	50	50
4	66	34	63	37
5	84	16	76	24
6	99	01	99	01

Table 2:

Repellency at 30% concentration

Duration of Repellency in hours	Seaweed Extract
	Codium flabellatum		Caulerpa taxifolia
	Biting rate in %	Repellency rate in %	Biting rate in %	Repellency rate in %
0	00	100	00	100
1	00	100	03	97
2	00	100	10	90
3	00	100	14	86
4	01	99	19	81
5	06	94	26	74
6	15	85	35	65
7	35	65	50	50
8	50	50	63	37
9	64	36	84	16
10	86	14	99	01
11	99	01	-	-

As presented in Table (4), considering the LT₅₀ values (the time required for 50% of the mosquitoes to repel), it is evident that the extract of C. flabellatum at a concentration of 40% (LT₅₀ value: 12 hours) exhibited notable effectiveness against Aedes aegypti.This was followed by the extract of Ctaxifolia at the same concentration, with an LT₅₀ value of 10 hours. Conversely, the extracts of C. flabellatumand Ctaxifoliaat a concentration of 20% demonstrated the most negligible efficacy, with an LT₅₀ value of 3 hours (Khattaba & Temraz 2017)

Table 3:

Repellency at 40% concentration.

Duration of Repellency in hours	Seaweed Extract
	Codium flabellatum		Caulerpa taxifolia
	Biting rate in %	Repellency rate in %	Biting rate in %	Repellency rate in %
0	00	100	00	100
1	00	100	00	100
2	00	100	00	100
3	00	100	00	100
4	00	100	00	100
5	00	100	01	99
6	00	100	02	98
7	02	98	06	94
8	06	94	17	83
9	13	87	30	70
10	22	78	50	50
11	38	62	66	34
12	50	50	78	22
13	65	35	99	01
14	77	23	-	-
15	99	01	-	-

Table 4:

LT50 value (lethal time at which 50% of mosquitoes repel)

Extract Concentration	LT50 (in hours)
Codium flabellatum 20%	3 hours
Codium flabellatum 30%	8 hours
Codium flabellatum 40%	12 hours
Caulerpa taxifolia 20%	3 hours
Caulerpa taxifolia 30%	7 hours
Caulerpa taxifolia 40%	10 hours

During the evening hours, individuals typically employ various methods such as coils, pads, lotion, smoke, mist, mosquito nets, Mospel, King, etc., to deter mosquitoes, commonly Culexand Anopheles, but hardly Aedes. However, these methods are often ineffective against Aedes mosquitoes, which are diurnal and active during daylight hours in schools, universities, colleges, factories, and offices. Synthetic repellents, although effective for up to 3 hours (Movahhedin et al., 2014; Tariq & Qadri, 2001; Khalid et al., 2018), pose safety concerns, particularly for infants who tend to put their hands in their mouth, posing a significant risk.

Interests in plant-based repellents diminished with the advent of synthetic alternatives (Vimaladevi et al., 2012), despite earlier reports indicating the strong insecticidal properties of Caulerpin and Caulerpinic acid found in genus Caulerpa (Alarif et al., 2010). Similar repellent activity against Aedes aegypti was observed with Caulerpa racemosaw hen prepared in acetone extract (Esteves et al., 2019; Madhusudan et al., 2011). However, research on repellent activity against mosquitoes using Codium flabellatum has been scarce. Previous reports have indicated that Ctaxifolia possesses nematicidal activity, while C. flabellatum exhibits anti-leishmanial and antibacterial properties (Khan et al., 2012). The biological activities of marine seaweed extracts (Ahmed et al., 2011) may be attributed to compounds such as saponin, flavonoids, phenolics, terpenoids and alkaloids present in seaweeds (El Gamal, 2010; El-Din 2015; Etcherla & Rao 2014; Sundar et al., 2013; Kolanjinathan et al. 2014). These findings underscore the potential of seaweeds as an eco-friendly approach to mosquito control (Govindarajan et al., 2011).

