Highlights in BioScience ISSN:2682-4043 DOI:10.36462/H.BioSci.202508 Research Article Open Access 1 Department of Biochemistry, School of Life Sciences, Federal University of Technology, Minna, Niger State, Nigeria. 2 Department of Microbiology, School of Life Sciences, Federal University of Technology, Minna, Niger State, Nigeria. 3 Department of Animal Biology, School of Life Sciences, Federal University of Technology, Minna, Niger State, Nigeria. 4 Africa Centre of Excellence for Mycotoxin and Food Safety, Federal University of Technology, Minna, Niger State, Nigeria. * To whom correspondence should be addressed: eogbadoyi@futminna.edu.ng Editor: Morad Mokhtar, University Mohammed VI Polytechnic, Ben Guerir, Morocco. Reviewer(s): Alsayed Alsoudy, Mohammed VI Polytechnic University, Ben Guerir, Morocco. Kamal Yadav, School of Agricultural Sciences, Nagaland University, Nagaland. Nagesha S. Narasimhappa, Department of Biotechnology, College of Agriculture, Hassan, Karnataka, 573225, India. Received: April 22, 2025 Accepted: August 26, 2025 Published: September 22, 2025 Citation: Nwosu OK, Ogbadoyi EO, Babayi H, Olayemi IK. The Potentials of Bacillus thuringiensis Isolate and Its Extracellular Cuticle-Degrading Enzymes Activity in the Biological Control of Callosobruchus maculatus (Cowpea Weevil) in Stored Vigna unguiculata L. Walp (Cowpea) . 2025 Sept. 22;8:bs202508 Copyright: © 2025 Nwosu OK et al.. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduc- tion in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and supplementary materials. Funding: The authors have no support or funding to report. Competing interests: The authors declare that they have no competing interests. The Potentials of Bacillus thuringiensis Isolate and Its Extracellular Cuticle-Degrading Enzymes Activity in the Biological Control of Callosobruchus maculatus (Cowpea Weevil) in Stored Vigna unguiculata L. Walp (Cowpea) Onyeka Kingsley Nwosu1 ><�, Emmanuel Olofu Ogbadoyi*1,4 ><�, Hausatu Babayi2 >< , Israel Kayode Olayemi3 ><� Abstract This study assessed the pesticidal effect of Bacillus thuringiensis isolate against Callosobruchus maculatus (cowpea weevil) infesting stored cowpea seeds. B. thuringiensis isolate was obtained through liquid fermentation using sucrose water as a substrate, then transferred onto talc powder for solid formulation. A six-month shelf-life study of the solid formulation was conducted using total microbial plate count. Insect mortality bioassays were performed by applying liquid and solid B. thuringiensis formulations to cowpea seeds containing first-generation adult C. maculatus, with mortality recorded over 12 and 7 days, respectively. Enzyme activity was assessed using specific enzyme substrates. Results showed a gradual decline in microbial count over time in the solid formulation. The bioassay revealed 100% mortality for the liquid medium and for the solid medium of the B. thuringiensis isolate. The B. thuringiensis isolate also significantly delayed the first-generation emergence of C. maculatus. Enzyme analysis indicated the production of cuticle-degrading enzymes—protease, lipase, and exochitinase with varying activity levels. This study concludes that B. thuringiensis isolate effectively controls C. maculatus, likely due to its cuticle-degrading enzymes, and has potential as a bio-control agent for stored cowpea pest management. However, higher concentrations are necessary in talc-based formulations to maintain an adequate shelf life. Keywords: Cowpea, cowpea weevil, shelf-life, biological control, enzyme activity. Introduction In the relentless pursuit of sustainable agricultural practices, the search for effective and eco- friendly pest management strategies remains paramount. Among the myriad of challenges faced by farmers, infestation of stored grains and seeds by pests stands as a persistent threat, leading to significant post-harvest losses and economic repercussions. Cowpea (Vigna unguiculata L. Walp) is an important leguminous crop in many tropical and sub-tropical regions, serving as a significant source of protein and income for many people, especially in Nigeria, which is considered the largest producer of cowpea [1]. However, its production and post-harvest storage are severely hampered by the cowpea weevil, Callosobruchus maculatus [2]. Infestation by C. maculatus causes considerable losses in yield and quality, posing a challenge to food security and economic stability in the affected regions [3]. The conventional methods of controlling C. maculatus, such as synthetic chemical pesticides, have drawbacks, particularly regarding the food safety of cowpea, as the produce contains high levels of chemical pesticides considered dangerous to human health [4; 5]. Other drawbacks include environmental pollution, biodiversity disruption due to toxicity to non-target organisms, and the development of pesticide resistance [6]. As a result, there is an increasing interest in exploring alternative eco-friendly approaches, such as biological methods, for managing post-harvest cowpea pest populations of C. maculatus. Among these biological methods is the use of microbial agents, notably bacteria and fungi, offering health- and environmentally-benign alternatives to conventional chemical pesticides [7]. Bacillus