Comparative evaluation of the effect of phytochemicals of garlic ( Allium sativum ) ethanolic extract against Aedes albopictus and Culex quinquefasciatus mosquitoes in Sri Lanka

: Botanical extracts offer sustainable and eco-friendly alternatives to synthetic insecticides for managing insect pests, including mosquitoes. This research focuses on the potential of ethanolic garlic extract as a larvicide against Aedes albopictus and Culex quinquefasciatus mosquito species in Sri Lanka. Third to early fourth instar larvae were exposed to six concentrations of ethanolic garlic extract (ranging from 5 to 250 ppm) for 72 hours to assess efficacy. The experiment, repeated four times with controls, monitored daily mortalities. Lethal concentrations required to eliminate 50% (LC 50 ) and 90% (LC 90 ) of larvae at 24, 48, and 72 hours were determined through regression analysis. A phytochemical analysis was conducted to assess the compounds present in the garlic extract. Positive correlations were observed between garlic concentration and mortality percentages during each exposure period for both Ae. albopictus and Cu. quinquefasciatus larvae. LC 50 values for Ae. albopictus larvae were 45.5 ppm, 28.0 ppm, and 14.4 ppm at 24, 48, and 72 hours respectively, with corresponding LC 90 values of 140.0 ppm, 91.0 ppm, and 42.9 ppm. For Cu. quinquefasciatus larvae, LC 50 values were 26.3 ppm, 9.4 ppm, and 4.4 ppm, while LC 90 values were 169.8 ppm, 30.7 ppm, and 17.6 ppm for the same exposure periods. The garlic ethanolic extract retained its flavonoids, saponins, and reducing sugars even after a year of extraction, as revealed by phytochemical analysis. The study underscores the potent toxicity of garlic extract against mosquito larvae, with LC 50 values below 50 ppm. These findings highlight the potential of garlic extracts as effective larvicides for combating mosquito vectors, contributing to environmentally friendly pest management strategies.