CONCLUSION

Our findings demonstrate that treatments utilizing seaweed extracts, specifically C. flabellatum and C. taxifolia, show significant potential for mosquito-repellent activity against Aedes aegypti while ensuring safety. Further investigations are warranted to explore the broader biological activities of seaweeds.

ACKNOWLEDGEMENT

We acknowledge the support of the Department of Botany, University of Karachi and the Department of Zoology, University in supporting this study.

CONFLICT OF INTERESTS

There is no conflict of interest among authors.

REFERENCES

1. Abbassy, M.A., Marzouk, M.A., Rabea, E.I. and Abd-Elnabi, A.D., (2014). Insecticidal and fungicidal activity of Ulva lactuca Linnaeus (Chlorophyta) extracts and their fractions. Annual Research & Review in Biology, 4(13), 2252-2262.
2. Afzal Rizvi, M. and Shameel, M., (2005). Pharmaceutical biology of seaweeds from the Karachi coast of Pakistan. Pharmaceutical biology, 43(2), 97-107.
3. Ahmad, R., Yu, K.X., Wong, C.L. and Jantan, I., (2016). Larvicidal and adulticidal activities of Malaysian seaweeds against Aedes aegypti (L.) and Aedes albopictus Skuse (Diptera: Culicidae). Southeast Asian Journal of Tropical Medicine and Public Health, 47(4), 719-730.
4. Ahmed, R.A., Ibrahim, A.M., Salem, T.A. and Hamza, H.A.,(2011). Anticancer activity of some Egyptian Marine Algae extracts. The Egyptian Society of Experimental biology., 7(1):9-13.
5. Alarif, W.M., Abou‐Elnaga, Z.S., Ayyad, S.E.N. and Al‐lihaibi, S.S., (2010). Insecticidal metabolites from the green alga Caulerpa racemosa.CLEAN–Soil, Air, Water,38(5‐6), 548-557.
6. Ali, M.Y.S., Ravikumar, S. and Beula, J.M., (2013). Mosquito larvicidal activity of seaweeds extracts against Anopheles stephensi, Aedes aegyptiand Culex quinquefasciatus. Asian Pacific Journal of Tropical Disease, 3(3),196-201.
7. Amerasan, D., Murugan, K., Kovendan, K., Kumar, P.M., Panneerselvam, C., Subramaniam, J., William, S.J. and Hwang, J.S., (2012). Adulticidal and repellent properties of Cassia tora Linn. (Family: Caesalpinaceae) against Culex quinquefasciatus, Aedes aegypti, and Anopheles stephensi. Parasitology research, 111(5), 1953-1964.
8. Ananth, Thangamathi, Wilson, Lavanya and Gnanasoundari, (2017). “Nutraceutical and pharmaceutical biology of green seaweed Ulva fasciata”, International journal of current research, 9(12), 63103-63111.
9. Anuradha, V., Syed, A. M.,and Yogananth, N., (2016). Efficacy of mosquito repellent and adulticidal activities of Halophila ovalis extract against filaria vectors. Journal of Tropical Diseases & Public Health. 4, 1000191
10. Arumugam, N., Chelliapan, S., Kamyab, H., Thirugnana, S., Othman, N. and Nasri, N., (2018). Treatment of Wastewater Using Seaweed: A Review. International journal of environmental research and public health, 15(12), 2851.
11. Asadollahi, A., Khoobdel, M., Zahraei-Ramazani, A., Azarmi, S. and Mosawi, S.H., (2019). Effectiveness of plant-based repellents against different Anopheles species: a systematic review. Malaria Journal,18(1), 436.
12. Ashrafi, S.H, Muzaffar, S. A. and Anwarullah, M. (1966). Mass rearing of mosquitoes, houseflies and coakroaches in PCSIR laboratories for insecticide testing. Sci. Indy 4:312-316
13. Chan, P.T. and Matanjun, P., (2017). Chemical composition and physicochemical properties of tropical red seaweed, Gracilaria changii. Food chemistry, 221, 302-310.