thuringiensis, a naturally occurring bacterium, is a Gram-positive, spore-forming bacterium known for its insecticidal properties against a wide range of agricultural Highlights in BioScience Page 1 of 10 September 2025|Volume 8 https://doi.org/10.36462/H.BioSci.202508 https://creativecommons.org/licenses/by/4.0/ mailto:nwosuonyeka6@gmail.com https://orcid.org/0000-0003-1197-6810 mailto:eogbadoyi@futminna.edu.ng https://orcid.org/0000-0001-6461-2362 mailto:acadbabayi@futminna.edu.ng mailto:isreal.olayemi@futminna.edu.ng https://orcid.org/0000-0001-7116-0720 http://bioscience.highlightsin.org/ Nwosu OK et al., 2025 The potentials of Bacillus thuringiensis isolate and its extracellular cuticle-degrading enzymes pests [8]. Its efficacy, specificity to target pests, and minimal impact on non-target organisms, coupled with its favorable safety profile for humans and the environment, make it an attractive candidate for integrated pest management (IPM) programs. B. thuringiensis produces pesticidal toxins through several molec- ular mechanisms, most of which are from the Cry family of crystalline proteins produced in the parasporal crystals and en- coded by the cry genes [9]. These Cry proteins are responsible for the death of insect pests when they ingest the Cry protoxins or absorb them through the insect body cell membrane. The protoxins solubilize, releasing a protease-resistant biologically active endotoxin [10]. This disrupts the osmotic balance through the formation of transmembrane pores, eventually causing cell lysis in the gut wall and leakage of gut contents [11]. Microbial isolates that have insecticidal potential, such as B. thuringiensis, can also be pathogenic to their host by penetrating the insect cuticle and sporulating on desiccated cadavers, thereby facilitating the onset and spread of epizootics [12]. Penetration of the insect cuticle is typically the initial stage of infection, rely- ing on mechanical pressure, cuticle-degrading enzymes (lipase, chitinase, protease, etc.), and specialized infection structures (ap- pressoria) produced by the hyphae, which breach the host cells and subsequently proliferate [13]. Cuticle-degrading enzymes are likely key virulence factors in entomopathogens, as their substrates contain structural components of the insect cuticle that provide carbon and nitrogen sources to support microbial growth and facilitate host penetration [14]. The spore propagates in response to chemical signals on the cuticle and subsequently develops an appressorium, the specialized structure responsible for penetration. For many years since identifying the pesticidal properties of B. thuringiensis, its preparations have usually been sprayed directly on plants to shield them against different orders of insect pests [10]. In current pest management innovations, the genes encoding the B. thuringiensis pesticidal proteins have been successfully transferred into plants through genetic engineering; such plants are now referred to as transgenic plants [15]. These transgenic plants containing B. thuringiensis genes, specifically for Lepidoptera and Diptera insect pests, were produced to pro- vide protection against pre-harvest pests without the need for spraying [16]. The potential of B. thuringiensis based bio-pesticides in con- trolling Lepidopteran field pests has been demonstrated in several studies, but its effectiveness against Coleoptera: bruchids such as C. maculatus remains relatively unexplored. While the molecular mechanism of B. thuringiensis in infecting hosts through Cry proteins, inducing cell lysis in the gut wall and resulting in leak- age of gut contents, has been extensively studied, the production of extracellular cuticle-degrading enzymes by B. thuringiensis isolates for degrading the cuticle of Coleoptera: bruchids like C. maculatus-a critical barrier to infection and death—remains markedly underexplored. This knowledge gap is especially strik- ing given that adult C. maculatus do not require food, unlike the larvae that feed and develop exclusively on seed legumes, thereby providing a scientific opportunity to assess the potential of cuticle-degrading enzymes to breach the adult C. maculatus ex- oskeleton and cause pathogenicity. Hence, the cuticle-degrading pathway may represent a pivotal, yet overlooked, component of the B. thuringiensis entomopathogenic arsenal, particularly against coleopteran species. This study aimed to evaluate the bio-pesticidal capabilities of a B. thuringiensis isolate and the implications of its extracellular cuticle-degrading enzyme activ- ity on the control of C. maculatus under laboratory conditions. The study hypothesized that B. thuringiensis isolate, applied in both liquid and solid talc-based formulations, would cause sig- nificant mortality in adult C. maculatus; its efficacy would be concentration-dependent; the solid formulation would be more effective; and the pesticidal activity is correlated with the pro- duction of extracellular cuticle-degrading enzymes like protease, lipase, and chitinase. Considering the relevance of this study in addressing the pressing issue of C. maculatus infestation in stored cowpea, it aims to contribute to the development of sustainable and environmentally friendly strategies for cowpea protection and storage, thereby