INTRODUCTION
Vector-borne diseases (VBDs) account for approximately 17% of the global burden of infectious diseases, resulting in over 700,000 deaths annually (WHO, 2014(WHO, , 2020)).Mosquito-borne diseases (MBDs) pose a significant threat to public health worldwide, and in Sri Lanka, with its tropical warm climate providing ideal conditions for mosquito breeding and disease transmission (Sirisena et al., 2017).The country is home to approximately 142 species of mosquitoes, representing high diversity and abundance (Chathuranga et al., 2018).These mosquitoes act as vectors for major MBDs such as dengue, chikungunya, malaria, and filariasis, which have caused significant morbidity and mortality in Sri Lanka.Aedes aegypti and Ae.albopictus are the principal vectors of dengue and chikungunya viruses in Sri Lanka (Sirisena & Noordeen, 2014).Dengue has been endemic in the country since the mid-1960s and continues to contribute to high fatality rates (Messer et al., 2002).In 2022, there were 42,729 reported dengue cases, with 39 fatalities, surpassing the number of cases in 2021 (33,000 cases and 26 deaths) (WHO, 2020).Chikungunya re-emerged in Sri Lanka in 2006 after a four-decade absence, affecting around 40,000 individuals in 2006 and 2007 (Hapuarachchi et al., 2010).Culex quinquefasciatus mosquitoes are major vector for filariasis.Sri Lanka successfully eradicated lymphatic filariasis as a public health issue in 2016, but the presence and abundance of the vector pose a risk of re-emergence (Chandrasena et al., 2018).Additionally, wild animals, such as birds, can act as reservoir hosts for these pathogens, which can be transmitted to humans via zoophilic mosquitoes (Chathuranga et al., 2018).
Considering the limited availability of specific drugs or vaccines for MBDs, vector control measures remain the most effective method for prevention (Erlanger et al., 2008;Achee et al., 2015).However, chemical interventions have drawbacks such as resistance development, environmental persistence, and adverse effects on non-target organisms.As a result, plant extracts have gained attention as promising alternatives owing to their bioactive properties and cost-effectiveness.Numerous plant species, including peppermint, lemongrass, rosemary, clove, citronella, thyme, spearmint, sweet orange, catnip, and basil, have shown insecticidal and repellent effects against various pests, including mosquitoes (Das et al., 2007;Hikal et al., 2017;Koul & Walia, 2009).
Garlic extracts (Allium sativum) has been identified as an effective larvicide against mosquitoes in several studies, particularly in South Asian and African regions.The earliest study reported was by Amonkar & Reeves (1970), where the efficacy of garlic extract was tested on larvae of Aedes sp. and Culex sp.Several examples include studies by Bilal et al. (2012), Rahmah et al. (2019), Yarsi & Munawaroh (2021) and Aminu et al. (2022) in which the efficacy of organic solvent extracts of garlic was demonstrated against larvae of Aedes sp.Other than that, Iqbal et al. (2018) revealed in their study that an aqueous extract of garlic was the most potent larvicide against Cu.quinquefasciatus among several other plant extracts.Kalu et al. (2010) also indicated that ethanolic extract of garlic is highly efficacious against the second, third and fourth instar stages of Cu. quinquefasciatus.
The reported active components in garlic cloves are allicin, diallyl disulfide, S-allyl cysteine, and diallyl trisulfide (Mikaili et al., 2013;Rahmah et al., 2019).Allicin, the most important compound, can interfere with RNA synthesis and lipid production in organisms, without affecting mammalian cells (Yarsi & Munawaroh, 2021).Garlic extracts are also claimed to contain flavonoids, which act as respiratory inhibitors (Rahmah et al., 2019).These properties make garlic a best candidate as a biopesticide for mosquito control.
Given Sri Lanka's tropical climate and high susceptibility to mosquito-borne diseases, it's crucial to assess the efficacy of plant extracts as replacements for synthetic insecticides.Despite the abundance of plant species in Sri Lanka, research on botanical biopesticides for mosquito control is limited compared to other pest management applications.Although there are several reported studies on application of garlic extracts to control pests of stored products and agricultural pests such as Callosobruchus maculatus (Niranjana & Karunakaran, 2019) and Tribolium castaneum (Karunakaran & Niranjana, 2019) in Sri Lanka, there are no reported studies conducted against mosquitoes up to date.Similar to synthetic insecticides, botanicals can inhibit acetylcholinesterase (the target-site of organophosphates and carbamates), GABA gated chloride channels and, disrupt sodium and chloride channels of the nervous system (target-sites of organochlorines and pyrethorids).Therefore, the response of mosquitoes to any biopesticide can vary depending on the insecticide exposure history, the target population and also on the environmental factors (Glunt et al., 2011;Gosh et al., 2012;Nkya et al., 2013;Owusu et al., 2017).Therefore, it is essential to conduct laboratory studies before implementing these botanical extracts in the field.Therefore, this study was conducted to evaluate the larvicidal activity of ethanolic garlic extracts against Ae.albopictus and Cu.quinquefasciatus mosquitoes in Sri Lanka under laboratory conditions and identify the secondary metabolites present in the extracts.These findings can serve as a foundation for further research and the development of novel mosquito control strategies using bioactive ingredients from plant materials.

Mosquito larvae for larval bioassays
Mosquito larvae for bioassay experiments were obtained from Ae. albopictus and Cu.quinquefasciatus mosquito colonies established at the insectary of the Department of Zoology, University of Peradeniya.The colony was maintained at room temperature (28 ± 2) °C under 12L:12D photoperiod and ± 70% Relative Humidity (RH).Late third and early fourth instar larvae were used for the bioassay experiments.

Preparation of the ethanolic crude extract from garlic
Manually separated 1 kg of garlic cloves (Allium sativum), collected from the local market of Digana on 5th of January, 2022, were washed and hot air dried at 75-85°C.Then, the dried cloves were ground in an electrical grinder to obtain a fine powder.The fine garlic powder was mixed with 100% ethanol in 1:5 ratios and loaded to a bottle shaker and was shaken for 72 h at room temperature (28 °C ± 2), at 100 rpm.The garlic-ethanol mixture was filtered using a double layer of cotton cloth and a Whatman No. 1 filter paper.Ethanol was removed using a vacuum rotary evaporator (HS-2005V-N, Hanshin Scientific, South Korea) under the inner temperature of 40-41°C and 130 rpm speed to obtain a semisolid crude extract.The crude extract was stored at 4 °C temperature until later use.

Preparation of the concentration series
Stock solution of 5000 ppm was prepared by dissolving 50 mg of the crude garlic extract in 10 mL of hot (40 °C) distilled water, stored in screw-cap glass vials, and kept in the refrigerator at 4 °C.A series of concentrations determined by preliminary experiments; 250 ppm, 100 ppm, 50 ppm, 25 ppm, 10 ppm, and 5 ppm was prepared.The bioassays for Ae.albopictus and Cu.quinquefasciatus were conducted in a final volume of 5 mL and 20 mL by mixing the appropriate volume of the stock solution with distilled water.

