The rice weevil Sitophilus oryzae (L.) is widely recognized as one of the most destructive pests, particularly inflicting damage on cereal grains, especially rice, and related products (Baloch, 1992). These pests thrive in warm climates, reproduce vigorously, and pose significant threats to the quality and quantity of unprotected stored grains. In tropical nations like India, damage to stored grains and grain products ranges between 20-30%, compared to only 5-10% in the temperate zones (Trivedi et al., 2018). Spices have been employed since ancient times to enhance the sensory appeal of food as culinary enhancements (Amalraj and Gopi, 2017). These natural ingredients, besides adding taste, have diverse applications like preserving food, medical purposes, religious practices, cosmetics, and perfumes (Cardoso-Ugarte and Sosa-Morales, 2022). Utilizing spices is economically friendly and accessible, especially for developing regions, and presents minimal risks, making them a secure choice without endangering the products (Diniz do Nascimento et al., 2020). Clove has been a highly regarded spice, valued not only as a food preservative but also for its extensive medicinal applications. It exhibits effectiveness against a wide spectrum of other bacteria, including Escherichia coli, Listeria monocytogenes, and Salmonella enterica (Friedman et al., 2002). Previous studies have also reported its antibacterial and antifungal properties (Hiwandika et al., 2021; Kaur et al., 2019), potential as an anti-inflammatory and anticarcinogenic agent (Han and Parker, 2017), antiallergic effects (DjidjouTagne et al., 2018), and antimutagenic activity (Ashafani et al., 2023). Eugenol, the primary component of clove oil, exhibits antioxidant properties and acts as an insecticide (Aara et al., 2020). Traditionally, fenugreek has been employed for its antidiabetic (Al-Habori and Raman, 1998), antioxidative (Bhanger et al., 2008), antineoplastic, anti-inflammatory (Almatroodi et al., 2021), antipyretic (George et al., 2016), immunomodulatory, and antitumor effects (Almalki and Naguib, 2022).

Furthermore, mature fenugreek seeds contain an array of bioactive components, including amino acids, fatty acids, vitamins, saponins, galactomannans, alkaloids, and polyphenols along with fibers, flavonoids, and various polysaccharides (Jayaweera, 1981; Gavahian et al., 2023). Asafoetida has found application in the treatment of a variety of ailments, including conditions like whooping cough, asthma, ulcers, epilepsy, stomachaches, flatulence, bronchitis, intestinal parasites, spasms, weak digestion, and influenza (Takeoka, 2001). Asafoetida was found to be an effective remedy for numerous stomach-related disorders (Mahendra and Bisht, 2012). Based on this background, the current research work was undertaken to study the efficacy of spice extracts of Syzygium aromaticum, Trigonella foenum-graecum and Ferula assa-foetida against Sitophilus oryzae at different concentrations and hours of exposure, in individual as well as combined formulations. The study was based on the analysis of its life cycle stages, morphological abnormalities in the egg, larval, and pupa stages of development, adult mortality bio-assay, and qualitative and quantitative assessment of α-amylase activity of rice weevil following standard techniques.

Dried clove buds, fenugreek seeds and asafoetida powder were purchased from the local retail market of Guwahati (26°09′15.9“N 91°44′22.2“E; 26°09′17.1“N 91°44′18.0“E). A taxonomist from the Department of Botany, Cotton University identified the collected spices as Syzygium aromaticum (clove), Trigonella foenum-graecum (fenugreek), Ferula assa-foetida (asafoetida). Methanolic, ethanolic, and aqueous extracts were prepared following Furniss et al. (1980) and four concentrations- 0.1, 0.3, 0.5, and 0.7% of the crude extracts of each were prepared. Phytochemical screening of extracts was conducted in accordance with the methods described earlier (Silva et al., 2017; Kunatsa et al., 2020). The adult weevils were collected from infested rice grains, obtained from a Fairprice shop, Binowa Nagar, Guwahati (26°09′15.9“N 91°44′22.2“E; 26°09′17.1“N 91°44′18.0“E). The collected insects were identified using key of Gorham (1987) and classified with the help of ICAR-National Bureau of Agricultural Insect Resources and ITIS taxonomy database. Adult rice weevils were reared following standard procedure at 26-30ºC and 70-80%RH (Romano et al., 2016).

To assess the lifestages of S. oryzae in the presence of food, 20 male and female adults were selected from the stock culture and placed separately into observation and the incubation period was ascertained as described by Floyd and Newsom (1959). The effect of spice extracts on the life cycle of S. oryzae at various doses were investigated by separately spraying over the treated sets infested with rice grains containing eggs, larvae, and pupa and periodically studied by dissecting infected rice grains and observing them under a microscope. The fundamental basis for the observations is the detection of morphological abnormalities in the egg, larval, and pupa stages of development. Using a small hand sprayer, different doses of each spice methanolic extracts were sprayed over the infested grains and the mean number of dead insects and percentage adult mortality was calculated after 1, 5, 24, 72 and 168 hr of exposure (Batta, 2004). The rice weevils were considered dead if their appendages did not move when stimulated with a fine brush. In the treated sets, the doses of spice extracts were applied individually, and the 0.5% dose of combined extract was applied and mortality was assessed. For detection of α-amylase activity, both qualitative and quantitative analysis were performed. Qualitative analysis was carried out using Benedict’s test (Kunatsa et al., 2020) and quantitative was carried out following the dinitro-salicylic acid (DNS) method, as outlined by Ravan et al. (2009). All the assays were replicated three times. The data was presented in terms of Mean ± SE and subjected to analysis by two-way ANOVA. P-value of less than 0.05 was considered to be statistically significant. All the analysis were made by using GraphPad Prism Version (8.0.2) and Microsoft Office Excel (Version 2007).

