Bioactivity of some aqueous plant extracts against larval and adult stages of Callosobruchus maculatus (Fabricius) (Coleoptera: Bruchidae)

Introduction

Grain legumes as mung bean (Vigna radiata L. Wilczek) constitute an important part of a healthy diet as it source of dietary protein, micronutrients, and caloric energy to millions of families (Hanus, 2025) [9] . Losses of agricultural products, including grain legumes before and after harvest, lead to losses that may threaten food and nutritional security (Shahbazi et al., 2025) [23] . Like awise, using post-harvest loss reduction mechanisms can help increase the shelf life of agricultural products and enhance their contribution to achieving food security (Vutula, 2024) [26] . Postharvest insect infestations cause substantial losses in stored legumes worldwide. Under traditional storage conditions without adequate protection, grain damage in legumes may range from 25.2% to 100%, with average weight losses reaching up to 32.3% (Stathers et al., 2024) [25] . The pulse beetle, or Cowpae beetle, Callosobruchus maculatus (Fabricius) (Coleoptera: Bruchidae), is globally recognized as the most economically damaging primary pest of stored grain legumes. It is cosmopolitan in distribution and has been reported infesting 28 genera of leguminous plants, including: V. unguiculata, Vigna radiata, Cajanus cajan, Cicer arietinum and Phaseolus vulgaris (Hajam & Kumar, 2022) [8] . This bruchid is capable of infestation in the field prior to harvest, subsequently completing its entire development cycle within the grain seed under storage conditions (Ebadi et al., 2025) [5] . Under optimal conditions of temperature (25-35°C) and relative humidity (40-70%), a single female can oviposit 50-150 eggs directly onto seed surfaces, and emerging larvae bore into individual seeds to complete development through four instars before emerging as adults. Through successive generations, infestation densities can produce up to 90% seed damage within three to six months of unprotected storage, rendering infested grain unfit for human consumption or commercial trade (Revathy et al., 2025; Alhoqail, 2025) [22,2] . The conventional management strategy for stored product insects has relied heavily on synthetic chemical fumigantsprincipally phosphine (PH₃) and methyl bromide -and contact insecticides. Although chemical pesticides are effective in the short term, however their repeated use may lead to several negative effects, including the emergence of pesticide-resistant individuals, the accumulation of pesticide residues on stored foods, impacts on non-target organisms, and due to the effects of fumigant on the ozone layer, increasing restrictions on their use (Ogbonnaya et al., 2025; Fan et al., 2025; Muhammad & Rashid, 2024) [18,6,15] . These effects have catalyzed global interest in the development of more safe alternatives as botanical insecticide that are both efficacious and suitable with food safety and environmental sustainability objectives (Li et al., 2025) [12] . Several studies have investigated the ability of plant extracts to control the C. maculatus. Akbar et al. (2024) [1] found that plant extracts showed great effectiveness as insecticides against Callosobruchus maculatus. Among the five plant extracts evaluated, Azadirachta indica (neem) proved to be the most effective, recording the lowest infestation rate, minimum seed weight loss, and least adult emergence. The turmeric Curcuma longa extracts at 20% concentration have been documented to completely suppress adult emergence www.extensionjournal.com of Tribolium castaneum and achieve substantial mortality in Callosobruchus maculatus (Revathy et al., 2025) [22] . Farhadi et al. (2025) [7] found that peppermint essential oil (EO) lead to the effect in male and female adults of C. maculatus, as well as its beyond direct lethality, sublethal concentrations significantly suppressed fecundity. Although neem, turmeric, and mint extracts have been tested individually against various stored grain pests, studies that compare all three together against both larval and adult stages of C. maculatus on mung bean, using different concentrations and time periods, are still scarce. This study was therefore conducted to compare the effectiveness of aqueous extracts of: Neem (Azadirachta indica A. Juss), turmeric (Curcuma longa L.) and mint (Mentha spp.) against both larval and adult stages of Callosobruchus maculatus at three exposure periods (24, 48, and 96 hours)under controlled laboratory conditions.

Materials and Methods 1. Insect Culture

A colony of Callosobruchus maculatus was established and maintained under controlled laboratory conditions (27 ± 2 °C and 60 ± 5% relative humidity), with a 12:12 h (Light:Dark) photoperiod. Initial colony stock was sourced from naturally infested mung bean grain and reared through successive generations on cleaned, sterilized mung bean seeds (Vigna radiata L.) prior to the study, following the rearing protocol described by Singh et al. (2024) [24] .