offering valuable insights for ensuring cowpea food safety, improving food security, and guaranteeing liveli- hoods in major cowpea-growing regions. Materials and methods Collection of B. thuringiensis isolate B. thuringiensis isolate was obtained from the Reference Laboratory section of Biocrops Biotechnology Limited, Abuja, Nigeria, originally sourced from the soil within the Abuja envi- ronment and identified and confirmed through molecular analy- sis. Bacterial cell culture was performed using Potato Nutrient Agar containing penicillin G. Culture plates were incubated at room temperature (28◦C ± 2◦C) for 72 hours. The emerging colonies after the incubation period were discretely isolated and sub-cultured repeatedly on freshly prepared Nutrient Agar to obtain pure isolates. The pure isolates were subjected to morpho- logical and biochemical tests for confirmation. The confirmed pure isolates were maintained on agar slant bottles, stored at 4◦C, and subsequently subjected to liquid fermentation to en- hance microbial proliferation, with sucrose water serving as the carbon-rich substrate and liquid growth medium for the isolate. Collection of cowpea seeds and C. maculatus Cowpea seeds (Black-eye pea, Sampea 10-Nigeria) were harvested directly from the experimental farm at the National Biotechnology Research and Development Agency (NBRDA), Abuja, Nigeria, to eliminate the possibility of prior pesticide ex- posure during storage. The seeds were aseptically sealed in sterile bags, placed in tightly covered containers, and stored at freezing temperature until further use. Cowpea seeds already infested with cowpea weevils were collected locally from cowpea storage facilities in the Federal Capital Territory, Abuja, Nigeria, and also from traders of the cowpea commodity. Adult weevils were isolated from visibly infested seeds through selective picking for subsequent experimental use. Highlights in BioScience Page 2 of 10 September 2025|Volume 8 http://bioscience.highlightsin.org/ Nwosu OK et al., 2025 The potentials of Bacillus thuringiensis isolate and its extracellular cuticle-degrading enzymes Table 1. Shelf-life of B. thuringiensis isolate on talc powder medium Concentration of B. thuringiensis in Talc Powder CFU count of B. thuringiensis (per g) (×104) Months (June to November) 1 2 3 4 5 6 10 ml/50 g 63.5±0.14a 53.4±0.07a 37.9±0.05a 18.7±0.21a 6.15±0.09a 2.45±0.02a 15 ml/50 g 213.4±0.81b 92.5±0.18b 55.6±0.31b 26.2±1.18b 7.87±0.22b 4.54±0.05b 20 ml/50 g 272.3±0.24c 143.3±0.40c 80.9±0.44c 47.9±0.56c 12.8±0.06c 8.16±2.11c Values are in mean ± S.E. Values between experimental treatments on a column bearing the same superscript are not significantly different at the 5% level (P > 0.05). S.E = Standard error of mean. CFU: Colony Forming Unit, X: concentration. Figure 1. Percentage (%) mortality of C. maculatus treated with the liquid medium of B. thuringiensis isolate Culturing of the C. maculatus Collected cowpea weevils were reared on freshly collected cowpea seeds under laboratory conditions to facilitate acclima- tization, following the method described by [17]. Batches of about one thousand seeds were each distributed into five pre- washed, sterilized, and dried containers. Thirty weevils were introduced into each transparent plastic container and covered with mesh nets fastened tightly with rubber bands and masking tape to permit ventilation and prevent escape of the pest. The cowpea seed-weevil mixtures were incubated under laboratory conditions for 10 days to permit mating and oviposition. Parent weevils were then removed, and rearing continued until adult emergence. First filial generation (F1) adult weevils were used in the subsequent experiments. Inoculation of the B. thuringiensis isolate onto solid medium (talc powder) and shelf-life study The talc stones were collected from Ejiba town in Yagba West Local Government Area of Kogi State, Nigeria and were processed into fine powder using the mechanical grinding ma- chines. The talc powder was packed into tightly sealed water- proof nylons and then sterilized appropriately using autoclave with a holding time of 15 minutes at 121 o C. The sterilized talc powder was stored at room temperature. Talc powder was used as the solid medium because of its cost-effectiveness considering its local availability, ability of being chemically inert as it doesn’t react with other substances and its good adhesion capacity due to its fine particle size and high surface area. The B. thuringiensis isolate with microbial load of 3.66× 106 CFU/ml was at different quantity (10 ml, 15 ml and 20 ml) each inoculated onto 50 g of sterilized talc powder, appropriately sealed and stored at room temperature. The talc powder containing B. thuringiensis isolates at varying concentrations, were used for mortality study after one month to allow for sporulation. The shelf-life of the B. thuringien- sis isolate on the talc powder at different concentrations were assessed at one-month interval for a period of six months using Total Microbial Plate Count (Colony Forming Unit per gram) as described by [18]. Effect of B. thuringiensis isolate on mortality of C. maculatus Ten-fold serial dilution and calculation of Total Microbial Plate Count of the microbial isolates was done as described by [18]. a) In