The results of the qualitative phytochemical analysis of spice extracts are summarized in Table 1. The most common phytochemicals identified in Syzygium aromaticum include flavonoids, glycosides, alkaloids, terpenoids, and phenolic compounds, in addition to which tannins and triterpenoids were also abundant in Trigonella foenum-graecum. It was observed that Ferula assa-foetida had a comparably lower amount of phytochemicals than the other two plant extracts. Methanolic extract was more potent due to the presence of higher amounts of phytoconstituents. Therefore, the methanolic extract of the spices was used in dose formulation for conducting different bioassays. The duration of the entire lifecycle of S. oryzae in normal conditions (with no treatment) ranged from 35-50 days. Adult insects were reddish-brown to black with four reddish or lighter-hued spots situated at the corners of their elytra. The prothorax displayed distinctive pitting characterized by round or irregularly shaped depressions. The incubation period on rice grains spanned for 5-7 days. The larval stage persisted for 21-29 days. Fully developed larvae underwent pupation within the rice grains, where they remained for 7-10 days before emerging as adult weevils. The pupal stage closely resembled the adult. The lifecycle results are in conformity with reports of earlier workers like Choudhury and Chakraborty (2014). Syzygium aromaticum, Trigonella foenum-graecum, and Ferula assa-foetida methanolic extracts were tested for their toxic effects. On Sitophilus oryzae across their life cycle stages and were found to be harmful et al does concentrations.

The toxicity of these extracts renders the egg stage inactive or prevents the insect from developing into larva. In the early and late instar stage, deformation is observed at lower concentrations which leads to larval death at higher concentrations. However, in adults, although lower concentrations had no effect, higher concentrations caused shock and, ultimately death (Fig. 1). Morphological aberrations such as distorted shape, inactivity in larval stages, larval, and pupal death with no further development into adulthood were observed. The effects increased with increase in the dosage. The mortality data indicated that clove buds possess substantial pesticide properties. Eugenol, a constituent of clove oil has been noted for its involvement in photochemical reactions (Mihara and Shibamoto, 1982) and its robust antioxidative capabilities (Ogata et al., 2000), as well as its potential to counter aflatoxinogenesis (Caceres et al., 2016). Secondary metabolites such as phenolic compounds, alkaloids, terpenoids, saponins and flavonoids have been identified to exhibit strong activities against several pathogens and insect pests (Rami et al., 2021). Plant secondary compounds, responsible for the unique aroma of essential oils have insecticidal properties, rendering them effective against a broad spectrum of insects (Sharma et al., 2023; Sulhath et al., 2024). The insecticidal activity of saponins has been studied by Chaieb (2010). Saponins, found in healthy plants, are secondary metabolites that possess the ability to combat pathogens and act as chemical defenses against them (Zaynab et al., 2021). The enhanced effectiveness observed in the combined formulations may be attributed to the presence of terpenoids, a group of phytoactive compounds, which is common among all the spices selected. Terpenoids act on insects in various ways, mainly as chemical defences, insect repellents, attractants, insecticides, and antifeedants and their derivatives can show enhanced pesticidal activity (Deka et al., 2022). Terpenes exhibit bacteriostatic, bactericidal, and virucidal properties. They have demonstrated effectiveness against house flies, storage grain insects, and medical pests. Terpenes represent potent antiparasitic agents, offering potential for formulating orally delivered treatments. With rapid absorption in the stomach, they enable dosage reduction and mitigate toxic side effects effectively (Yadav and Kant-Upadhyay, 2022; Liu et al., 2023).

At 24, 72 and 168 hr post-treatment, dose formulations of 0.5%, and 0.7% of clove, fenugreek and asafoetida showed mean mortality of very high statistical significance (p<0.0001). The mean % mortality of adult S. oryzae treated with a 0.5% combined dose was the highest, reaching 100% (Fig. 2). The resuts of the mortality bioassay affirm the insecticidal efficacy of spice extracts. The bioassay also demonstrated a pronounced effect of Syzygium aromaticum (clove) extract on the adult S. oryzae mortality rate when compared to Trigonella foenum-graecum and Ferula assa-foetida extracts. Clove, clove powder, and clove oil have been subjects of investigation in addressing urban and agricultural pest issues, and research has revealed their pesticidal characteristics (Kafle and Shih, 2013). The observed mortality of weevils in this study exhibited an upward trend corresponding to the dosage of clove extract administered. This increase is attributable to the presence of secondary metabolites, which exert potent insecticidal effects (Forim et al., 2012). El El Gohary et al. (2021) reported that the aqueous extract of S.aromaticum essential oil (EO) and EO encapsulated in nanoparticles show potential as effective larvicides for managing Culex pipiens. Batubara et al. (2023) indicated that the polar fraction of S. aromaticum extract exhibited highest mortality in termites.

