Evaluation of different high doses aqueous plant extracts for the sustainable control of Aedes aegypti mosquitoes under laboratory conditions

2.1 Collection and preparation of plant extracts

The plants; Parthenium hysterophorus, Nicotiana tabacum, Fagonia indica and Melia azedarach were collected from the local area of Peshawar. The plants were washed and air-dried for 48 h at room temperature. The selection of Parthenium hysterophorus, Nicotiana tabacum, Fagonia indica, and Melia azedarach, obtained from local sources in Peshawar, due to their geographical accessibility and potential historical utilization as botanical assets. The chosen doses (0.5–0.9 % and 1–3 %) are likely indicative of an effort to examine a spectrum of dilutions in order to accurately evaluate the impacts of the treatment. The plant materials leaf, flower, and seed were taken and ground into small pieces with the help of an electric blender and filtered with stainless steel mesh (0.01 mm). Out of the selected plants, 100 gm of powder was mixed with 1 L of water to prepare a 10 % stock solution. The solution was then left for 48 h. The stock solution was filtered with muslin cloth and then was serially diluted in different concentrations (0.5–0.9 % and 1–3 %) to evaluate the Treatment effect. Serial dilution was done by using the following formula: $$\mathrm{C}1\mathrm{V}1=\mathrm{C}2\mathrm{V}2$$

Required concentrations; 0.5 %, 0.6 %, 0.7 %, 0.8 % 0.9 %, and 1 %, 1.5 %, 2 %, 2.5 %, 3 %. $$\mathrm{V}1=0.5\times 90/10=4.5\mathrm{m}\mathrm{l}$$

4.5 ml of the stock solution were taken and diluted in 90 ml of water to make a 0.5 % concentration. $$\mathrm{C}1\mathrm{V}1=\mathrm{C}2\mathrm{V}2$$ $$\mathrm{V}1=3\times 90/10=27\mathrm{m}\mathrm{l}$$

Similarly, for 3 % concentration, 27 ml were taken from the stock solution and dissolved in 90 ml water.

2.2 Bioassay

Aqueous-based extracts of various plants were used against the various stages of Aedes aegypti Mosquito according to standard WHO (World Health Organization) procedures.

2.3 Ovicidal trails

Aedes aegypti mosquito eggs, collected from NIFA's Medical Entomology laboratory in Peshawar, were exposed to various plant extract concentrations in plastic cups, with about 100 eggs per cup. The control group was subjected to tap water. Each concentration was replicated thrice. Data on egg hatching was recorded, with hatched eggs separated using a plastic dropper. The toxicity of plant extracts was evaluated by counting hatched and unhatched eggs at 24, 48, and 72-hour post-exposure intervals. The following formula was used to calculate Percent egg hatching: $$\mathrm{P}\mathrm{e}\mathrm{r}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{o}\mathrm{v}\mathrm{i}\mathrm{c}\mathrm{i}\mathrm{d}\mathrm{a}\mathrm{l}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y}=\mathrm{N}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r}\mathrm{o}\mathrm{f}\mathrm{h}\mathrm{a}\mathrm{t}\mathrm{c}\mathrm{h}\mathrm{e}\mathrm{d}\mathrm{e}\mathrm{g}\mathrm{g}\mathrm{s}/\mathrm{e}\mathrm{x}\mathrm{p}\mathrm{o}\mathrm{s}\mathrm{e}\mathrm{d}\mathrm{e}\mathrm{g}\mathrm{g}\mathrm{s}\times 100$$

2.4 Larvicidal potential

For larval mortality hundred numbers of the third instar, uniform size larvae were taken from the Medical Entomology laboratory of NIFA, Peshawar. The Larvae were then placed in disposable plastic cups containing different concentrations of plants extracts. Each tested concentration was repeated three times and the control only with tap water. During data recording, the dead larvae were separated from alive larvae with the help of a small plastic dropper. The plant extracts toxicity effect was determined by carefully counting the number of dead and alive larvae after 24, 48, and 72-hours’ post-exposure time. Percent larval mortality was recorded from the average of three replications. The percent mortality was corrected using the following formula (Abbott, 1925). $$\%\mathrm{M}\mathrm{o}\mathrm{r}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}=[\mathrm{P}0-\mathrm{P}\mathrm{C}]/[100-\mathrm{P}\mathrm{C}]\times 100$$

Whereas P0 = Larval Mortality in treated Treatment PC = Larval Mortality in the control Treatment.

