Novel insecticidal properties of bioactive zoochemicals extracted from sea urchin Salmacis virgulata

2.1 Collection and extraction of zoo-extract from the test and spines of S. virgulata

Sea urchin Salmacis virgulata were collected from Manora, Thanjavur (Bay of Bengal, southeast coast), and the collected specimens have been identified the key Venkatraman and Padmanaban [18] and stored at the Zoological Museum of the PG and Research Department of Zoology, RSGC, Thanjavur (Voucher No. RSGC-Z-K01). The sea urchin tests and spines from 20 various-sized sea urchin S. virgulata were washed thoroughly using water and ethanol. Then, the test and spines were separated and mashed together using a mortar and pestle. After 24 h, 10 g powder was extracted with 100 ml of ethanol and water in a 1:1 ratio. Following 24 h, the extract was filtered and refrigerated under 4°C for further use.

2.2 Zoochemical assessment

The zoo extract underwent zoochemical analysis utilizing gas chromatography–mass spectrometry (GC–MS) techniques, specifically the S. virgulata test, and a 50% ethanolic extract was conducted with the Shimadzu 2010 Plus, which includes an AOC-20i autosampler and GC–MS. The Wiley and NIST libraries were utilized for component identification, and their retention indices were compared. The components were identified by comparing them to those in the computer libraries associated with the GC–MS instrument. The results were subsequently aggregated [19].

2.3 Insect

Tribolium castaneum (red flour beetles) was employed to assess the insecticidal efficacy of zoo extract. T. castaneum was cultured in a lab setting free from insecticide exposure. The insect-reared method, as described [20,21], was used to raise the red flour beetles on wheat flour at room temperature.

2.4 In vitro AChE inhibitor assay

The AChE biochemical assay was administered to the tested insects. The extraction method for the AChE enzyme from the T. castaneum insect, as described by Hematpoor et al. [22], and the evaluation of AChE inhibition activity, as conducted [23], with minor modifications.

2.5 In vitro insecticidal efficacy (cytotoxic impact) of the zoo extract on the Sf-9 cell line utilizing the (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assay

The cell viability trial adhered to the methodologies outlined [24,25]. The Sf-9 insect ovarian cell line (NCCS) was utilized to cultivate cell suspensions in 100 μl per well of 96-well culture plates. Various concentrations of zoo extract (50, 100, 150, 200, and 250 μg/ml of S. virgulata 50% ethanolic extract) were administered and incubated for 48 h following an initial 24-h incubation period. A control medium was utilized, comprising cells without the experimental drug. Cellular morphological features such as membrane blebbing and cellular shrinkage or swelling were observed using an inverted microscope [25].

2.6 Repellency assays

The repellency assays of S. virgulata 50% ethanolic extract were detailed [26,27], with minor modifications. Test solutions were formulated at concentrations ranging from 50 to 250 µg/ml/cm² in an aqueous solvent. A micropipette was utilized to apply each concentration of zoo extract to one-half (30 mm²) of Whatman filter paper, which had been divided into two 90 mm² discs. The sole treatment administered to the remaining half of the filter paper was an aqueous solution. The solvent was completely evaporated by drying the halves treated with zoo extract and water. The treated and untreated halves were then placed at the bottom of the petri dish and secured with cellophane tape. Following the introduction of 20 adult T. castaneum into the center of the filter paper disc, the Petri plates were sealed with parafilm and stored in darkness. The quantity of insects in both the treated and untreated sections was documented after 4 h in subdued illumination. Classes were classified on a scale from 0 to 5, and percent repellency (PR) was calculated [28]. The repellency classes are classified as follows: class 0 (PR ≤ 0.1%), class I (PR = 0.1–20%), class II (PR = 20.1–40%), class III (PR = 40.1–60%), class IV (PR = 60.1–80%), and class V (PR = 80.1–100%). Percent repellency (PR) is calculated as (Nc − Nt)/(Nc + Nt) × 100, where Nc represents the number of insects on untreated paper and Nt denotes the number of insects on treated paper.