Evaluation of larvicidal activity
Ten healthy late-third instar larvae of Ae. albopictus and Cu.quinquefasciatus were separately exposed to each test concentration.The larvae were provided with ground fish food as the food source.Four replicates were conducted for each test concentration.Six controls were performed simultaneously with distilled water.All the tests were conducted at room temperature 28± 2 °C and a photoperiod of 12L:12D.Larval mortalities in treatments and control experiments were recorded at 24 h, 48 h, and 72 h exposure periods.Dead larvae were identified when they failed to move after probing with a glass rod in the siphon or cervical region.All the experiments were repeated if control mortality was more than 20%.

Data analysis
Percentage mortality values were calculated and corrected using Abbott's formula if the mortality value for controls were more than 5%.Mean percentage mortality values corresponding to each concentration were calculated and converted to probit values using Finney's table (Hamidi et al., 2014).The data were subjected to probit-log (dose) regression analysis using Minitab Software (version 18.0) to calculate lethal concentrations; LC 50 and LC 90 at 24 h, 48 h and 72 h.
Corrected mortality was calculated as follows using Abbott's formula; Rajapaksha et al.
Correlation analyses were conducted using Minitab software (version 18.0) for each exposure period to determine the correlation between the mean percentage mortalities and concentrations of garlic extract.

Phytochemical analysis of ethanolic garlic extract
The qualitative phytochemical analysis was carried out as shown in Table 1, to identify different phytochemicals in ethanolic garlic extract following the protocols described by Soni & Sosa (2013), Banu & Catherin (2015), Esienanwan et al. (2020), Nazir & Chauhan (2019), Priska et al. (2019), and Singh & Kumar (2017).For all the tests, a 10 000 ppm stock solution of the ethanolic crude extract was prepared using distilled water.

Larvicidal effect of garlic extract on Aedes albopictus and Culex quinquefasciatus
The results indicated a gradual increase in Ae. albopictus and Cu.quinquefasciatus larval mortalities with increasing concentration and exposure period.
A positive correlation was observed between the concentration of garlic extract and the percentage mortalities of Ae. albopictus larvae at 24 h (r= 0.983, p<0.05) and 48 h (r = 0.859, p<0.05) exposure periods.Although Ae. albopictus mosquito larvae showed positive correlation after 72 h (r= 0.732, p>0.05) exposure period, this correlation was not significant.A non-significant positive correlation was observed between the concentration of garlic extract and the percentage mortalities of Cu. quinquefasciatus larvae at 24 h (r=0.707,p=0.116),48 h (r=0.592,p=0.216) and 72 h (r=0.676,p=0.140) exposure periods.
The results derived from the regression analysis of Ae. albopictus and Cu.quinquefasciatus larvae are presented in Figure 1 with the obtained regression equations.The results revealed similarities and differences in Ae. albopictus and Cu.quinquefasciatus larvae responses to the treatments over different time intervals.For Ae. albopictus larvae, the LC 50 values decrease progressively as the exposure time increases.At 24 hours, the LC 50 value is 45.5 ppm, which decreases to 28.0 ppm at 48 hours and further decreases to 14.4 ppm at 72 hours.Similarly, the corresponding LC 90 values for Ae.albopictus larvae show a decreasing trend, with values of 140.0 ppm, 91.0 ppm, and 42.9 ppm for 24 hours, 48 hours, and 72 hours, respectively.The concentration required to kill 90% (LC 90 ) of the population at all three-time points was approximately three times higher than that of the concentration needed to kill 50% of the larvae (LC 50 ).
In contrast to Ae. albopictus larvae, the LC 50 values for Cu.quinquefasciatus larvae decrease significantly with each successive time interval.The LC 50 values for Cu.quinquefasciatus larvae at 24 hrs, 48 hrs, and 72 hrs were 26.3 ppm, 9.4 ppm, and 4.4 ppm, respectively.The LC 50 values for Cu.quinquefasciatus at all three time points were considerably lower than that for Ae.Table 1: Procedures followed for phytochemical assays.
Quinones 5 mL of the stock was mixed with a few drops of diluted sodium hydroxide and observed for blue, green or red colouration (Soni & Sosa, 2013).Flavonoids 3 mL of the stock was treated with 2 mL of diluted sodium hydroxide.The formation of intense yellow colour, which becomes colourless upon the addition of a few drops of Hydrochloric acid was observed (Singh & Kumar, 2017;Esienanwan et al.,2020).
Cardiac glycosides 1 mL of the stock was treated with 1.5 mL of glacial acetic acid and 5% ferric chloride.Thereafter, about 2 mL of conc.Sulphuric acid was added carefully from the side of the test tube and the appearance of a brown ring at the interphase or formation of blue colouration in the acetic acid layer was observed (Singh & Kumar, 2017;Nazir & Chauhan,2019;Esienanwan et al., 2020) Terpenoids About 5 mL of 1% stock was mixed with 2 mL of chloroform and 3 mL of conc.Sulphuric acid was added carefully to form a layer.A reddish-brown colouration was observed for the presence of Terpenoids (Soni & Sosa, 2013).