triplicate, the B. thuringiensis isolate was assayed for pesticidal activity using the method described by [19]. Twenty gram of the cowpea seed were introduced each into sterilized transparent containers with small openings on the cover to allow ventilation. Ten cowpea weevils were introduced each into the containers followed by spraying 1 to 4 ml of the liquid formulation of the B. thuringien- sis isolate with 3.66 × 106 CFU/ml, 7.32 × 106 CFU/ml, 11.0 × 106 CFU/ml and 14.6 × 106 CFU/ml concentrations respectively using small (10 ml) plastic spray bottles. b) Two gram from each of the prepared 10 ml/50 g, 15 ml/50 g, 20 ml/50 g talc powder-B. thuringiensis isolates mixture with 8.99 × 105 CFU/g, 10.5 × 105 CFU/g, 12.6 × 105 CFU/g concentrations, respectively, were introduced into a sterilized transparent container with small openings on the Highlights in BioScience Page 3 of 10 September 2025|Volume 8 http://bioscience.highlightsin.org/ Nwosu OK et al., 2025 The potentials of Bacillus thuringiensis isolate and its extracellular cuticle-degrading enzymes cover that allows ventilation, containing 20 g of cowpea seed and 10 cowpea weevils by dusting over the seeds. c) Distilled water treatment was used as the control for the B. thuringiensis isolate liquid medium assay while distilled water-talc powder mixture was used as control for the B. thuringiensis isolate solid medium assay. Mortality study of the C. maculatus Adult cowpea weevil mortality was recorded at 24-hour in- tervals over an exposure period of 12 and 7 days for the liquid and solid formulation of the B. thuringiensis isolates respectively. Dead weevils were carefully removed with the help of a magni- fying lens and forceps to reveal the hidden pests in the grains. Weevils that failed to respond to gentle prodding with the forceps were considered dead and subsequently removed. The percent mortality was calculated using the following formula: % Mortality = Number of Dead Weevils Number of Weevils Introduced × 100 (1) Assessment of the percentage of emergence of first generation of C. maculatus Emergence of first-generation (F1) adult C. maculatus was determined after 40 days in the same samples used to assess per- cent mortality. The percentage of emergence (PE) was estimated using: PE = X Y × 100 (2) where: X = Number of insects that emerged in the treatment Y = Number of insects that emerged in the control treatment Treatments where first-generation emergence is ≤ 50% are con- sidered as promising [20]. Cuticle-degrading enzyme activity assay Liquid culture for the extracellular enzyme production of the B. thuringiensis isolate The nutrient broth was prepared according to the manufac- turers specifications. For the production of extracellular en- zymes, 1 mL of the liquid medium of the isolate containing 3.66× 106 CFU/mL was inoculated into the nutrient broth and in- cubated at room temperature (28 ± 2◦C) for 7 days. After incuba- tion, the contents of the test tube were centrifuged at 10,000 rpm for 20 minutes at 4◦C. Finally, the supernatants were used in enzymatic assays. Protein concentration assay The total soluble protein concentration in B. thuringiensis isolate was determined using the method of [21], with bovine serum albumin (BSA, 1 mg/mL) as the standard. A stock BSA solution was prepared by dissolving 0.25 g of BSA in distilled water and making up the volume to 250 mL in a volumetric flask. Aliquots of 0.2, 0.4, 0.6, 0.8, and 1.0 mL of the BSA solution were dispensed into test tubes and made up to a volume of 1 mL with distilled water. From each tube, 0.5 mL was transferred to a new tube, followed by the addition of 0.5 mL of reagent (50 mL of 2% Na2CO3 in 0.1 M NaOH) and 1.0 mL of 0.5% CuSO4 in 1% sodium-potassium (Na–K) tartrate. The mixtures were thoroughly mixed and allowed to stand for 10 minutes at room temperature. Subsequently, 0.5 mL of Folin–Ciocalteu reagent was rapidly added to each tube and mixed immediately. The tubes were again allowed to stand for 10 minutes at room temperature, after which absorbance was measured at 625 nm using a UV–VIS spectrophotometer (1800-series, Shimadzu). A calibration curve was generated by plotting absorbance against BSA concentration. For the determination of sample protein concentration, 0.5 mL of the enzyme solution was processed in the same manner, and protein concentrations were extrapolated from the calibration curve based on the corresponding absorbance values. Cuticle-degrading enzyme assay The cuticle-degrading enzymes; total protease, exochitinase, and lipase assays were performed as described by [14] in trip- licates. The total protease was assayed using casein as the sub- strate, while the exochitinase was assayed using p-nitrophenyl-N- acetyl-β-D-glucosaminide as the substrate, and the lipase activity was assayed using p-nitrophenyl butyrate as the substrate. Calculation of the specific enzyme activity The specific protease, exochitinase, and lipase activity of the B. thuringiensis isolate was calculated using the following formula: Specific Activity (U/mg) = Enzyme Activity (U/mL) Protein Concentration (mg/mL) (3) Statistical Analysis Data were expressed as mean value ± standard error of the mean (S.E.M) and analyzed using ANOVA. Significant differences between the control and treatment