1 Introduction

Mosquitoes, the major arthropods, are lethal disease carriers and a severe menace to millions of people worldwide. They serve as vectors for fatal parasites and diseases such as filariasis, dengue fever, malaria, West Nile Virus, yellow fever, and Chikungunya (Ramos et al., 2019; WHO, 2020; WHO, 2014). Mosquito-borne illnesses damage human and animal life and have a significant economic effect, particularly in nations with tropical or subtropical climates that are hotspots for these types of epidemics or pandemics (Liu et al., 2021). Aedes mosquitoes, in particular, are worrying in terms of public health since they are the primary vectors of arboviruses such as malaria, filariasis, dengue, chikungunya, and Zika, which cause fatal viral illnesses (Matthews et al., 2018). Synthetic chemical pesticides have long been used to control mosquitoes. Still, their indiscriminate use has resulted in well-known and significant issues, such as genetic resistance, rising application costs, handling risks, and environmental contamination (Bharathithasan et al., 2021). This has prompted the need to study and develop ecologically safe, biodegradable, and low-cost indigenous vector control methods that individuals and communities may employ with little care in certain situations.

Plant products are emerging as a viable alternative to synthetic pest management/control chemicals. Phytochemicals from different chemical classes, including steroids, alkaloids, terpenes, and phenolic components, have previously been studied for pest control potential and have shown to be promising agents (Senthil-Nathan, 2020). Artemisia absinthium, commonly known as wormwood is an important medicinal plant of ethnopharmacological interest. It is indigenous to temperate regions of Europe, South Africa, North America, and Asia. This aromatic has been extensively studied for its antifungal, antimicrobial, and antiprotozoal properties. Traditionally, its leaf powder is used to treat gastrointestinal disorders and intestinal worms (Liu et al., 2019; Rizvi et al., 2018). The capacity to control insects varies with plant age, species, extracted component, collecting place, and extraction solvent. Thus the present study is aimed at investigating the larvicidal activity of Artemisia absinthium leaf extracts against the 4th instar larvae of Aedes aegypti and its detoxifying enzymes under laboratory conditions.

2 Materials & methods

2.1

2.1 Chemicals

Methanol, ethanol Fast blue –B salt and ACh (Iodide) were procured from Hi-media (Mumbai). α- and β-naphthol and DTNB were purchased from Sisco Research Laboratories (Mumbai, India). All other chemicals and glassware used were of analytical grade.

The Artemisia absinthium plant was collected from the Daksum area of Anantnag, Jammu and Kashmir, India at an altitude of 2438 m above sea level (Fig. 1 and Fig. 2). The identification and authenticity process of the plant was completed in the Kashmir University (CBT-botany) vide voucher specimen number 3070-(KASH) Herbarium. The leaves of the plant were shade dried in hygienic conditions for at least fifteen days. Thereafter the leaves were crushed into a coarse powder in an electrical grinder machine and packaged carefully for further processing.

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Fig. 1

Artemisia absinthium plant.

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Fig. 2

Collection site Daksum Anantnag Jammu and Kashmir, India. Latitude 33°36′43″N & longitude 75°26′6″E.

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2.4

2.4 Mosquito larvicidal bioassay

Mosquito larvae were collected from residential areas of Chennai, Tamil Nadu (Fig. 3). The early fourth instar larvae were chosen for the larvicidal bioassay, which was carried out according to the (WHO standard with slight modifications, 1996). In brief, batches of ten early fourth instar larvae were placed in 100 ml of tap water containing each solvent extract at final concentrations of 200, 400, 600, 800, and 1000 ppm. As a control, ordinary tap water with 1 % DMSO was utilized. The larvae were fed a 3:1 combination of dog biscuits and yeast and kept in the laboratory at a temperature of 27 2 °C and an ideal photoperiod (12 h light:12 h dark). The larvicidal bioassays were replicated five times, and the percentage of larvae death was calculated after 24 h of exposure.

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Fig 3

Aedes aegypti larvae.

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3 Results

Analysis of the mass Spectra of the obtained phytocompounds through gas chromatographic separation by comparing with the references in the Wiley and NIST led to the identification of 11 different bioactive phytocompounds (as shown in Fig. 4 and Table 1). Out of these, 3 compounds were dominant with a total percentage of 77.66 %. Concentration of Tris(2,4-di-tert-butylphenyl) phosphate (43.86 %) was highest, followed by Cycloisolongifolene,8,9-dehydro-9-formyl- (25.50 %), and 9,12,15-Octadecatrienoic acid, methyl ester, (8.30 %).

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Fig. 4

Chromatogram of ethanolic leaf extract of Artemisia absinthium.

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4 Discussion

Since arthropod-borne illnesses are predominantly treated by insecticide-based vector activity, the development of resistance is a critical issue (Gan et al., 2021). Insecticide resistance is rising in Southeast Asia, matching the global trend of other kinds of resistance. Given their availability and safety, natural herbs may serve as possible insecticides in controlling disease vectors, minimizing the demand for synthetic pesticides (Lengai et al., 2020).

Our study evaluated the potential larvicidal action of Artemisia absinthium. Ethanolic and Methanolic leaf extracts against the 4th instar larvae of Aedes aegypti. After 24 h of exposure, larval mortality was found to be 92 ± 1.99 % in ethanolic extract and 68 ± 1.99 % in the methanolic extract at 1000 ppm. The overall activity of the crude plant extracts is attributed to a diverse mixture of active compounds.