Coumarin
About mL of 10% sodium hydroxide and chloroform was added to a small amount of the stock.A yellow colour was observed for the presence of Coumarin (Soni & Sosa, 2013).
Saponin About 1 mL of the 1% stock was mixed with 5 mL of distilled water in a test tube and it was shaken vigorously.The formation of stable foam was taken as an indication of the presence of Saponins (Banu & Cathrine, 2015;Singh & Kumar, 2017;Esienanwan et al., 2020) Reducing sugar About 1 mL of the 1% stock was mixed with a small amount of Benedict's reagent and heated in a water bath for 5-10 minutes.The appearance of green, yellow or red colouration was observed for the presence of reducing sugar (Singh & Kumar, 2017)  larvae were significantly lower than that for Ae.albopictus larvae.However, LC 90 for Cu.quinquefasciatus after 24 h was slightly higher than that for Ae.albopictus.The concentration required to kill 90% (LC 90 ) of the population after a 24 h exposure period is approximately seven times higher than that of the concentration required to kill 50% (LC 50 ) of the population whereas, the LC 90 value at 48 h and 72 h exposure is approximately three-four times higher than the corresponding LC 50 value.

Phytochemical analysis of ethanolic garlic extract
The garlic extract was tested for the presence of phenolic compounds, quinones, flavonoids, terpenoids and steroids, cardiac glycosides, coumarin, saponins and reducing sugar.
The results revealed the presence of three phytochemicals; flavonoids, saponins and reducing sugar in the ethanolic garlic extracts used for the study.

DISCUSSION
The importance of vector control in mitigating the spread of mosquito-borne diseases cannot be neglected.Although traditional chemical insecticides have been successful in keeping mosquitoes under control, their prolonged usage poses challenges that urge for innovation of eco-friendly and sustainable alternatives.One such alternative is the use of biological insecticides derived from plant sources.This study evaluated the potential larvicidal activity of garlic extract against Ae.albopictus and Cu.quinquefasciatus mosquito in Sri Lanka and the results confirmed the larvicidal properties of garlic extracts against Ae.albopictus and Cu.quinquefasciatus larvae in Sri Lanka.
The concentration-response curves clearly demonstrated the significant effectiveness of garlic extracts, even at lower concentrations.Previous studies conducted by Aminu et al. (2022) in Nigeria and Amonkar & Reeves (1962) in California, USA, also reported similarly high efficacy against Aedes larvae following 24-hour exposure.Despite the geographical differences in these studies, the consistent results underscore the potential of garlic as an effective biopesticide, provided further research is conducted.Laojun et al. (2020) reported that an experimental material is highly effective if the LC 50 value is less than 50 ppm.
In our study, we received, low LC 50 values (<50ppm) for Ae.albopictus and Cu.quinquefasciatus larvae confirming the high effectiveness of the ethanolic extract of garlic as a potential larvicide.Aminu et al. (2022) 2019) have also identified these phytochemicals in garlic extracts.Flavonoids are water-soluble polyphenolic molecules, belonging to the polyphenol family, that can act as respiratory inhibitors by inhibiting the electron transport system and ATP production causing possible larvicidal effects (Singh & Kumar, 2017;Yarsi & Munawaroh, 2021).Xu & Lee (2001) stated in their study that flavonoids can disrupt bacterial growth by inhibiting protein synthesis.This antibacterial property of flavonoids could also contribute to the larvicidal activity of garlic.
Saponins are steroid or triterpenoid glycosides distinguished by their bitter or astringent flavour and foaming effects (Singh & Kumar, 2017).Studies conducted to test the efficacy of several plant extracts, which are positive for saponins as the major compound, have demonstrated high larvicidal activity against Ae.aegypti (Jawale, 2014;Bagavan et al., 2008) and Cu.quinquefasciatus (Bagavan et al., 2008) larvae.These studies further demonstrate that the saponin content of garlic accounts for its larvicidal effect against Ae.albopictus and Cu.quinquefasciatus mosquitoes.In future investigations, the phytochemical analysis may be used to assist in further analyzing the components of garlic extracts and developing new, efficient insecticides.
To further enhance the understanding of garlic extract components and develop more efficient insecticides, future investigations could involve in-depth phytochemical analyses.Additionally, comparative studies on extraction methods and solvents can be conducted to determine the most effective approach for obtaining garlic extracts with optimal larvicidal properties against Ae.albopictus and Cu.quinquefasciatus species.The knowledge gained from this study can also be extended to combat agricultural and stored product pests.However, further comprehensive research and continuous surveillance, such as quasi-field or pilot studies, are essential before applying garlic extracts to natural ecosystems.

