1. INTRODUCTION:

Infectious diseases spread by insects are still a leading global cause of illness and demise. Especially in subtropical and tropical countries, vector and vector-borne diseases have a social and economic impact, which has made them a challenging public health issue¹. Over 700,000,000 people worldwide, including 40,000,000 Indians, have been harmed by illnesses conveyed by mosquitoes, which are prevalent in more than 100 countries². Mosquitoes are among the most well-known insect disease vector groups, spreading pathogens that result in more than 400,000 fatalities globally each year^3,4. Millions of people die each year from diseases that impact both people and livestock, such as Japanese encephalitis, filariasis, chikungunya, dengue fever, and malaria and are primarily spread by mosquitoes^5,6.

1.1. Transmission and eradication of Culex spp.

WHO in 2019 estimated 68000 clinical cases of Japanese encephalitis each year, affecting many Asian nations where the primary carrier was Culex quinquefasciatus. Mosquito larvae and pupae feed in water, and mosquito eggs are laid in areas with standing water or in places where there is little movement. Larvae consume both living and dead aquatic plants and animals. Adult females feed on the vertebrate’s blood^7,8.

In many tropical countries, Culex quinquefasciatus (Diptera: Culicidae) is the predominant mosquito that rests within homes. It breeds in water that is primarily contaminated by organic debris. The other common house mosquito, Culex pipiens (L.), is a widely dispersed species^9,10. It can spread a variety of arboviruses, including the ones that cause avian malaria and lymphatic filariasis as well as West Nile, Japanese encephalitis, Rift Valley fever, St. Louis encephalitis, Eastern equine encephalitis and Sindbis viruses^11,12. The World Health Organization (WHO) released a manual on mosquito vector control, including Culex spp., emphasizing the importance of preventing their reproduction and dispersal. To eradicate the mosquito population, chemical compounds such as hexachlorobenzene (BHC), dichlorodiphenyl trichloroethane (DDT), and dieldrin were used^13,14. These chemical insecticidal or larvicidal agents exacerbate a variety of environmental hazards. Finding an effective biocontrol agent that not only lowers mosquito populations but also protects the environment is crucial¹⁵.

1.2 Use of plant extracts as biocontrol agents against Culex spp.

Most commonly, synthetic pesticides like organophosphates, organochlorines, carbamates, and pyrethroids are used to control mosquitoes^16–18. Insect populations have become resistant to pesticides as a result of the extensive use of chemical sprays for mosquito control which had detrimental impact on people, other non-target organisms, and the environment¹⁹. Scientists have proposed new alternatives to conventional pesticides in response to the growing concern about pesticide residue levels in various environmental sites, particularly food²⁰. The use of plant-derived extracts or essential oils as an alternative for controlling the spread of diseases by mosquitoes has become popularized. This is possible due to the presence of bioactive components of plants and also do not cause any harm to non-target organisms^21–26. Natural insecticides have been derived from plants, and they are excellent sources for the creation of green pesticides²⁷. Numerous studies have examined botanicals as efficient insecticides and larvicides for eradicating various species of pests^28–30. It has been revealed in many studies that phytochemicals derived from plants of families Asteraceae, Rutaceae, Labiatae, Miliaceae, Solanaceae, Oocystaceae, and Cladophoraceaeexhibit larvicidal, repellant properties against various mosquito species³¹.It is known that more than 2000 plant species emit metabolites and chemical components that can be utilized to regulate pests^32,33.

Sauromatum venosum Kunth (Voodoo Lily) (Synonyms: Sauromatum venosum Kunth, Arumvenosum Aiton, Typhonium venosum) belongs to the family Araceae or the Aroid family. It is found in forests and riparian meadows in Asia and Africa. It has antibacterial, antifungal, and insecticidal properties³⁴. Sauromatum toxicity was revealed against Bactrocera cucurbitae (melon fly). It was noticed that lectins can affect different developmental parameters by reporting reductions in pupation and emergence of 44% and 7.9%, respectively. The current study investigates the role of Sauromatum venosum tuber extract as a control agent against Culex quinquefasciatus.