The index of repellency (IR) was calculated by the following formula: IR = 2 G/G + P. The repellency index was classified by Mazzonetto and Vendramim [29] as: values <1 repellency; 1 neutral; >1 attractant. The percentages of insects present in treated (G) and control/untreated (P) areas were recorded [27].

2.7 Larvicidal efficacy of S. virgulata extracts from test and spines

The larvae of A. aegypti (Linnaeus, 1762) were identified [30] and reared in deionized water enriched with yeast powder and glucose. The standard methodology [31,32] was employed, with slight modifications, to sustain the A. aegypti colony in the laboratory at 27°C, 75 ± 5% relative humidity, and a photoperiod of 14 h. Experiments were performed to evaluate the efficacy of S. virgulata test and spine extract in exterminating fourth-instar A. aegypti larvae. The larvicidal activity was assessed through a bioassay following the standard guidelines established by the WHO [33].

2.8 In silico insecticidal efficacy

2.9 Statistical analysis

Each experiment was conducted thrice, and the statistical methods employed included probit analysis, chi-square test, one-way ANOVA, and post hoc test, all analyzed using IBM SPSS Version 20.0. The data was statistically significant if *p < 0.05, while NS indicated statistical non-significance (p > 0.05).

3.1 Zoochemical profile of 50% ethanolic extract of S. virgulata test and spines

This study investigated the insecticidal properties of zoo-waste extracts from deceased and dried S. virgulata sea urchins, specifically from the test and spines, against T. castaneum, A. aegypti, and the Spodoptera frugiperda cells (sf-9) cell line. The sea urchin samples were extracted using a 50% ethanolic solution over 24 h, yielding a light greenish-brown extract (Figure 1), which was then concentrated for further zoochemical and insecticidal analysis. The zoochemical profile of the S. virgulata extract, detailed in Table 1 and Figure 2, revealed the presence of various zoochemicals. GC–MS analysis identified 40 zoochemical components by comparing them to the NIST and Wiley databases. Notably, Table 2 lists 11 bioactive zoochemicals, including four pesticide compounds: geraniol, 9-octadecenoic acid, n-hexadecanoic acid, and squalene.

Figure 1

Zoochemical extraction from S. virgulata test and spines.

Figure 2

GC–MS chromatogram of S. virgulata test and spines 50% ethanolic extract.

Table 2

Identification of bioactive compounds, from S. virgulata test and spines 50% ethanolic extract using GC–MS techniques with literature review

Bioactive compound	PubChem structure	Biological activity	References
Geraniol		Antitumour, anti-inflammatory, antioxidant, antimicrobial, hepatoprotective, cardioprotective, neuroprotective effects, insecticidal and repellent properties	[37,38,39, 40]
9-Octadecenoic acid		Anti-inflammatory, antiandrogenic cancer preventive, dermatitigenic hypocholesterolemic, 5-alpha reductase inhibitor, anemia genic, Insectifuge, and flavor	[41]
Tetratetracontane		Antioxidant and cytoprotective activities	[42,43]
Nonanoic acid, 9-oxo-, methyl ester		Antifungal, antioxidant, and antimicrobial	[44]
n-Hexadecanoic acid		Antioxidant, hypocholesterolemic nematicide, pesticide, anti-androgenic flavor, and hemolytic	[45]
Dibutyl phthalate		Antimicrobial and antifouling	[46]
Tetradecanoic acid		Antioxidant, cancer preventive, nematicide, lubricant, and hypocholesterolemic	[41]
Squalene		Antibacterial, antioxidant, pesticide, antitumor, cancer preventive, immunostimulant, chemopreventive, and lipoxygenase-inhibitor	[47]
7-Hexadecenal		Antiviral activity	[48]
9-octadecenoic acid, 1,2,3-propanetriyl ester		Antioxidant, antibacterial and inflammation-suppressing effects	[49,50,51]
Eicosane		Antifungal activity	[52]

Geraniol, a terpene alcohol frequently found in essential oils, can be synthesized via recombinant microorganisms or extracted from natural sources [37]. This compound, valued for its pleasant fragrance, is present in numerous aromatic plants and is noted for its insecticidal and repellent properties, making it a low-toxicity natural pesticide [38]. The present study identified geraniol in sea urchin extracts using GC–MS, suggesting its presence may be linked to the sea urchin's aromatic nature and its repellent effects. Geraniol is also recognized for its hepatoprotective, cardioprotective, antitumor, anti-inflammatory, antioxidant, and neuroprotective activities [37,38,39], in addition to its insecticidal and repellent properties [40]. Additionally, dodecanoic acid, identified as the oviposition pheromone for the sand fly Lutzomyia longipalpis [53], was also found in S. virgulata extracts, along with its undecyl ester.