Artemisia absinthium ethanolic leaf extract has strong larvicidal activity when compared to methanolic extract. As a result, Artemisia absinthium ethanolic leaf extract was subjected to GC–MS analysis to identify the chemical components of the extract. The GC–MS profile of the chemicals revealed some essential biologically active metabolites with very strong insecticidal properties. Neophytadiene, 9,12,15-Octadecatrienoic acid, methyl ester, have been reported to have larvicidal or insecticidal activity (Ravi et al., 2018; Evanjaline and Vr, 2018; Kannathasan et al., 2008; Hikal et al., 2017). Cycloisolongifolene, 8,9-dehydro-9-formyl is a bicyclic monoterpenoid, and such compounds have been seen to inhibit acetylcholinesterase (AChE) activity (Miyazawa and Yamafuji, 2005). The synergistic action of the identified compounds may also be responsible for the death of the larvae.

Knowing the specific mechanism of botanicals' interaction with the physiological system of target insects is critical for developing better insecticides. The systemic effect of Artemisia absinthium crude methanol and ethanol extract was studied at the threshold period (24 h) for its fatal effect utilizing specific lethal concentration (LC50) tests on 4th instar larvae with regard to alterations in Aedes aegypti biochemical components.

Proteins, such as chitin production and cuticle development, play a crucial role in insect metamorphosis. When insects are subjected to plant-based biopesticides, their whole-body protein levels may vary due to downregulation or upregulation (Parthiban et al., 2021; Parthiban et al., 2019). In our investigation, the extract-exposed fourth instar larval protein level was seen to be significantly lower than the control larvae. Similar effects were also observed by (Parthiban et al., 2020). Acetylcholinesterase is a neurotransmitter enzyme that plays an important role in hydrolyzing its substrate acetylcholine into acetate and choline. The acetylcholine is continuously released from the pre-synaptic neuron into the synaptic cleft, where it binds to the receptor /ligand-gated sodium channels, allowing the heavy movement of sodium ions to pass through this channel (Jankowska et al., 2018; Oni et al., 2019). Once the action has been completed, acetylcholinesterase, a special enzyme, is released to break acetylcholine into choline and acetate. The choline is reabsorbed and combined with acetyl CoA to take part in regular cycles to synthesize acetylcholine (Wilkinson, 1976). AChE has been reported to be carbamate and organophosphorus resistant, and it is generally known that AChE alteration is one of the primary resistance mechanisms in insect pests (Ghosh et al., 2012). In our investigation, the extract-exposed fourth instar larval protein level was seen to be significantly lower than the control larvae. Similar effects were also observed by (Koodalingam et al., 2011; Parthiban et al., 2020). AChE inhibitory properties of plant extracts have been reported (Silvério et al., 2020). The death of insects due to plant extract treatment suggested that the chemicals present in the extracts may interfere with the cholinergic synapse and disrupt the communication network from one exonic end to the other, thereby stopping nerve impulse transmission. Thus, the deadly impact may be due to a build up of acetylcholine (ACh), a neurotransmitter, at synaptic junctions, disrupting coordination between the nerve and muscle junctions (Jankowska et al., 2018; Oni et al., 2019).

The key esterase enzymes in the insect physiological system, acetylcholinesterase, α and β-carboxylesterase, detoxify xenobiotics and other exogenous compounds that enter the insect body (Lushchak et al., 2018). Insecticide-resistant mosquitoes develop due to enhanced detoxifying enzyme activity brought on by increased gene amplification or upregulation after exposure to synthetic pesticides (Şengül Demirak and Canpolat, 2022). Plant extracts and derivatives have been shown to reduce carboxylesterase (α-β-carboxylesterase) levels in Aedes aegypti larvae (Koodalingam et al., 2014; Lija-Escaline et al., 2015). As a result, several researchers began to report diverse plant metabolites with esterase inhibitory activities tested on insect models, including mosquitoes. They then investigated how esterase inhibitors target enzyme binding sites in insects to develop effective pesticides (Al-Solami, 2021). According to our findings, when Aedes aegypti fourth instar larvae were exposed to plant extract for 24 h, the activity of and α and β-Carboxylesterase enzymes was lowered due to the downregulation of these enzymes. Similar results were observed for β-Carboxylesterase upon exposure of the aqueous extract of Sapindus emarginatus to the fourth instar larva Aedes aegypti (Koodalingam et al., 2011). This demonstrates that the ethanolic extract of Artemisia absinthium can suppress the tested organism's particular enzyme activity without acquiring resistance.

Using chemical pesticides in water sources endanger both humans and the environment. Natural pesticides derived from plants are thus potential agents, particularly for mosquito larvae management (Alois et al., 2022).

5 Conclusion

To summarize, the leaf extract of the plant Artemisia absinthium included in the research has significant larvicidal activity, with ethanol extract exhibiting the highest activity. Ethanol extract significantly lowered the activity of AChE, α and β-carboxylesterase enzymes in the larvae. The study also needs in-depth phytochemical analysis using different solvents and extraction methods that will reveal more bioactive compounds in the leaves having larvicidal properties and can assist us in devising novel larvicidal formulations with minimal toxicity risks to the environment and life. In the meantime, the crude extract of Artemisia absinthium or isolated phytocompounds could be employed in stagnant water bodies, which are known mosquito breeding grounds. Besides, more research on the active principles involved and their mode of action and field testing is typically required before recommending this plant as an anti-mosquito product in a mosquito control program.