14. da Costa, K. S., Galúcio, J. M., da Costa, C. H. S., Santana, A. R., dos Santos Carvalho, V., do Nascimento, L. D., ... & Lameira, J. (2019). Exploring the potentiality of natural products from essential oils as inhibitors of odorant-binding proteins: A structure-and ligand-based virtual screening approach to find novel mosquito repellents. ACS omega,4(27), 22475-22486.
15. El Gamal, A.A., (2010). Biological importance of marine algae. Saudi Pharmaceutical Journal, 18(1), 1-25.
16. El-Din, S.M.M., (2015). Utilization of seaweed extracts as bio-fertilizers to stimulate the growth of wheat seedlings. The Egyptian Journal of Experimental biology Botany, 11(1), 31-39.
17. Esteves, P. O., de Oliveira, M. C., de Souza Barros, C., Cirne-Santos, C. C., Laneuvlille, V. T., & Palmer Paixao, I. C. (2019). Antiviral effect of caulerpin against chikungunya. Natural Product Communications, 14(10), 1934578X19878295.
18. Etcherla, M. and Rao, G.N., (2014). In vitro study of antimicrobial activity in marine algae caulerpa taxifolia and Caulerpa racemosa (c. agardh). International journal of applied biology and pharmaceutical technology, 5(2). 57-62.
19. Govindarajan, M., Mathivanan, T., Elumalai, K., Krishnappa, K. and Anandan, A., (2011). Mosquito larvicidal, ovicidal, and repellent properties of botanical extracts against Anopheles stephensi, Aedes aegypti, and Culex quinquefasciatus (Diptera: Culicidae). Parasitology research, 109(2), 353-367.
20. Gurunathan, A., Senguttuvan, J. and Paulsamy, S., (2016). Evaluation of mosquito repellent activity of isolated oleic acid, eicosyl ester from Thalictrum javanicum. Indian journal of pharmaceutical sciences,78(1), 103.
21. Hane, G.K. and Raj, T.S., (2018). Screening the efficacy of Seaweed extracts against sheath blight of rice caused by Rhizoctonia solani(Kuhn). International Journal of Tropical Agriculture,36(2), 463-470.
22. Hira, A., Tabassum, A., Ali, T.M., Qaisar, S. and Aliya, R., (2018). Comparison in biodegradable films formulated from two red seaweeds of Karachi coast. Pakistan Journal of Marine Sciences, 27(2), 87-92.
23. Hira, K., Tariq, R.M., Sultana, V., Ara, J. and Ehteshamul-Haque, S., (2017). Effect of seaweeds occurring at Karachi coast on mosquito larvae and liver function in rats. Pakistan journal of pharmaceutical sciences, 30(2), 387-391.
24. Kabasenche, W. P., & Skinner, M. K. (2014). DDT, epigenetic harm, and transgenerational environmental justice. Environmental Health, 13, 1-5.
25. Khalid, S., Abbas, M., Saeed, F., Bader-Ul-Ain, H. and Suleria, H.A.R., (2018). Therapeutic Potential of Seaweed Bioactive Compounds. In Seaweed Biomaterials. IntechOpen.
26. Khan, A.M., Ameen, M., Naz, S. and Noureen, S., (2012). Bio screening of marine organisms from the coasts of Pakistan. Journal of the Chemical Society of Pakistan, 34(1), 184-194.
27. Khattaba, R.A. and Temraz, T.A., (2017). Mangrove Avicennia marina of Yanbu, Saudi Arabia: GC-MS constituents and mosquito repellent activities. Egyptian Journal of Aquatic Biology and Fisheries, 21(3), 45-54.
28. Kolanjinathan, K., Ganesh, P. and Saranraj, P., (2014). Pharmacological importance of seaweeds: a review. World Journal of Fish and Marine Sciences,6(1), 1-15.
29. Kumaresan, R., Vinitha, K., & Kannan, K. (2015). Scientometric analysis of seaweed research with reference to web of science. Library Philosophy and Practices,1, 1-15.