Recent studies have utilized GC–MS techniques for the screening and identification of zoochemicals from various animal sources (Table 2). Furthermore, chemical analysis of the test and spines of the sea urchin S. virgulata revealed compounds including hexadecanoic acid methyl ester, tetradecanoic acid, and (Z)-9-octadecenoic acid methyl ester. Interestingly, these compounds were also identified in the gonads of the sea urchin D. setosum, highlighting a shared biochemical profile across these species [54]. The findings confirm the presence of valuable zoochemicals in S. virgulata, highlighting their potential contributions to biomedical research and environmental applications and affirming their role as an eco-friendly resource.

3.2 AChE inhibitory and cytotoxicity activity of S. virgulata test and spines

AChE is a molecular insecticide target, and inhibiting by natural or synthetic compounds proves death for the target insects – one of the primary targets of insecticide development. In vitro, T. castaneum AChE enzyme is inhibited by the S. virgulata test and spines 50% ethanolic extract (IC₅₀ = 143.41 µg/ml) to cause toxicity in the target insect T. castaneum. The zoo extract of S. virgulata exhibited strong dose-dependent insecticidal activity (R² = 0.99), as shown in Figure 3a, with effective AChE inhibition that resulted in the mortality of target insect species, indicating its potential as a natural insecticide. Georgiev et al. [55] studied methanolic extracts and essential oils from species of Asteraceae, Lamiaceae, Brassicaceae, and Amaryllidaceae were evaluated in vitro for AChE inhibitory activity using the method by Ellman et al. [23]. Between 45.67 and 58.38 μg/ml, the essential oils of Callicarpa candicans exhibited encouraging activity. IC₅₀ values ranging from 28.71 to 54.69 μg/ml indicated good activity for the essential oils of Callicarpa sinuata, C allicarpa petelotii, Callicarpa nudiflora, Callicarpa erioclona, and Vitex ajugifolia. The IC₅₀ values of 81.34 and 89.38, respectively, indicated modest activity for the essential oils Vitex trifolia subsp. trifolia and Capsella rubella [56]. In the marine environment, seaweed produces a variety of naturally occurring compounds that are both chemically and physiologically active. At 200 μg/ml, ethanolic extracts made from 47 macroalgae species were tested against tyrosinase (TYR), butyrylcholinesterase (BChE), and AChE. Dictyota dichotoma var. intricata was the only algae extract that showed a significant inhibition of BChE (72.0 ± 0.07%). In contrast, the other macroalgae showed no or minimal inhibition of AChE (2.20 ± 0.39%–14.20 ± 2.16%), BChE (0.62 ± 0.07%–41.20 ± 3.07%), and TYR (<10%) [57]. Recently, in vitro insecticidal activity of zoo-extract against AChE enzyme by Karnan et al. [58] used marine sponge Hyattella intestinalis zoo extract (141.10 µg/ml). Hung et al. [56] have reported that the obtained results indicate the potential utility of these essential oils AChE inhibitory activities of essential oils from Vietnamese traditional medicinal plants for the management of mosquito vectors. Mattar et al. [59] reported the essential oil of Schinus areira against Rhipibruchus picturatus and its inhibitory effects on AChE (IC₅₀ 0.62 mg/ml). AChE activity tests showed that S. virgulata extract had an excellent inhibitory effect on AChE and was inhibited by zoo insecticides due to the toxicity of T. castaneum. Exposure to organophosphorus compounds (OP) raises ROS levels, which attack proteins, lipids, and DNA. This leads to damage to membranes, inactivation of enzymes, DNA damage, and cell death [60]. Inhibitors of the esterase activity of AChE continued to promote cell death. The mechanism by which extracellular AChE triggers apoptosis is still unknown, even though preventing AChE's cytotoxic effect at low temperatures suggested enzyme-driven processes [61]. The discussions demonstrate the potential use of S. virgulata test and spines with 50% ethanolic extract in the development of new insecticides and further contributors to the fumigant toxicity activity and repellent properties against T. castaneum.