2. MATERIALS AND METHODS:

2.1 Collection and rearing of mosquito:

The larvae of vector Culex spp. were collected from stagnant water from the Bajhol Valley Solan, 30.86°N, 77.11°E, situated between 1300 and 1500 meters above sea level. Larvae were collected by using the net and placed in a bottle. Mosquito larvae were reared in the laboratory as shown in Figure 1. They were maintained at 25° to 30° temperature. The larvae were reared on dog biscuits and brewer’s yeast powder mixture in ratio 3:1³⁵. After 5 days, adult male mosquitoes were fed with a 10% sucrose solution. The emerging female mosquitoes acquired blood meal from albino rats for 2-3 hours for egg production³⁶. To deposit eggs, a small piece of filter paper that had been conically wrapped was used. The filter paper containing the eggs was placed in a plastic petri plate with 300mL of distilled water for larval emergence. The recurring production of larvae was maintained by keeping all emerged adults in separate cages and using an 3 analogous method.

a) Larvae collection site b) Rearing of mosquito

Figure 1. Collection and rearing of mosquito

2.2 Collection of plant samples:

The samples were acquired from Bajhol valley Solan, situated in the latitudinal range of 30.86° north and longitudinal ranges from 77.11° East at an elevation ranging from 1300-1500m above sea level. The plant specimen was photographed, given in Figure 2. The tuber portion was then collected for further study.

Figure 2. Sauromatum venosum

2.3 Extraction:

Collected plant tubers were washed firstly with plain water and then with distilled water. After that, the tuber were grinded into pieces and then shade dried for 2-3 weeks. The shade dried tuber was ground into a powder as shown in Figure 3. Extraction was done with ethanol by using the rotary shaker method³⁷. The extract was shaken at 120rpm for two days³⁸. After filtering, the extract was kept in the hot air oven for drying. The crude extract was then maintained at 6˚C in Eppendorf.

a) Tuber pieces b) Tuber in powdered form c) Plant Extract

Figure 3. Collection and extraction of plant material

2.4 Larvicidal activity:

Larvae were treated with the stock solution. Different concentrations (100ppm, 50ppm, 25ppm 5ppm) were prepared from the stock solution to check the larvicidal activity. The mortality was checked on fourth instar larvae and was assessed with protocol given by WHO with some modifications. The number of dead larvae was counted after 24, 48, 72, and 96hours of exposure. Larval mortality percentage was calculated by using the Abbott formula (Abbott, 1925). Probit analysis was used for data analysis. Larvae were categorized into five groups having 10 larvae in each group and were exposed to different concentrations of the botanical.

2.5 Phytochemical analysis of plant extract:

One of the most important steps in determining the bioactive components is preliminary phytochemical screening. The occurrence of carbohydrates, tannins, phenols, and triterpenoids was checked by the Fehling test, ferric chloride test, and Salkowski test respectively.

2.6 Statistical analysis:

LC₅₀ and further data such as chi-square values, and upper and lower fiducial limits with a 95% confidence interval were calculated by probit analysis using SPSS (Statistical Package of Social Sciences) software version 16.0.

2.7 FTIR Spectroscopy:

For the identification of functional groups Fourier Transform Infrared Spectrophotometer (FTIR) was performed. It is the best technique to identify the functional groups present in the plant extract.

3. RESULTS AND DISCUSSIONS:

3.1 Phytochemical constituents of plant extract:

Phytochemical screening was done for the ethanolic extract of plant tuber. Table 1 represents the results of the plant metabolites screening. The presence of carbohydrates, triterpenoids, tannins, and phenolic compounds in the plant extract is mainly responsible for the larvicidal activity of the Sauromatum venosum,and shown in Figure 4. Alkaloids exhibits analgesic and bactericidal effects³⁹.

Figure 4. Preliminary phytochemical screening of plant extract

Table 1: Preliminary phytochemical screening of tuber extract of S.venosum

S. No.	Constituents	Ethanolic extract of S.venosum
1.	Carbohydrates	+
2.	Tannin and phenolic compounds	+
3.	Triterpenoids	+

3.2 Larval Mortality:

At different concentrations, the ethanolic extracts of plant tuber extracts showed strong larvicidal action against Culex spp. larvae in the fourth instar. Our findings were expressed as percentage mortality against third larval instars at various concentrations for 24, 96, 48, and 72hours.The percentages of larval mortality at 24hours, 48hours, 72hours, and 96hours following the treatment with the various concentrations of tuber extract are shown in Table 2.

Table 2: Mortality at different concentrations

	Number of Dead Larvae (Out of 10 in each group)
Time	Group 1 (control)	Group 2 (100ppm)	Group 3 (50ppm)	Group 4 (25ppm)	Group 5 (5ppm)
24hrs	0	0	0	0	0
48hrs	0	-2	-1	-1	0
72hrs	0	-2	-1	-1	0
96hrs	0	-3	-2	-1	-1

No mortality was observed in Group 1 which included larvae in double distilled water as well as after 24hrs of exposure. After 48hours of exposure, two larvae were found dead in group 2, one in group 3 and 4, and no mortality was found in group 5. After 72 hours of exposure, two larvae in group 2, one in group 3 and 4 were found dead. Group 5 remained unaffected. Three larvae in group 2, two larvae in group 3, and one larva in groups 4 and 5 were found dead after 96 hours of exposure.An increase in mortality was observed with an increase in the concentration of the extract. Abbot formula was applied for the calculation of mortality percentage at different concentrations as given in Table 3 and 4.