Abbott’s formula was used to correct the mortality data, and the lethal concentration values of LC50 were calculated by using a probit analysis program.

2.5 Pupicidal potential

In the case of the pupal trial hundred numbers of uniform size, pupae were taken from the Medical Entomology laboratory of NIFA, Peshawar. It was then placed in plastic cups containing varied concentrations of the extracts. Each tested concentration was repeated and undertaken three times and the control was treated only with tap water. The motionless pupae were considered dead and unable to emerge into adults. During data recording, the dead pupae were separated from alive pupae with the help of a dropper. The plant extracts toxicity effect was determined through carefully counting the number of emerging and dead pupae after 24, 48 and 72-hours’ post-exposure time. Percent adult emergence was recorded by using the formula: $$\mathrm{N}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r}\mathrm{o}\mathrm{f}\mathrm{a}\mathrm{d}\mathrm{u}\mathrm{l}\mathrm{t}\mathrm{s}\mathrm{e}\mathrm{m}\mathrm{e}\mathrm{r}\mathrm{g}\mathrm{e}\mathrm{d}/\mathrm{e}\mathrm{x}\mathrm{p}\mathrm{o}\mathrm{s}\mathrm{e}\mathrm{d}\mathrm{p}\mathrm{u}\mathrm{p}\mathrm{a}\mathrm{e}\times 100$$

2.6 Adult deterrence activity

The adult deterrence test was performed following Rahuman et al. (2009) procedure with slight modification. The experiment was performed in the Medical Entomology laboratory of NIFA, Peshawar. In the presence of various Treatments in varying concentrations of aqueous extracts, the adult's ovipositional deterrent effect and the number of eggs deposited were investigated. During the pupal stage, about 20 male pupae and 20 female pupae were separated and placed in rearing cages (24.5 × 24.5 × 24.5 cm) at a temperature of 27 ± 2 °C and relative humidity of 75 ± 5. In the cages, the pupae were let to develop into adults. Adult males were given a 10 % sugar solution in disposable cups with cotton wicks regularly, while females were fed with Bovine blood/Albino mice after the fifth day of their emergence. Five cups holding 30 ml of different extracts solution with the same concentration were placed in each cage in the various treatments test, along with a 9-cm piece of filter paper for egg ling. The sixth (06) cup, devoid of extracts, was used as a control. Three replications of various treatments with the same concentration for each bioassay were done in cages put side by side. Under a stereomicroscope, the data was counted based on the number of eggs laid in Treatment and control cups after 24, 48, and 72 h of exposure time. The following formula was used to calculate effective repellency for each Treatment and concentration: $$\mathrm{E}\mathrm{F}=\mathrm{N}\mathrm{C}-\mathrm{N}\mathrm{T}/\mathrm{N}\mathrm{C}\times 100$$

Where the EF is the effective repellency, NC is the number of eggs in control, and NT is the number of eggs in Treatment.

2.7 Statistical analysis

The percentage of larval mortality was calculated by dividing the number of dead and inactive larvae by the total number of larvae that were tested. Microsoft Excel and IBM SPSS Statistics for Windows software were used for data analysis. The Pearson fit test was used to determine whether Probit’s model adequately fit the data provided by the experiment. The percentage of egg hatchability at each dose was calculated by dividing the number of eggs hatched by the total number of eggs inserted, multiplied by 100. The average hatchability at each dose was calculated, including the standard deviation. One-way analysis of variance was used to determine whether there was a significant difference in the mean hatchability between the different doses.

4.1 Percent egg hatching of Aedes aegypti mosquitoes in various extracts at high dose concentrations under different times (24, 48, and 72 h)

Plant extracts have been extensively studied for their mosquito control potential. Early research of Qualls and Xue (2009) demonstrated how plant-derived compounds disrupt mosquito life cycles, leading to reduced egg hatching. More recently, Govindarajan and Benelli (2017) confirmed the ovicidal activity of Feronia elephantum extracts against Aedes aegypti, reducing egg hatching. Our study aligns with these findings, showing the effectiveness of high-dose plant extracts in decreasing egg hatching over time. However, the impact varies with plant type, extraction method, and concentration (Kumar et al., 2012; Li et al., 2022, Zhang et al., 2022). Specific extracts displayed stronger effects, highlighting the need for comprehensive exploration of plant species and concentrations.