Figure 3

In vitro insecticidal activity of S. virgulata test and spines 50% ethanolic extract: (a) in vitro AChE inhibitory activity and (b) in vitro cytotoxicity activity. *Statistically significant (p < 0.05), compared with control. The dotted line in (a) exhibits the mean correlation coefficient (R ²).

The Sf9 insect cell line from S. frugiperda was used for an in vitro insecticidal model, and its cytotoxic activity was investigated using an MTT assay. The present study revealed that S. virgulata test and spines 50% ethanolic extract were highly toxic based on dose dependence (R ² = 0.986) against the Sf-9 cell line (EC₅₀ = 194.68 µg/ml), respectively. Figure 3b shows significantly inhibited Sf-9 cell growth at various concentrations between 50 and 250 µg/ml of zoo extract, compared to control (100% cell viability). The morphology of S. virgulata test and spines 50% ethanolic extract treated Sf-9 cells indicate cell alterations, such as cell shrinkage and cell fragmentation, and the control group indicates cell aggression (Figure 4), were observed at 48 h. Present cytotoxicity properties based on the presence of bioactive zoochemicals in the S. virgulata test and spines. Pandya et al. [25] and Saleh et al. [62] recently utilized the S. frugiperda Sf9 insect cell line to evaluate insecticidal efficacy through in vitro assays. According to Pandya et al., toxicity bioassays revealed that ammonia was hazardous for Sf9 (IC₅₀ 350 μg/ml) and profenofos was harmful for Sf9 (IC₅₀ 400 μg/ml) [25]. Additionally, morphological changes to the cell, such as shrinkage and fragmentation, were detected. Currently available S. virgulata test and spines 50% ethanolic extract is environmentally safe and effective zoological insecticides that destroy Sf-9 cells, using the MTT assay.

Figure 4

The morphology of normal Sf-9 cells and S. virgulata test and spines 50% ethanolic extract treated cells. The red mark indicates cell alterations, such as cell shrinkage and cell fragmentation. (a) control, (b) and (c) S. virgulata test (50–250 µg/ml). (d), (e) and (f) S. virgulata test and spines 50% ethanolic extract (150–250 µg/ml).

The zoochemicals identified in the test and spines of S. virgulata are involved in AChE inhibition, which leads to the overstimulation of AChE receptors, resulting in symptoms of zoochemical poisoning in T. castaneum (Table 3 and Figure 5). Table 3 shows that zoochemical are binding to the insecticidal target AChE (PDB ID: 1DX4), and highest insecticidal activity was observed in squalene (−33.89 kJ/mol), compared with 9-octadecenoic acid (−27.614 kJ/mol), n-hexadecanoic acid (−26.359 kJ/mol), and geraniol (−23.849 kJ/mol). Tyr 71 amino acid residue was involved in all zoo-insecticide binding interactions, which indicates S. virgulata zoochemicals have similar modes and actions of insecticidal activity, as represented in Figure 5. The standard AChE inhibitor Aldicarb commonly interacts with acetylcholinesterase (AChE) (PDB ID: 1DX4) through key amino acid residues, including Tyr370 and Gly150. Similarly, docking analysis revealed that 9-octadecenoic acid, n-hexadecanoic acid, and squalene exhibited comparable interactions with these residues. These findings suggest that the zoochemicals derived from S. virgulata and spines share a mode of inhibitory action similar to that of Aldicarb. Similarly, computational insecticidal development of secondary metabolites against molecular target AChE (PDB ID: 1DX4) was used [63,64,65], whose recommended AChE inhibitions promising potential insecticidal development against the harmful insect. Recently, bioactive compounds against AChE inhibition used molecular modeling approaches, which resulted in the identification of new cholinesterase inhibitors and also insect control [66,67,68].