Preparation of Plant Extracts

Three plant materials were prepared (obtained from local markets) including: A-Neem (Azadirachta indica A. Juss): The fresh leaves were shade-dried for 14 days, then into powdered. The 3%, 6% and 10% (w/v) concentrations were prepared (Akbar et al. 2024) [1] . B-Turmeric (Curcuma longa L.): The dry rhizomes powdered, the 3%, 6% and 10% (w/v) concentrations were prepared (Alhoqail, 2025) [2] . C-Mint (Mentha spp.): The fresh leaves were air-dried (ambient temperature), the 3%, 6% and 10% (w/v) concentrations were prepared (Farhadi et al.2025) [7] . According to Akbar et al. (2024) [1] , the plant extracts attended freshly (on the day of bioassay) then maintained at 4 °C (to minimize phytochemical degradation).

Bioassay Design

The insecticidal activity of plant extracts was evaluated separately against larval and adult stages of C. maculatus using a filter paper contact bioassay method. Petri dishes (9 cm diameter) were lined with Whatman No. 1 filter paper and treated with 1 mL of each plant extract at the designated concentrations, following the standard contact bioassay procedure described by Akbar et al. (2024) [1] . Control dishes received 1 mL of distilled water. Treated filter papers were air-dried for 30 minutes before introducing test insects. For larval bioassays, mung bean seeds were surface-treated with extracts at each concentration and infested with 10 adult pairs of C. maculatus for oviposition over 48 hours. Following oviposition, adults were removed and the seeds with eggs were maintained under the same controlled conditions. Larval mortality was assessed by surface examination and seed dissection at 24, 48, and 96 hours post-treatment, as described in Alhoqail (2025) [2] . For adult bioassays, 10 newly emerged adult C. maculatus (5 male, 5 female) were introduced into treated Petri dishes and mortality was recorded at 24, 48, and 96 hours. An insect was considered dead when it failed to respond to a gentle probe stimulus at the thorax (Farhadi et al.,2025) [7] . Each treatment was replicated three times in a Completely Randomized Design (CRD).

Statistical Analysis

Percentage corrected mortality was calculated using Orell and Schneider equations (Püntener, 1981) to adjust for natural mortality in control groups. All data were subjected to analysis of variance (ANOVA) using a two-factor factorial design (Plant type × Concentration) at each exposure period. Means were compared using the Least Significant Difference test (LSD) at the 5% probability level (P ≤ 0.05). Statistical analyses were performed using SAS 9.4 software.

Results

Efficiency of plants aqueous extracts in the C. maculatus Larval Stage Mortality 1.Effect of plants aqueous extracts in the C. maculatus

Larval Stage Mortality at 24 hours exposure After 24 hours of exposure, all the treatment, the plant extract, the concentration and the interaction between them have a statistically significant effects in larval mortality (Table 1). The lowest Mean corrected larval mortalities averaged were 20.52% for neem and the highest 24.43% for turmeric. The highest average concentration-dependent mortality was recorded at 10% concentration (29.62%), while the 3% concentration recorded the lowest average mortality, it᾿s reached to 18%. As the interaction between the plant extract and the concentration, the highest and the lowest mortality-corrected average reached to 30.55, 15% in the neem extract (10% and 3% concentration respectively).

Effect of plants aqueous extracts in the C. maculatus Larval Stage Mortality at 96 hours exposure:

The 96-hour exposure period yielded the most pronounced larval mortality outcomes, with neem achieving complete larval mortality (100%) at the 10% concentration, while the mint at 3% and 6% concentrations reduced the larval mortality to the least, it᾿s reached to 25.99% (Table 3). Overall plant-averaged mortality at this period reached 59.89% for neem, while the least was 30.99% for mint. Average mortalities at 3% and 10% concentrations were the least (30.55%) and the highest (65.99%), respectively, underscoring the critical role of concentration in determining protectant efficacy. All treatments effects remained highly significant

Effect of plants aqueous extracts in the C. maculatus adult Stage Mortality at 48 hours exposure:

After 48 hours of adult exposure, mint maintained its superiority with a highest average mortality of 42.66%, while the lowest average mortality at neem (35.66%) (Table 5). As the interaction between plant and concentration, the highest average mortality was 53.54% in 10% concentration at mint treatment, while the neem, 3% achieved the lowest average, it᾿s 23.99%. Average concentration-dependent mortality increased from 27.59% at 3% to 50.51% at 10%. All factorial effects remained significant.

Effect of plants aqueous extracts in the C. maculatus adult Stage Mortality at 96 hours exposure

The most adult mortality outcomes were recorded at the 96hour exposure period, where both neem and mint achieved complete adult mortality (100%) at the 10% concentration (Table 6), while neem and turmeric at the 3% concentration recorded least average mortality, it᾿s reached 30%. Mean plant-highest and lowest averaged adult mortality at 96 h was 60.14% for mint and36.81% for turmeric at respectively -with mint exhibiting the highest overall efficacy against adult C. maculatus in this test. Concentration-dependent means increased from 31.67% at 3% to 80.85% at 10%. All factorial effects were statistically significant.