CONCLUSION
The present study revealed the high efficacy of garlic ethanolic extract as a larvicide against mosquitoes of Ae. albopictus and Cu.quinquefasciatus in Sri Lanka with LC 50 values lower than 50 ppm.Moreover, the garlic ethanolic extracts proved to contain several important secondary metabolites with larvicidal potential such as flavonoids and saponins.In conclusion, garlic could be considered a promising eco-friendly biopesticide to be used in mosquito control programmes in the future.

Figure 1 :
Figure 1: Log-probit dosage curves obtained for (A) Aedes albopictus and (B) Culex quinquefasciatus larvae exposed to six different concentrations (250 ppm-5 ppm) of garlic extract for 24 h, 48 h and 72 h albopictus indicating relatively higher susceptibility of Cu. quinquefasciatus larvae to garlic crude extract.The corresponding LC 90 values for Cu.quinquefasciatus larvae also demonstrate a similar decreasing trend, with values of 169.8 ppm, 30.7 ppm, and 17.6 ppm for 24 hours, 48 hours, and 72 hours, respectively.Similar to the LC 50 , the LC 90 at 48 h and 72 hours for Cu.quinquefasciatus sp.
Iqbal et al. (2018)alue of 42.5 ppm for Aedes larvae exposed to aqueous garlic extract for 24 hours which was almost similar to what was observed in the present study.Ghosh et al. (2012)conducted a comparative analysis of over 150 plant extracts against Aedes, Culex, and Anopheles mosquitoes and stated that the efficacy of phytochemicals against mosquito larvae may also vary depending on the solvent used for extraction, which is attributed to differences in solvent polarity.This statement is further confirmed with the differences in LC 50 values between the present study and study byAmonkar & Reeves (1962), where methanol is used to prepare the garlic extract.The later study demonstrated a LC 50 of 33.7 ppm against Aedes larvae which is lower than the present study.Moreover, significant disparities in the LC 50 and LC 90 values of garlic extract against Aedes larvae have been reported from different parts of the world.For example, Susheela et al. (2016) observed much higher LC 50 values ranging from 10 000 ppm to 15 000 ppm for fourth instar stages of Ae. aegypti.The extraction method followed by these authors is, however, different as they have used simple cold extraction for garlic extraction.These variations highlight the influence of extraction methods on the efficacy of garlic extract.Numerous studies have discovered that garlic's aqueous and ethanolic extracts are both very efficient against Cu.Rajapaksha et al.quinquefasciatus larvae.Kalu et al. (2010)reported LC 50 values of 144.54 ppm, 165.70 ppm, and 184.18 ppm for second, third, and fourth instar Culex larvae, respectively, when exposed to ethanolic garlic extract.In contrast,Iqbal et al. (2018)reported a significantly higher LC 50 value of 13,700 ppm for Culex larvae exposed to cold aqueous garlic extract.The lower LC 50 values obtained in the present study indicate a higher susceptibility of Cu. quinquefasciatus larvae to garlic extract compared to these already published data.This could be due to the relatively lower resistance levels observed in the Sri Lankan population of Cu.In comparison to the LC 50 values obtained for Ae.albopictus of the present study, Cu. quinquefasciatus shows significantly lower LC 50 values for all the exposure periods.Furthermore, 86.25% of the tested Cu. quinquefasciatus larvae were dead by the third day of exposure to the treatments.The cumulative larval mortality for Ae.albopictus after third day of exposure was 66.67%.This confirms that Cu. quinquefasciatus larvae are relatively more susceptible to garlic extract compared to Ae. albopictus larvae.