Figure 5

In silico AChE inhibitory activity of S. virgulata test and spines zoochemicals (A: 9-octadecenoic acid; B: geraniol; C: n-hexadecanoic acid; D: squalene; E: Aldicarb; a: 3D docked view and b: 2D view).

3.3 Repellence impact of S. virgulata test and spines

S. virgulata 50% ethanolic extract repellency assays were conducted using 50–250 µg/ml/cm² in an aqueous solvent. A 90 mm² Whatman filter paper disc was halved. One-half of the filter paper (30 mm²) was uniformly treated with each S. virgulata zoo extract concentration, while the other half was treated with an aqueous solution. The aqueous-treated halves increased insect populations, while the zoo-extract-treated halves decreased insect populations due to repellent properties on T. castaneum adults (Figure 6a). Zoo extract repellency was dose-dependent (R ² = 0.976). The repellence effect is statistically significant as determined by the chi-square test (p < 0.05). Each concentration between the zoo-extract-treated and aqueous solvent-treated samples was statistically significant, confirming the reduction of insect populations on zoo-extract-treated filter paper halves, while insect populations increased on aqueous solvent-treated filter paper halves (Table 4). The percentage repellency value varied from 20.00 ± 10.00% to 93.33 ± 5.77% at doses of 50–250 µg/ml/cm² of S. virgulata test and spines within 4 h of exposure (Figure 6b). It identified repellency classes I, II, IV, and V based on the mean repellency rate. The index of repellency (IR) values range from 0.80 to 0.06, with concentrations below 0.80 (Figure 6c), indicating the repellent efficacy of S. virgulata extract.

Figure 6

Repellence affect of S. virgulata test and spines 50% ethanolic extract against adults T. castaneum: (a) repellency assays; (b) percentage repellency; (c) index of repellency (IR).

Using S. virgulata zoochemicals against OBP (PDB ID: 3K1E), Table 5 demonstrated the in silico identification of zoo-repellents by ligand-based screening and OBP structure-based molecular docking. Since they transport the chemicals to odorant receptors through the sensillum lymph, the globular proteins known as OBP are crucial to insect olfaction. Because the OBPs and olfactory receptors (ORs) of a species are an integral part of the olfactory system and are a valuable resource for downstream translational research, which eventually aims to improve insect control, accurately identifying them is imperative. The amino acid residues Phe 123, Leu 80, Leu 76, Leu 73, Met 91, Gly 92, and Ala 88 were involved in all zoo-insecticide binding interactions. This indicates that the zoochemicals of S. virgulata exhibit similar modes and mechanisms of insecticidal activity, as illustrated in Figure 7.

Figure 7

In silico repellence impact of S. virgulata test and spines zoochemicals (A: 9-octadecenoic acid; B: geraniol; C: n-hexadecanoic acid; D: squalene; E: diethyltoluamide; a: 3D docked view and b: 2D view).

OBP plays a protective role by shielding odorants from degradation by enzymes. Venthur and Zhou [69] have highlighted recent research exploring OBP as potential targets in the insect peripheral nervous system for developing eco-friendly pest control strategies. OBP is believed to facilitate the transport of odorants to ORs, contributing to the signal transduction processes that regulate behaviourally significant odors. A study by Ramsha et al. [70] evaluated the repellent effects of plant extracts from clove, coriander, neem, and mint against the red flour beetle, demonstrating that these natural extracts exhibit significant repellent activity, positioning them as promising sources of biological insect repellents. Murugesan et al. [71] reported a mean repellency of 82% for ethyl acetate leaf extract at a concentration of 1,500 µg/ml/cm² after 1 h of exposure. The methanol (52%) and hexane (28%) leaf extracts showed lower repellency. In addition, extracts from R. stricta, S. persica, and R. chalepensis at 500 ppm demonstrated repellent effects on adult O. surinamensis, with values of 91.7, 83.3, and 78%, respectively [72]. In the current study, the zoo extract of S. virgulata, comparable to plant extracts, was found to have repellent properties due to its rich composition of zoochemicals, including geraniol, 9-octadecenoic acid, n-hexadecanoic acid, and squalene. Molecular docking studies identified four key zoochemicals with high binding affinities for OBP (PDB ID: 3K1E), showing that S. virgulata triggered significant behavioral responses and potent repellency. The standard insect repellent diethyltoluamide (DEET) commonly interacts with the OBP target (PDB ID: 3K1E) through key amino acid residues, including Met91, Ala88, Phe123, Leu80, and Leu76. Notably, similar interactions were observed in the docking analysis of geraniol, 9-octadecenoic acid, n-hexadecanoic acid, and squalene, suggesting that the zoochemicals derived from S. virgulata and spines exhibit a mode of action comparable to that of DEET. The olfactory system plays a crucial role in regulating insect behaviors like host-seeking, mating, oviposition, toxin avoidance, and negative taxis [73]. Recent molecular docking studies have confirmed the binding interactions of secondary metabolites with OBP (PDB ID: 3K1E), as reported [74,64].

