Description:Description:
1. Background of the Invention:
In agriculture, particularly in post-harvest storage, pest infestations in grains result in large economic losses (Rajashekar et al., 2025). The World Bank estimates that 12 to 16 million metric tons of food grains are lost after harvest in India annually, which is enough to feed one-third of the country's impoverished (Nagpal and Kumar, 2012). There are more than 600 insect species known to infest grains that have been kept worldwide, although only around 100 of them result in substantial financial losses. Secondary pests like Tribolium castaneum, Oryzaephilus surinamensis, and Cryptolestes ferrugineus feed on broken or previously damaged grains, while stored grain pests of primary pests like Callosobruchus chinensis, Sitophilus oryzae, Rhyzopertha dominica, and Trogoderma granarium infest whole grains (Rajashekar et al., 2025; Anukiruthika et al., 2021). The most dangerous and cosmopolitan pest is the red flour beetle, Tribolium castaneum, which is the main stored grain pest of green grams (Sekar et al., 2025). That beetle is a significant pest affecting stored grains, leading to considerable economic losses globally.
Synthetic pesticides, such as insecticides, repellents, antifeedants, and fungicides, are potential agents for controlling pests in agriculture, but they have some adverse effects on non-targeted animals, humans, phytotoxicity (reduced seed germination), and environmental pollution, such as air, water, and soil. In pest control alternative techniques are used; natural sources, such as plants, microbes, and animal metabolites, are cost-effective and eco-friendly pesticide agents.
In experiments evaluating the insecticide properties of plant extracts, Tribolium castaneum is essential. As a cosmopolitan pest, it inflicts damage by feeding externally on various grains and processed foods, highlighting the importance of effective pest management strategies for controlling its population. An environmentally responsible and sustainable replacement for synthetic pesticides are botanical insecticides, which are made from organic compounds such as flavonoids, alkaloids, and essential oils (Rajashekar et al., 2025).
Recently, animal-derivative biomaterials have been used for insect control, such as marine sponge (Karnan et al., 2023), sea urchin (Karnan et al., 2025), and snail shell (Elumalai et al., 2025). Animal-derivative bio-material insecticides are similar to botanical insecticides, with a close relationship to the presence of bio-metabolites such as alkaloids, terpenoids, steroids, and polyphenols (Karnan et al., 2025).
In the present invention, an eco-friendly antifeedant of marine crab Portunus pelagicus shell bioinspired extract against storage grain pests has dual roles of Tribolium castaneum repellent and non-phytotoxicity. That invention delivered cost-effective, eco-friendly pesticide agents of antifeedant and repellent against storage grain pests,directly enhancing seed germination and reducinganimal waste management.

2. Methodology
2.1 Portunus pelagicus shell bioinspired extraction
5 kg of Portunus pelagicus crab was collected from the Thanjavur fish market, and the collected crab were carefully removed from their shells, and the shells were washed several times with tap water and dried under room temperature. The dried crab shell is used to make fine powder using a mortar and pestle. Briefly, 20 grams of the finely powdered crab shell of Portunus pelagicus was first extracted with hexane solvent (100 mL) for 48 hours in a dark environment to prevent degradation of light-sensitive compounds, and after extraction, it was filtered and collected. The remaining residual shell powder was subjected to a second solvent of 100 mL chloroform, extracted for 48 hours in a dark environment, and after extraction was filtered and collected. The final solvent of 100 mL of ethanol was used for ethanol extraction of the remaining residual shell powder under a dark environment for 48 hours. These subsequent extractions yielded a colorless solution, indicating the depletion of zoochemicals. The three organic extracts were combined and concentrated under reduced pressure at 40°C using a rotary evaporator (Figures 1 and 2). The resulting crude extract was stored at 4°C and used for subsequent pest control in post-harvest storage.
2.2 Adult mortality
The toxic effect of crab shell extract was tested against adults of T. castaneum by adult toxicity methods was followed by Chaubey, (2007) with some modification. Ten adults taken from the laboratory culture were placed with 1gm of wheat flour in 90 mm petri plate. Flour was spread uniformly along the whole surface of the petriplate. A treated with solutions of different concentrations of crab extract (50 to 250µg/ml) was pasted on the inner surface of the covered and sealed with parafilm of each petri plate. All the closed petri plates were kept in dark. After 24 h of espouser, adult mortality was recorded.
2.3 Repellency assays
Repellency assays of 1/10 LC50 adult dose of crab shell extract was described by Obeng-Ofori et al., 1998; Chaubey, (2007); Padin et al. (2013) with some modification. Whatman filter paper was cut into two halves of 90 mm discs and 1/10 LC50 adult dose of crab shell extract per cm3 was applied to a filter paper half as uniform, using a micropipette. The other half of the filter paper was treated with aqueous solution (water) only. The crab shell extract treated and aqueous treated halves were dried to evaporate the solvent completely. After that both treated and untreated halves were attached with cellophane tape and placed at the bottom in the petri plate. 20 adults of Tribolium castaneum were released at the center of the filter paper disc and then Petri plates were covered and sealed with parafilm while kept in dark, during 12m hours. Number of the insects on both the treated and untreated halves was recorded every two hours in mild light Chaubey, (2007).
The percentages of insects present on treated (G) and control/ Untreated (P) areas were recorded (Padin et al., 2013). Index of repellency (IR) was calculated by following formula: IR = 2 G / G + P. The repellency index was classified by Mazzonetto, (2002) as: values <1 repellency; 1 neutral; >1 attractant.