Our study aligns with recent research Benelli (2020) that shows increased egg hatch deterrence over time following exposure to Ocimum canum extract. Extended exposure to high-dose botanical extracts disrupts the Aedes aegypti mosquito life cycle, holding promise for vector management amid mosquito resistance to synthetic insecticides (Liu, 2015). Plant extracts present eco-friendly, renewable, and biodegradable alternatives for population control by inhibiting egg hatching. However, identifying specific bioactive compounds responsible for ovicidal activity and assessing their safety for non-target organisms is crucial, as emphasized in previous studies (Pavela and Benelli, 2016; Korunić et al., 2016). Our findings support the documented potential of plant extracts in mosquito control, with larvicidal and ovicidal properties well-established (Ghosh et al., 2012; Pavela and Benelli, 2016).

4.2 Percent larval mortality of Aedes aegypti mosquitoes in various plants extracts at high dose concentration in different time (24, 48, 72 h)

Our study demonstrated a significant increase in larval mortality over time for all plant extracts. Parthenium showed the highest efficacy within 24 h, resulting in mortality rates between 45.6 % and 94 % at 1 %-3% concentrations. Tobacco, Fagonia indica, and Melia azedarach extracts also exhibited notable larval mortality, but lower than Parthenium, consistent with Ghosh et al.'s research (2012). At 48 h, the plant extracts maintained their lethal impact, with Parthenium showing persistent efficacy (55 %-91 %). This trend aligns with Yang et al.'s findings (2020), attributing prolonged impact to continuous larval exposure to plant compounds. After 72 h, Parthenium's efficacy increased further (100 % mortality at 3 %), while other extracts showed substantial larval mortality, similar to Saliva et al.'s report (2008).

Importantly, our research showed negligible larval mortality in the control group across all time frames, which underscores the substantial larvicidal efficacy of the plant extracts. The standard check concurrently displayed high larval mortality, affirming the accuracy of our testing protocol. The escalating larval mortality with increasing extract concentration and exposure duration substantiates the larvicidal potential of these botanicals. Our results, indicating a dose-dependent response in mosquito larvae to plant extracts, agree with Govindarajan et al.'s findings (2016). Additionally, we corroborate Kumar et al.'s research (2012) showcasing the effectiveness of Parthenium extract in causing Aedes aegypti larval mortality. Our results are consistent with Isman's 2020 review, suggesting that variations in the efficacy of plant extracts can be attributed to the intricate interactions of bioactive compounds present within each extract.

4.3 Percent lethal concentration of Aedes aegypti mosquitoes exposed to various plant extracts of high dose after 24, 48 and 72 h

As early as 24 h into the exposure period, Parthenium extract emerged as the most potent, with the lowest LC50 value (0.975 %), an indication of its higher toxicity towards Aedes aegypti larvae. This is consistent with the findings of Sharma et al. (2018), who observed a significant larvicidal effect of Parthenium hysterophorus against Aedes aegypti. Other plant extracts, Tobacco (1.573 %), Fagonia indica (1.931 %), and Melia azedarach (2.640 %), also displayed considerable larvicidal activities but at varying degrees, thereby illustrating their potential as botanical insecticides.

Upon extending the exposure to 48 h, there was a noticeable decrease in LC50 values for all plant extracts, with Parthenium maintaining its lead, showing an LC50 value of 0.420 %. Previous studies have also reported the enhanced larvicidal effects of plant extracts with prolonged exposure (Ghosh et al., 2012). In this context, our findings align with the existing literature, as increased exposure led to increased mortality in mosquito larvae.

At the final time point of 72 h, the trend continued, with Parthenium exhibiting an LC50 value as low as 0.120 %, further establishing its prominent larvicidal properties. Fagonia indica and Melia azedarach also demonstrated a substantial decrease in LC50 values to 1.104 % and 1.703 %, respectively, signifying their larvicidal potential.