3.4 Larvicidal activity of S. virgulata test and spines

The larvicidal activity of 50% ethanolic crude extracts from the test and spines of S. virgulata was evaluated against A. aegypti fourth instar larvae using concentrations of 50, 100, 150, 200, and 250 ppm over 24 h. The LC₅₀ (lethal concentration for 50% mortality) is 153.205 µg/ml (95% confidence interval: 123.048–196.122 µg/ml), and the LC₉₀ (lethal concentration for 90% mortality) was 414.374 µg/ml (288.595–945.734 µg/ml (Figure 8). The larvicidal efficacy of S. virgulata extract increased in a dose-dependent manner (R² = 0.943), suggesting it to be a highly effective natural insecticide and a promising alternative for mosquito control. This aligns with other research, such as Kamaraj et al. [75], which proposed plant extracts as viable substitutes for synthetic insecticides. Similarly, Bharathi and Suseem reported larvicidal properties in the aqueous extract of Phaseolus vulgaris against A. aegypti mosquitoes [76].

Figure 8

Larvicidal activity of S. virgulata test and spines 50% ethanolic extract against A. aegypti 4th instar larvae.

Further studies by Hasaballah et al. [77] demonstrated the larvicidal potential of zinc oxide nanoparticles (ZnO-NPs) synthesized using marine sponge extract (Spongia officinalis), showing LC₅₀ values of 31.823 µg/ml for Culex pipiens and 12.634 µg/ml for Anopheles pharoensis. These findings indicate that ZnO-NPs derived from marine sources exhibit strong larvicidal properties. Similarly, the larvicidal activity of S. virgulata test and spine extract reinforces its role as an eco-friendly, natural insecticide. Both studies underscore the potential of natural and marine-derived compounds as alternatives for mosquito control, offering environmentally sustainable solutions.

This study aims to investigate the larvicidal activity of pesticide zoochemicals that were identified from S. virgulata tests and spines. Table 6 presents the results of this investigation, and Figure 9 illustrates the interactions between amino acids and their effects on mosquito growth regulator of JH and larvicidal development. In comparison to 9-octadecenoic acid (−33.054 kJ/mol), n-hexadecanoic acid (−31.38 kJ/mol), and geraniol (−22.594 kJ/mol), squalene (−46.861 kJ/mol) exhibited the highest larvicidal activity. The standard drug Aldicarb, an AChE inhibitor, commonly interacts with the JHBP target (PDB ID: 5V13) through key amino acid residues, including Val68, Tyr64, Tyr129, Tyr33, Trp50, Trp53, and Val51. Notably, similar interactions were observed in the docking analysis of 9-octadecenoic acid, n-hexadecanoic acid, and squalene, indicating that the zoochemicals derived from S. virgulata and spines exhibit a mode of inhibitory action comparable to that of Aldicarb. Recently reported JHBP (PDB ID: 5V13), which is a molecular target for larvicidal activity [78,79,80,81]. According to Zalewska et al. [12], JH regulates the development, reproduction, and metamorphosis of insects. Through an in silico model with effective mosquito vector control of sea urchin S. virgulata, the current S. virgulata zoo extract was involved in controlling insect development, such as inhibiting larvae growth of zoochemicals.