Discussion

General Insecticidal Efficacy and Concentration Effects of aqueous extracts in C. maculatus larval and adult stages

The results of the present investigation unambiguously establish that aqueous extracts of neem, turmeric, and mint exert significant, concentration-and time-dependent insecticidal activity against both larval and adult stages of Callosobruchus maculatus on mung bean. The clear positive relationship between 3% and 10% concentrations with corrected mortality for three botanicals and life stages, it is corroborates that mortality increased by extract concentration increasing (Mustafa & Ghani, 2026; Muhammad, 2025) [17,16] . The corrected mortality increasing with exposure time, it is www.extensionjournal.com mechanistically compatible with the botanical insecticides mode of action (contact-toxicity) (Benyan & Khalaf, 2026) [4] . The sustained contact of bioactive compound leads to penetrate the insect cuticle, specifically in aqueous extracts where the concentration of bioactive molecule at the cuticle surface is lower than in organic solvent extracts (Oksari et al., 2024) [19] . This effect pattern was particularly clearly for neem, where the mortality of larvae increased from 20.52% at 24 h to 59.89% at 96 h.

The Insecticidal Effect of plants aqueous extracts in C. maculatus larval and adult stages:

The highest larval mortality reached to 100% after 96 hours of treatment with 10% concentration of neem extract. These results are in similar with Ogbonnaya et al. (2025) [18] , who recorded 100% mortality of C. maculatus after 96 h treatment using 0.0015 µg/mL azadirachtin, confirming the strong effect of neem's active constituents. The efficacy of neem against C. maculatus larvae reflects the primary mechanism of azadirachtin -interference with ecdysone signalling, disrupting the ecdysis process critical to larval moulting and development (Jenkins et al., 2003; Kutcherov, 2020) [10,11] . Turmeric exhibited intermediate efficacy in larval bioassays and relatively lower performance in adult bioassays compared to neem and mint. These results were consistent with what Lithi et al. (2019) [13] mentioned, as they confirmed that substances extracted from turmeric-derived materials as effective stored grain protectants at specific concentrations but generally of lower efficacy than neemderived materials in direct insecticidal bioassays. The insecticidal mechanisms of turmeric against C. maculatus encompass both contact toxicity through curcuminoid activity and potential fumigant activity from volatile terpenoid constituents of the essential oil (Anas et al., 2024; Revathy et al., 2025) [3,22] . The most notable finding in the adult bioassays was the superior performance of mint extract relative to both neem and turmeric at the 24-and 48-hour exposure periods, with mint consistently producing higher adult mortality at equivalent concentrations and time periods. At 96 h and 10% concentration, mint and neem co-achieved 100% adult mortality, yielding plant-averaged adult mortalities of 60.14% (mint) and 56.48% (neem). This stage-specific pattern -where mint performed less effectively against larvae than neem but outperformed neem against adultspoints to differential susceptibility of larval versus adult cuticular composition and target-site sensitivity to mint bioactive constituents Rajkumar et al. (2019) [21] . Menthol and menthone, the principal bioactive terpenoids of Mentha essential oil, are well-characterised acetylcholinesterase inhibitors in arthropod systems. Adult C. maculatus, which possess a fully chitinised adult cuticle distinct in composition from larval cuticle, may present enhanced penetration accessibility to lipophilic menthol molecules through spiracular openings and thinner inter-segmental membranes (Wu et al., 2023; Madrid et al., 2026) [27,14] .

Practical Implications for Integrated Post-Harvest Management:

The achievement of 100% larval and adult mortality by neem (10%, 96 h) and mint (10%, 96 h) establishes both botanicals as candidate grain protectants for integration into IPM programmes for mung bean storage. From a practical point of view, the 10% concentration represents available application rate for storage seed surface treatment by aqueous botanical extracts in small storage systems, requiring neither specialized equipment nor hazardous chemical handling. Treating seeds before storage, instead of treating them after pest infestation in the storage, provides sufficient concentration of biologically active compounds on the seed surface during the early durations of insect infestation (Muhammad & Rashid, 2024) [15] .

Conclusion

The current study finding, that aqueous extracts of neem, turmeric and mint have significant effects, concentrationand time-dependent insecticidal activity against Callosobruchus maculatus larvae and adult stages. The conclusions including: 1. The neem and mint aqueous extracts were the highest effective botanical treatments, both leading to 100% of C. maculatus larvae and adults mortality at 10% concentration after 96 hours, whereas turmeric showed moderate efficacy. In addition, a differences was observed between the two leading extracts, as neem was more affect against larvae whereas mint was more activity against adults, indicating stage-specific responses that can guide targeted application strategies. 2. These findings emphasizes the use of neem and mint extracts as safe and available alternatives to synthetic insecticides in post-harvest mung bean protection.

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