groups were determined by Duncan’s Multiple Range Test (DMRT) in SPSS (version 26). Results The six-month shelf-life study of the B. thuringiensis isolate inoculated onto talc powder as a solid medium (Table 1) revealed that B. thuringiensis experienced a gradual reduction in colony- forming units (CFU) per gram over time, with the initial CFU count in the range of 63.5 to 272.3 × 104 CFU/g and the final count in the range of 2.45 to 8.16 × 104 CFU/g, respectively. The toxicity assay of the B. thuringiensis isolate evaluated its effi- cacy against C. maculatus, a major pest affecting stored cowpea grains. This study assessed the effectiveness of B. thuringiensis in inducing mortality in C. maculatus when applied in liquid and solid forms. The results, presented in (Figures 1 and 2), illustrate Highlights in BioScience Page 4 of 10 September 2025|Volume 8 http://bioscience.highlightsin.org/ Nwosu OK et al., 2025 The potentials of Bacillus thuringiensis isolate and its extracellular cuticle-degrading enzymes Figure 2. Percentage (%) mortality of C. maculatus treated with the B. thuringiensis isolate in solid medium (Talc powder) the insecticidal activity exhibited by the B. thuringiensis isolates across different concentrations and durations of treatment. The results presented in (Tables 2 and 3) show the mean emergence of first-generation (F1) adult C. maculatus treated with the B. thuringiensis isolate in liquid and solid formulations, respectively, and further reveal the percentage emergence of F1 adults, highlighting the efficacy of each treatment concentration in reducing pest reproduction. Treatments where F1 emergence is ≤ 50% are considered promising for pest suppression [22; 20]. This study also evaluated the enzymatic activity of B. thuringien- sis to determine their contribution to the toxicity potential of the isolate against C. maculatus. The results presented in (Figure 3) illustrate the specific activities of protease, lipase, and exochiti- nase of the B. thuringiensis isolate, considering their potential to degrade the structural components of the insect exoskeleton [23]. Discussion The development of B. thuringiensis-based pesticides for the control of C. maculatus in stored cowpea represents a significant advancement in sustainable agricultural practices. Microbial- based pesticides, derived from entomopathogenic bacteria such as B. thuringiensis, offer an eco-friendly alternative with promising potential for pest management in cowpea production [24]. These microbial pesticides are target-specific, biodegradable, and pose minimal risks to non-target organisms and human health [25]. The shelf-life study of the B. thuringiensis isolate inoculated onto talc powder provides crucial insights into the long-term viability of the isolate and its potential as a bio-pesticide. (Ta- ble 1) revealed a significant decline in microbial counts over six months, with variations depending on the concentration, confirm- ing that the gradual decrease in microbial load was significantly influenced by the concentration formulations. The highest con- centration (20 mL/50 g) generally maintained higher microbial counts for a longer duration compared to lower concentrations, suggesting that a higher initial inoculation helps sustain viability. In other words, the higher the quantity of B. thuringiensis isolate inoculated onto talc powder, the higher the CFU count and the longer the shelf life. The declining trend observed is also similar to that reported by [26]. Other studies have shown a similar declining trend in micro- bial load of Bacillus species when inoculated onto talc-based powder. The study of [27] reported a talc-based powder formula- tion of Bacillus cereus strain B25 spores, in which CFU count of 1.1 × 109 gradually declined during 360 days of storage at room temperature. Similarly, [28] reported that talc-based formulations of Bacillus subtilis with an initial count of 2.0 × 108 CFU/g at zero day was reduced to 8.3× 106 CFU/g after 80 days of storage at room temperature. Any inorganic solid carrier material should offer a protective environment for microbial growth and, when applied for seed treatment, should possess strong adhesion capacity to maximize efficacy and guarantee successful release of bacterial cells after application [29]. In this study, the good efficacy of talc powder– B. thuringiensis isolate may have resulted from strong adhesion capacity to cowpea seeds. Talc powder as a solid carrier for microbial isolates has also been reported to be cost-effective, easy to process, chemically stable, with good moisture absorption and buffering capacity, non-toxic to both plants and microbes, and able to ensure bacterial cell viability for at least 2–3 months [30]. Studies have shown that Bacillus species in talc-based formulations remain viable at a density of 1.0 × 106 CFU/g after 45 days of storage [31]. In this study, despite the decrease in shelf-life of the B. thuringiensis isolate inoculated on talc powder as storage time increased, it still showed that B. thuringiensis cell viability was maintained even at the 4th month (120 days), with maximum viability at 47.9 ± 0.56 × 104 CFU/g and minimum viability at 18.7 ± 0.21 × 104 CFU/g, indicating that talc powder can be a veritable carrier for inoculating