Figure 9

In silico Larvicidal of S. virgulata test and spines zoochemicals, against JHBP (A: 9-octadecenoic acid; B: geraniol; C: n-hexadecanoic acid; D: squalene; E: Aldicarb; a: 3D docked view and b: 2D view).

The geraniol nanoemulsions killed 94% of H. armigera and S. litura larvae, respectively. Thus, the study identified geraniol's possible larvicidal properties, which may be used to formulate biocides for the efficient management of phytopathogenic insects [82]. Rahuman et al. [83] investigated the mosquito-larvicide properties of n-hexadecanoic acid, which was extracted from Feronia limonia leaves using acetone. It was found to be effective against fourth instar larvae of C. quinquefasciatus, A. stephensi, and A. aegypti, with LC₅₀ values of 129.24, 79.58, and 57.23 µg/ml, respectively. In vitro study observed the highest activity of larvicidal (LC₅₀ 153.205 µg/ml) compared to cytotoxicity (EC₅₀ 194.68 µg/ml); similarly, in silico study observed squalene, n-hexadecanoic acid, and 9-octadecenoic acid, where the highest activity was observed in molecular target on larvicidal (JHBP; PDB ID: 5V13) compared with AChE (PDB ID: 1DX4). One potential mechanism of action of natural insecticides that results in high insect pest mortality is inhibition of AChE activity [84].

Several novel classes of compounds have demonstrated insecticidal, herbicidal, and fungicidal properties, highlighting marine natural products as a promising source for the development of novel agrochemical agents [85]. Ethanolic extracts from marine sponges exhibited insecticidal activity against the fifth instar larvae of Culex quinquefasciatus, Achaea janata, and Pericallia ricini. Among the twelve sponges screened, larval growth inhibition ranged from 9–70% for A. janata and 10–96% for P. ricini. These findings indicate that sponge-derived secondary metabolites serve as effective biopesticides against C. quinquefasciatus larvae and lepidopteran pests [86].

In vivo studies utilizing methanolic extracts of crab shells were conducted on zebrafish (Danio rerio) to evaluate inflammatory responses in a CuSO₄-induced model. The results revealed that crab shell extract significantly increased oxidative stress enzyme mRNA expression and total antioxidant capacity by 1.3–2.15-fold. Furthermore, pro-inflammatory cytokine mRNA levels were downregulated by 1.3–2-fold compared to CuSO₄-induced zebrafish. The shell extract of the Nile crab (Potamonautes niloticus) was found to be rich in antioxidant and anti-inflammatory compounds [87]. Similarly, Galal-Khallaf et al. investigated the shell extract of Charybdis natator, testing both low and high doses for their ability to mitigate copper-induced oxidative stress and pro-inflammatory responses in adult Danio rerio [88]. The current study demonstrated that the zoo extract inhibited the AChE enzyme, which was confirmed by in silico studies and the target insects' death. The sea urchin S. virgulata also contained the bioactive substances geraniol and n-hexadecanoic acid. According to an in-silico study, the 50% ethanolic extract of sea urchin S. virgulata (test and spine) currently has insecticide qualities based on the presence of pesticide compounds.

3.5 Statistical examination of in silico insecticidal efficacy employing correlation methodologies

Table 7 presents the correlation matrix among the insecticidal molecular targets (N = 4 zoochemicals), indicating that the S. virgulata test and spines zoochemicals exhibit comparable involvement in insecticidal activity across all selected molecular targets: AChE (PDB ID: 1DX4), OBP (PDB ID: 3K1E), and JHBP (PDB ID: 5V13). The correlations are exceptionally strong, ranging from r = 0.977 to 0.995, underscoring a positive relationship between the insecticide molecular targets and robust evidence of the insecticidal properties of S. virgulata test and spines against detrimental pests via AChE enzyme inhibition, repellence, and larval toxicity. We advocate for the utilization of zoo-waste material from the sea urchin S. virgulata, specifically its test, and spines, as effective strategies for pest control through repellent and toxic properties.