2.4 In vivo efficacy of crab shell extract against storage pest Tribolium castaneum (Antifeedant activity)
In vivo practical efficacy of crab shell extract against Tribolium castaneum was performed following the method given by (Shukla et al., 2011 and Kiran et al., 2017) with mild modification. 100g uninfected green gram (Vigna radiata) were kept in the 200 ml jar and treated with crab shell extract at the concentration (1/10 of LC50). Thereafter, 50 adult of insect pests was released into the jar containing green gram (Vigna radiata) separately and the jars were made airtight and kept for three months of storage in a temperature-humidity control cabinet (27 ± 2˚C temperature and 75±5% relative humidity). A control set (without crab shell extract) was kept parallel to treatments sets. The emergence of whole in the green gram (Vigna radiata) was assessed as a damaged seed samples. The efficacy of Z-CuONPs in the protection of green gram (Vigna radiata) was determined based on the % grain damage and % weight loss of treated and control sets.
2.5 Seed germination/Phytotoxicity test of Portunus pelagicus shell
Seed germination test methods followed by Singh et al., (2023), with some modification. The green gram Vigna radiata (L.) seeds were surface sterilized using 1% sodium hypochlorite for 10 minutes and washed properly with autoclaved distilled water. The 20 sterilized seeds of green gram were separately kept on Whatman filter paper no. 1 already treated with 1/10 of LC50 in crab shell extract on T. castaneum, using adult toxicity assay (El-Bakry et al., 2016) and placed in glass petri plates. The filter paper was kept moist throughout the experimental period by spraying it with distilled water. The germination test was performed for 24 h to 3 days. In the control petri plates, the Whatman filter paper was only soaked with sterilized distilled water. The observation was taken after 24 h to 76 h and the germination percentage was calculated (Rajashekar et al., 2016).

3. Summary of the Invention
The present invention revealed that crab Portunus pelagicus shell bioinspired extract was highly toxic based on dose dependence (R2 = 0.955) against adults of T. castaneum. The mortality rate of adults in T. castaneum. In the present study, we reported LC50 (µg/ml) = LC50 = 187.5 µg/mL for Portunus pelagicus shell bioinspired extract, which was observed at 24 hrs (Figure 3).
The repellency activity of 1/10 of LC50 of Portunus pelagicus shell extract per cm3 was studied against the storage pest T. castaneum. The T. castaneum insect population reduction effect of Portunus pelagicus shell depended on the time interval in this study, which statistically agrees with the Chi-square test. The index of repellency (IR) was less than 1 (IR < 1) at 2 to 12 hours of filter paper assay and 8 to 12 hours of constant IR (IR = 0.2506 to 0.2584); however, Portunus pelagicus shell had an adverse effect on the target pest, which is a response to the significant reduction of the T. castaneum population (Figures 4a-b).
An absolute protection of V. radiata seed was observed at 1/10 LC50 concentration of grain protection, with a significant reduction of grain loss compared with the control (P < 0.05) during one month (Figure 5). Crab extract as adult toxicity at 1/10 LC50 doses exhibited strong feeding deterrents for T. castaneum insects during a one-month in vivo trial under laboratory conditions in the model storage grain of green gram V. radiate (Figure 6). Additionally, the beneficial effect of 1/10 LC50 of Portunus pelagicus shell had no phytotoxic effects on stored grain V. radiate germination (p>0.05). Further, the fact that crab shell may have no adverse effect on the germination of V. radiata seed strengthens its wide applicability as an insect adult toxicity test in the protection of stored grain seeds for storage purposes. According to current findings, our Portunus pelagicus shell can be seen as the development of a new environmentally friendly antifeedant to replace conventional antifeedants that respond to pest control in post-harvest grain storage with biodegradable antifeedants.
, C , C , Claims: Claim 1:
An eco-friendly bioinspired extract derived from Portunus pelagicus shell, obtained through sequential solvent extraction with hexane, chloroform, and ethanol, wherein the extract functions as an antifeedant for post-harvest storage grain protection against Tribolium castaneum adults.
 Claim 2:
The Portunus pelagicus shell bioinspired extract of Claim 1 acts as a repellent against Tribolium castaneum, thereby reducing adult insect infestation and grain loss during storage.
 Claim 3:
The Portunus pelagicus shell bioinspired extract of Claim 1 exhibits insecticidal activity with a dose-dependent adult toxicity effect against Tribolium castaneum, characterized by an LC₅₀ value of approximately 187.5 µg/mL under laboratory conditions.
 Claim 4:
The Portunus pelagicus shell bioinspired extract of Claim 1, when applied at sub-lethal concentrations (1/10 LC₅₀), provides absolute protection of stored grains, particularly Vigna radiata, by significantly reducing grain damage and weight loss compared to untreated controls.
 Claim 5:
The Portunus pelagicus shell bioinspired extract of Claim 1 is non-phytotoxic at effective concentrations, maintaining normal germination rates of Vigna radiata seeds, thereby ensuring preservation of seed viability and quality during storage.
 Claim 6:
The invention as claimed in any of the preceding claims provides a sustainable method for post-harvest grain protection, wherein the utilization of Portunus pelagicus shell as a raw material reduces marine biowaste while offering an eco-friendly alternative to conventional synthetic pesticides.