Indeed, the lower LC50 values of temephos across all exposure times (0.256 %, 0.104 %, 0.003 % for 24, 48, and 72 h respectively) underscore its proven efficacy as a larvicide. However, concerns about the development of resistance among Aedes populations and the environmental implications of synthetic insecticides drive the need for alternative methods (Luz et al., 2019). Furthermore, as pointed out by Wei et al. (2015), extracts from Tobacco and Melia azedarach have shown promise against a range of insect pests.

4.4 Percent adult emergence of Aedes aegypti mosquitoes in various plant extracts under different exposure times at a high dose concentration

Findings from previous studies align with the trend seen in our investigation, which show a marked decrease in adult emergence of Aedes aegypti mosquitoes under increased concentrations of plant extracts. This suppression of mosquito metamorphosis aligns with the findings by Rajkumar and Jebanesan (2008), who illustrated a decrease in adult emergence following treatment with plant-derived Eucalyptus globulus extracts, marking a precedent for the results observed in our study.

Our study concurs with Pavela's 2016 review emphasizing the potential of plant extracts, rich in secondary metabolites, in hindering mosquito larval growth and development. The tested extracts, including Parthenium, Tobacco, Fagonia indica, and Melia azedarach, likely introduced growth-inhibiting secondary metabolites into the aquatic environment, suppressing Aedes aegypti adult emergence.

Our findings of reduced adult emergence with increased extract concentration align with Nagoor Meeran et al.'s 2020 study, which observed significant inhibition of Aedes aegypti adult emergence using 2 %-3% Tephrosia purpurea and Cleistanthus collinus extracts. This reaffirms that elevated plant extract concentrations can disrupt Aedes aegypti maturation.

In another related study, Amer and Mehlhorn (2006) examined the impact of various plant extracts on the life cycle of Aedes aegypti and found significant reductions in adult emergence. This validates our results and further establishes the potential of botanical extracts as biocontrol agents in managing mosquito populations. However, not all investigations have yielded identical findings. Benelli et al. (2013) highlighted that the effectiveness of plant extracts could be species-specific.

4.5 Percent adult deterrence of Aedes aegypti mosquitoes in various extracts under different time intervals at high dose concentrations

Parthenium extract displayed the most potent oviposition deterrent effect among the tested plant extracts. This potent oviposition deterrence aligns with the findings of a study by Amir et al., (2017), which established the ovicidal activity of Parthenium hysterophorus against Aedes aegypti. The present study, however, extended this understanding by demonstrating that this activity persists across different time intervals and concentration ranges.

The Tobacco extract's efficacy against adult Aedes aegypti aligns with previous studies highlighting the larvicidal and repellent properties of Tobacco-derived products (Panneerselvam and Murugan, 2013). Our study extends existing knowledge by showcasing oviposition deterrence in mosquitoes subject to high-dose Tobacco extracts. Comparable deterrent effects were found with Fagonia indica and Melia azedarach extracts, in line with previous research (Chellappandian et al., 2015; Sujitha et al., 2015). The study suggests that various plant extracts might serve as broad-spectrum deterrents, presenting a promising strategy for expanded vector control. Distinguished oviposition rates among treated and untreated mosquitoes, and a standard temephos-treated group underscore plant extracts' potential as oviposition deterrents. A dose-dependent decrease in oviposition, aligning with previous studies (Warikoo et al., 2011), implies a potential dose-dependent toxicity or deterrent mechanism. The research seems to only examine particular plant extracts obtained from a single geographical location, perhaps constraining the applicability of the results to different areas or mosquito species. Moreover, this study evaluates the impacts of plant extracts within relatively brief time periods, specifically 24, 48, and 72 h. This observation may fail to encompass the enduring consequences or fluctuations in the life cycles of mosquitoes. Nevertheless, the primary emphasis of the study is on plant extracts with high-dose concentrations. It could be advantageous to investigate a more extensive array of concentrations in order to enhance comprehension of dose–response connections. The experiments appear to have been conducted within a controlled laboratory environment. In a similar vein, while this is crucial for maintaining scientific rigor, it may not comprehensively depict the intricate and ever-changing habitats in which mosquitoes flourish in their natural settings. However, it is worth noting that the research lacks field testing, a crucial component for evaluating the actual effectiveness and feasibility of plant-based mosquito control techniques in real-world scenarios.