Table 3: Mortality percentage at different plant extract concentrations

Time	Group 1 (control)	Group 2 (100ppm)	Group 3 (50ppm)	Group 4 (25ppm)	Group 5 (5ppm)
24hr	0	0	0	0	0
48hr	0	20%	10%	10%	0
72hr	0	20%	10%	10%	10%
96hr	0	30%	20%	20%	10%

Table 4: Total number of dead larvae exposed to different concentrations for calculation of LC₅₀

Concentration	Total no. of larvae	No. of Dead Larvae
0.00	10	0
100ppm	10	7
50ppm	10	4
25ppm	10	3
5ppm	10	1

Lethal concentration can be computed using the SPSS software by taking different concentrations and counting the number of larvae killed in each concentration. Log 10 values were calculated for all the different concentrations. The mortality percentage was determined using the probit transformation curve. LC₅₀ was determined by using the equation Y= mx+ c. ‘m’ denotes the slope line and c represents the y intercept of the line.The graph for LC₅₀is given in Figure 5.

LC₅₀ value is given in Table 5 i.e. 55.571ppm and the regression equation was 1.3032x+2.728

Table 5: LC₅₀ value with 95% confidence interval

LC	LC	95% Confidence interval
(%)	(ppm)	Lower interval	Upper interval
LC₅₀	55.571	26.347	117.212

Figure 5: Graphical representation for the mortality curve of larvae exposed to different concentrations.

3.3 FTIR spectroscopy (Fourier transform infrared spectroscopy):

For the identification of functional groups best method to perform is the FTIR. The presence of a functional group is depicted by the highest peak and results are given in Table 6.

Table 6: FTIR results for the ethanolic extract of S. venosum

Peak value/ wave no. cm^-1	Functional Group	Predicted compound (Sumayya and Murugan, 2017)
3312	OH stretching vibration	Phenol and alcohol
2113	C=O stretching	Carboxylic acid
1640	C=C stretch	Terpenes

FTIR for plant extract shows the absorption at wavelength 3312 cm^-1, 1640 cm^-1and 2113 cm ^-1which was due to the stretching of the hydroxyl group, C=C and C=O stretching respectively indicating the presence of phenolic, terpenoids and carboxylic compounds given in Figure 6. These outcomes supported the findings from a preliminary phytochemical analysis of the tuber extract.

3.4 Discussion:

Triterpenoids, phenolic compounds, and tannin are among the bioactive chemicals reported in S. venosum extract. The plant-based bio-pesticides are easily accessible, affordable, environmentally friendly, and have no adverse effects on people or animals. After 96 hours, the tuber extract of Sauromatum venosum had an LC₅₀ value of 55.571 ppm. The goal of this experimental investigation is to establish Sauromatum venosum as a promising source for future mosquito larvicides. To recognize, separate, purify, and characterize these components, more investigation is required.

4. CONCLUSION:

Plant extracts can be useful in controlling mosquito populations because they don't pose as great a risk to human health. The synthesized chemical is fairly inexpensive and easily accessible. However, using plants to control larvae also provides a more secure option. These extracts are less toxic, easier to use, and more affordable solutions for controlling mosquito larvae.

5. CONFLICT OF INTEREST:

The authors have no competing interests to declare that are relevant to the content of this article.

6. DATA AVAILABILITY STATEMENT:

Availability of data and material- Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study.

7. AUTHOR’S CONTRIBUTION:

Bindiya Barsola and Ishika Verma have contributed substantially to the conception and design, acquisition of data. Shivani Saklani and Vandna Bhardwaj have helped in the analyzing and explanation of data for this study. Dr. Priyanka Kumari has given the sanction for this current version of the paper to be published. Priyanka Kumari has played a pivotal role in the editing and final drafting the manuscript.

8. CONSENT FOR PUBLICATION:

All the authors agree to the publication of this manuscript.

9. ACKNOWLEDGMENT:

During the drafting of this research article, I have received complete backing and assistance. I would like to thank my guide Dr. Priyanka Kumari whose expertise was invaluable in framing this research paper.

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Received on 24.10.2023 Modified on 20.02.2024

Research J. Pharm. and Tech 2024; 17(8):3685-3690.

DOI: 10.52711/0974-360X.2024.00574