B. thuringiensis isolate for com- mercial production. This study has confirmed that talc powder effectively supports B. thuringiensis isolates for bio-pesticide pur- poses by providing a stable medium for storage and application. While talc-based formulations can sustain microbial viability for some months, a decline is unavoidable. Considering that the rate of decline varies among organisms, there is a need to explore options that could further enhance microbial longevity. For example, [29] reported that talc was an effective carrier, but additional stabilizers such as glycerol improved shelf life beyond six months. Therefore, this study emphasizes the importance of considering optimized storage conditions (e.g., low humidity, cool temperatures) and incorporating stabilizers such as glycerol, silica gel, or oil-based formulations to further enhance microbial persistence over time. The toxicity assay, which assessed the efficacy of B. thuringien- sis isolate against C. maculatus, as shown in (Figures 1 and 2), revealed that at all treatment concentrations, a significant percent mortality was recorded compared with the control, indicating the high pesticidal potential of the B. thuringiensis isolate against adult C. maculatus. The study further showed that the isolate caused mortality in a concentration-dependent manner, with ef- fectiveness increasing over time and at higher microbial concen- trations, and demonstrated stronger effects through solid medium exposure. Highlights in BioScience Page 5 of 10 September 2025|Volume 8 http://bioscience.highlightsin.org/ Nwosu OK et al., 2025 The potentials of Bacillus thuringiensis isolate and its extracellular cuticle-degrading enzymes Table 2. Percentage emergence of first generation of C. maculatus after treatment with liquid medium of the B. thuringiensis isolate Microbial isolate Treatment volume (ml) Treatment concentration (CFU/ml) Mean emergence of treatment (X) Mean emergence of control (Y) PE (%) Remarks B. thuringiensis 1 3.66 × 106 2.33 ± 0.01a 11.3 ± 0.04b 20.61 Promising 2 7.32 × 106 1.33 ± 0.03c 9.66 ± 0.11d 13.76 Promising 3 11.0 × 106 0.67 ± 0.02c 7.67 ± 0.05e 8.73 Promising 4 14.6 × 106 0.33 ± 0.01a 6.33 ± 0.03b 5.21 Promising Values are in mean ± S.E. Values between experimental treatments on a row bearing the different superscript are significantly different at the 5% level (P > 0.05). S E = Standard error of Mean. PE: Percentage emergence. CFU: Colony Forming Unit. X = Number of insects that emerged in the treatment, and Y = number of insects that emerged in the control treatment. Treatments where first-generation emergence is ≤ 50% are considered as promising. Figure 3. The cuticle-degrading enzyme activity of B. thuringiensis isolate The 12-day treatment with the liquid medium of B. thuringien- sis isolate showed 100% mortality on the 12th day at concen- trations of 7.32 × 106 CFU/mL and 11.0 × 106 CFU/mL, and on the 8th day at 14.6 × 106 CFU/mL. A similar toxicity trend was observed in the 7-day treatment study using the solid car- rier, where 100% mortality was recorded on the 6th day at the lowest concentration (8.99 × 105 CFU/g) and on the 5th day at higher concentrations (10.5 × 105 CFU/g and 12.6 × 105 CFU/g). These results indicate that for the commercial production of B. thuringiensis-based bioinsecticides for the control of C. mac- ulatus, concentrations above 106 CFU/g or CFU/mL will be required for faster, early, and effective pest suppression. The con- trol group, which was not treated with any microbial suspension, showed negligible mortality, confirming that the observed insect deaths were substantially due to the microbial treatments rather than external environmental factors. According to [11], insect mortality caused by B. thuringiensis is usually a result of insecticidal toxins (Cry toxins), often re- ferred to as δ-endotoxins, which are somewhat specific to certain insect families. The adult C. maculatus may have absorbed the B. thuringiensis isolates through its cuticle as a result of the direct application of the isolate in liquid and solid carriers. These find- ings are consistent with previous research on microbial biocon- trol agents against C. maculatus. For example, [32] reported that Bacillus flexus biofilms exhibited significant insecticidal effects on C. maculatus, suggesting that bacterial biofilm extracellular matrices could be leveraged as bio-pesticides for cowpea stor- age protection. Similarly, [33] examined the impact of Bacillus flexus S13 on C. maculatus and reported LC50 values indicat- ing moderate toxicity, supporting the effectiveness of bacterial isolates in insect pest control. In comparison, the current study suggests that B. thuringiensis exhibits slightly higher potency than B. flexus against C. maculatus, though further comparative analysis is needed. The high toxicity of B. thuringiensis isolates on adult C. mac- ulatus recorded with the solid medium treatment, where even the lowest concentration achieved 100% mortality before the 7th day, may be due to the increased surface area exposure pro- vided by talc powder as a carrier. This level of mortality was not achieved with liquid medium treatments, even at the highest concentration used in the study. Therefore, in microbial control of C. maculatus in