Trigonella foenum-graecum seed extract presented as a promising herbal insecticide candidate for managing mealy bug populations (Mohamed et al., 2021). Trigonella foenum-graecum leaf extract-derived iron nanoparticles (FeNPs) exhibit robust insecticidal efficacy, inducing significant mortality rates of 92.83% and 91.41% in Spodoptera litura and Helicoverpa armigera, respectively, after 72 hr of treatment. This environmentally friendly approach presents a promising strategy to combat pyrethroid resistance, with a synergism ratio of 1.38 and 1.36 for the FeNPs-fenvalerate combination, as revealed by probit analysis. (Muthusamy et al., 2023). Fotouhi et al. (2024) focused on evaluating the fumigant toxicity of essential oil from F. assa-foetida against the carob moth, E. ceratoniae, a significant agricultural pest. Results showed varying susceptibility between male and female moths, with males exhibiting higher sensitivity. This difference was linked to variations in enzyme activities related to detoxification, antioxidant defense, and intermediary metabolism, emphasizing the potential of F. assa-foetida essential oil as a promising and environmentally friendly insecticide. The essential oil derived from clove consists of 85 to 92% eugenol (Dorman and Deans, 2000), a compound recognized for its ability to entirely inhibit the development of eggs, larvae, and pupae within grains for species like S. granarius, S. zeamais, T. castaneum, and Prostephanus truncatus (Obeng-Ofori and Reichmuth, 1997). Adedire et al. (2011) stated that clove powders and oils inhibit the locomotion of adult insects or cause their death.

The qualitative estimation of α-amylase activity in S. oryzae was performed using Benedict’s test and it revealed a gradual decrease in insect amylase activity with increased duration of exposure (Table 2). Therefore, a DNS assay was performed in order to quantify the decrease in amylase activity of the samples, and the results describe the inhibiting effect of all of the spice extracts along with their 0.5% combination dose. The maltose standard curve was constructed by plotting the concentration of maltose (μg/ml) along the X-axis and absorbance recorded at 540 nm along the Y-axis. The slope of the graph was calculated by considering two points lying on the curve. This slope value (0.0012) was further used in determining the concentration of the unknown reaction mixture, for estimation of amylase activity. The combined methanolic extract of the selected spices (in the proportion of 1:1:1), as well as the individual S. aromaticum extract, caused the highest reduction in the alpha-amylase activity. The depletion of alpha-amylase activity by the spice extracts could be due to their secondary metabolite compounds (phytochemicals), which act as inhibitors of insect alpha-amylase. These chemical compounds can form complexes with the digestive enzymes of insect pests that are stable and dissociate slowly. Numerous natural plant and organic compounds employed for insect pest control are recognized for their impact on digestive enzymes. Plants produce secondary organic compounds that play a crucial role in safeguarding them against insect pests (Athanassiou et al., 2005). These secondary metabolites can either directly exhibit toxicity to insect pests or influence signalling pathways leading to the production of plant-based toxins. Additionally, certain plant secondary metabolites function through an antixenosis mechanism, inducing a lack of preference in host plants (War et al., 2020).

There appears to be a strong connection between the fluctuation in α-amylase activity within S. oryzae and its feeding behaviour. Sitophilus weevils, particularly S. oryzae, constitute major pests in stored product ecosystems, thriving on diets rich in polysaccharides and heavily reliant on alpha-amylases for sustenance (Mendiola-Olaya et al., 2000). Given that rice grains represent a primary source of starch for S. oryzae, this species depends on α-amylase to facilitate starch breakdown, resulting in elevated α-amylase activity, as observed in feeding insects (control group). However, the introduction of enzyme inhibitors disrupts the activity of digestive enzymes, including amylases and other essential components. This disruption hinders starch digestion, leading to diminished nutrient utilization, developmental delays, and, ultimately, insect mortality due to starvation (Isman, 2006; Hosseininaveh et al., 2007).

(‘4+’ denotes presence of chemical constituents in abundance; ‘3+’ and ‘2+’ denotes moderate presence; ‘+’ denotes presence; ‘-’denotes absence of chemical compound. These signs are based on colour intensity of the reactions in different phytochemical tests.)

‛5+’ indicates very high amylase activity (reddish precipitate during Benedict test);‛4+’ indicates high amylase activity (very dark green colouration); ‛3+’ and ‛2+’ indicate moderate amylase activity (dark green colouration); ‛+’ indicates low amylase activity (light green colouration); Values significant at p<0.05. Here,

indicates p<0.05;

indicates p<0.01;

indicates p<0.001;

indicates p<0.0001, compared to the values of the control set using Two-way ANOVA.

References