30. Madhusudan, C., Manoj, S., Rahul, K. and Rishi, C.M., (2011). Seaweeds: A diet with nutritional, medicinal and industrial value. Research Journal of Medicinal Plant, 5(2), 153-157.
31. Mahyoub, J.A., (2018). Mosquito Larvicidal activity of seaweed extracts against Anopheles d’thali with reference to its side effects on aquatic nontraget organisms. International journal of mosquito research, 5(6),34-38.
32. Movahhedin, N., Barar, J., Azad, F.F., Barzegari, A. and Nazemiyeh, H., (2014). Phytochemistry and biologic activities of Caulerpa peltatanative to Oman Sea. Iranian journal of pharmaceutical research: IJPR, 13(2), 515.
33. Nagaraj, S.R. and Osborne, J.W., (2014). Bioactive compounds from Caulerpa racemosa as a potent larvicidal and antibacterial agent. Frontiers in biology, 9(4), 300-305.
34. Pasdaran, A., Hamedi, A. and Mamedov, N.A., (2016). Antibacterial and insecticidal activity of volatile compounds of three algae species of Oman Sea. International Journal of Secondary Metabolite, 3(2), 66-73.
35. Pati, M.P., Sharma, S.D., Nayak, L.A.K.S.H.M.A.N. and PANDA, C.R., (2016). Uses of seaweed and its application to human welfare: A review. International Journal of Pharmacy and Pharmaceutical Sciences, 8(10), 12-20.
36. Qari, R., & Khan, E. (2018). Diversity of Green Seaweeds along the Coast of Karachi. International Journal of Marine Science, 8(22), 176.
37. Raj, T., Nishanthi, P., Hane Graff, K. and Ann Suji, H., (2018). Seaweed extract as a biostimulant and a pathogen controlling agent in plants. International journal of Tropical Agriculture, 36, 563-580.
38. Ramar, M., Ignacimuthu, S. and Paulraj, M.G., (2014). Mosquito knock-down and adulticidal activities of essential oils by vaporizer, impregnated filter paper and aerosol methods. International Journal of Mosquito Research, 3(1), 26-32.
39. Shameel, M., (2008). Change of divisional nomenclature in the Shameelian classification of algae. International Journal of Phycology and Phycochemistry (Pakistan), 8(1), 7-22.
40. Sundar, R., Sundar, V. and Sakkarapani, G., (2013). Toxicity of Gracilaria verrucosa methonal extract against selected medically important vector mosquitoes (Diptera: Culicidae) of Puducherry, India. International journal of current research, 5, 3388-3392.
41. Tabassum, A., (2016). Biofilms from agar obtained from an agarophyte of Karachi coast. Pakistan Journal of Marine Sciences, 25(1&2), 37-40.
42. Tan, K., Faierstein, G.B., Xu, P., Barbosa, R.M., Buss, G.K. and Leal, W.S., (2019). A popular Indian clove-based mosquito repellent is less effective against Culex quinquefasciatus and Aedes aegypti than DEET. Public library of science(PLOS ONE),14(11), e0224810.
43. Tariq, R.M. and Qadri, S.S., (2001). Repellant activity of some local plant’s oil, two commercial repellants, dimethyl phthalate and non-alcoholic itter against dengue vector mosquitoes. Pakistan Journal of Entomology Karachi, 16(1&2), 7-10.
44. Vimaladevi, S., Mahesh, A., Dhayanithi, B.N. and Karthikeyan, N., (2012). Mosquito larvicidal efficacy of phenolic acids of seaweed Chaetomorpha antennina (Bory) Kuetz. against Aedes aegypti. Biologia, 67(1), 212-216.
45. Yu, K.X., Wong, C.L., Ahmad, R. and Jantan, I., 2015. Mosquitocidal and oviposition repellent activities of the extracts of seaweed Bryopsis pennata on Aedes aegypti and Aedes albo Molecules 20(8): 14082-102.