stored cowpea, application methodologies that enhance surface area exposure of the microbial isolate should be adopted for effective control. These findings align with recent research demonstrating the efficacy of entomopathogenic bacteria on solid carriers for pest control. For instance, [34] reported that B. thuringiensis on a solid carrier significantly impacted stored- product insects, particularly Trogoderma granarium, through its endotoxins. The current study also found B. thuringiensis on talc powder to be highly potent against C. maculatus, with the highest mean mortality (over 80%) in the 20 mL/50 g treatment. While these results are consistent with past research, certain variations exist due to environmental factors, strain differences, and formu- lation methods. For instance, [35] emphasized that formulation type (liquid vs. solid) influences microbial persistence and vir- ulence, which may explain minor differences in mortality rates Highlights in BioScience Page 6 of 10 September 2025|Volume 8 http://bioscience.highlightsin.org/ Nwosu OK et al., 2025 The potentials of Bacillus thuringiensis isolate and its extracellular cuticle-degrading enzymes Table 3. Percentage emergence of first generation of C. maculatus after treatment with the B. thuringiensis isolate in solid medium (Talc powder) Microbial isolate Treatment volume (ml/50g) Treatment concentration (CFU/g) Mean emergence of treatment (X) Mean emergence of control (Y) PE (%) Remarks B. thuringiensis 10 8.99 × 105 2.00 ± 0.01a 9.33 ± 0.18b 21.43 Promising 15 10.5 × 105 1.67 ± 0.01a 7.67 ± 0.12b 21.77 Promising 20 12.6 × 105 0.67 ± 0.01a 5.33 ± 0.22b 12.57 Promising Values are in mean ± S.E. Values between experimental treatments on a column bearing the different superscript are significantly different at the 5% level (P > 0.05). S E = Standard error of Mean. PE: Percentage emergence. CFU: Colony Forming Unit. X = Number of insects that emerged in the treatment, and Y = number of insects that emerged in the control treatment. Treatments where first-generation emergence is ≤ 50% are considered as promising. observed across different studies. Adjuvants such as inert carriers, like talc powder for loading microorganisms as active ingredients of microbial pesticides, possess greater adsorption capacity and enhanced dispersion properties, enabling controlled release of active components at appropriate intervals [36]. When microbial isolates penetrate the host cell, they proliferate, providing a pathway for epizootics [13]. At relatively low quantities, these crystalline proteins exhibit high insecticidal activity [37]. The emergence of the first-generation (F1) adult C. maculatus serves as a crucial indicator of the long-term effectiveness of microbial bio-pesticides. While initial mortality rates provide in- sight into the immediate insecticidal effects of microbial isolates, F1 emergence evaluates their residual impact on pest populations by determining whether microbial treatments can suppress re- productive success and prevent re-infestation. In this study, F1 emergence was assessed 40 days after treatment application, fol- lowing the mortality evaluation. The study showed a significant delay in the F1 emergence period of adult C. maculatus at all treatment concentrations compared with the control. The signifi- cant delay observed in both liquid and solid medium treatments was expressed in a concentration-dependent manner, with greater delays recorded at higher concentrations. The percentage emer- gence further validated the efficacy of B. thuringiensis treatments. In both formulations, B. thuringiensis isolates at higher concen- trations resulted in F1 emergence percentages well below the 50% threshold. The results from this study align with previous research on microbial bio-pesticides for stored-product pest management. For instance, [38] confirmed the efficacy of B. thuringiensis in reducing weevil emergence and suggested that the persistence of B. thuringiensis spores in treated grains may provide long- term protection against re-infestation, reinforcing the results of this study. Similarly, [39] investigated Trichoderma spp. for cowpea beetle control and found that while microbial spores significantly reduced F1 emergence, they were not as effective as B. thuringiensis. Their findings align with the present study, where B. thuringiensis demonstrated high efficiency in reducing F1 emergence, suggesting that bacterial bio-pesticides such as B. thuringiensis may offer more immediate control compared to fungal alternatives. The findings of this study underscore the significance of bio- pesticide concentration, microbial strain selection, and storage conditions in optimizing pest suppression. Higher concentrations of B. thuringiensis isolate resulted in a significant reduction in F1 emergence, suggesting its potential as a leading candidate for the biological control of C. maculatus. Additionally, the superior performance of solid formulations in limiting F1 emergence sug- gests that formulation type plays a critical role in bio-pesticide effectiveness. Furthermore, the calculation of the percentage F1 emergence suggests that the concentration levels used in the study are promising for pesticidal activity. The assessment of cuticle-degrading enzyme activity in B. thuringiensis isolate demonstrated the presence of protease, li- pase, and exochitinase, suggesting their role in the toxicity mech- anisms of B. thuringiensis against C. maculatus. These enzymes are crucial for the pathogenicity of microbial bio-pesticides, as they degrade the structural components of the insect exoskeleton, facilitating microbial penetration and systemic infection [23]. Beyond mechanical pressure, enzymatic breakdown of insect cuticles can aid in the penetration of microbial isolates through the degradation of proteins, chitins, and lipids that make up the insect cuticle [40]. The present study revealed the secretion of different cuticle-degrading enzymes, such as proteases, lipases, and chitinases. The findings indicate that the B. thuringiensis isolate exhib- ited measurable enzymatic activity, although at varying levels, suggesting differential enzymatic strategies in their pathogenicity. Protease activity was observed to display the highest activity, exceeding that of lipase and exochitinase. The protease, exochiti- nase, and lipase enzyme activity of the B. thuringiensis isolate may have synergistically contributed to the pathogenicity against Highlights in BioScience Page 7 of 10 September 2025|Volume 8 http://bioscience.highlightsin.org/ Nwosu OK et al., 2025 The potentials of Bacillus thuringiensis isolate and its extracellular cuticle-degrading enzymes adult C. maculatus. The significant differences in the extracel- lular enzyme activities per milliliter of the liquid culture of the B. thuringiensis isolate could be attributed to variability in vir- ulence at different medium concentrations. Hence, this study suggests that the role of these cuticle-degrading enzymes in the pathogenicity of C. maculatus may be concentration-dependent. Proteases are regarded as pivotal catalysts in the infection processes by cleaving peptide bonds in proteins and breaking them into small peptides and amino acids [14]. Exochitinase plays a vital role in cleaving N-acetylglucosamine in cuticular layers, while lipases break down ester bonds in lipoproteins, fats, and waxes found in the interior part of the insect integument to release (un)saturated fatty acids [41]. The higher protease activity of the B. thuringiensis isolate may have acted as the key catalyst for the infection of adult C. maculatus. Support- ing the findings of this study, [42] reviewed hydrolytic enzymes in integrated pest management and highlighted the role of bac- terial entomopathogens such as B. thuringiensis in producing proteases that degrade insect cuticle proteins. This complements the current study, which observed significant protease activity in B. thuringiensis, reinforcing its role as an effective microbial pes- ticide. Similarly, [43] examined microbial hydrolytic enzymes and found that bacterial mechanisms of cuticle degradation differ from fungal entomopathogens, as bacterial enzymes often ex- hibit species-specific targeting, a factor that may influence their efficacy. The production of protease, exochitinase, and lipase by the B. thuringiensis isolate might be the key virulence factor against adult C. maculatus, considering that adult weevils do not require food or water within their limited lifespan and therefore could not have ingested the treated cowpea. Drawing on the results of this study, the relationship between cuticle-degrading enzymes and virulence could serve as a diagnostic criterion for selecting effec- tive microbial control agents for C. maculatus. Future studies are required to elucidate the mechanistic role of cuticle-degrading enzymes in the significant delay in adult C. maculatus emergence observed in this study. Conclusion This study demonstrated that Bacillus thuringiensis isolate is highly effective against adult Callosobruchus maculatus and holds significant potential as a bio-control agent within integrated pest management strategies for stored cowpea. Its application can promote food security and enhance cowpea safety by providing an alternative to toxic chemical pesticides. Furthermore, the study confirmed that the B. thuringiensis isolate secretes key cuticle-degrading enzymes-protease, exochitinase, and lipase which likely serve as major virulence factors. These enzymes facilitate cuticle degradation, enabling the bacterium to penetrate the insect host, ultimately causing infection and mortality. The findings underscore the promise of B. thuringiensis not only as a microbial bio-pesticide for controlling bruchids but also as a candidate for developing genetically engineered cowpea va- rieties with plant-incorporated protectants for bruchid resistance. 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Highlights in BioScience Page 10 of 10 September 2025|Volume 8 http://bioscience.highlightsin.org/ Abstract Introduction Materials and methods Collection of B. thuringiensis isolate Collection of cowpea seeds and C. maculatus Culturing of the C. maculatus Inoculation of the B. thuringiensis isolate onto solid medium (talc powder) and shelf-life study Effect of B. thuringiensis isolate on mortality of C. maculatus Mortality study of the C. maculatus Assessment of the percentage of emergence of first generation of C. maculatus Cuticle-degrading enzyme activity assay Results Discussion Conclusion