




































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

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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
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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.

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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

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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

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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.

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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

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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

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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.
For commercial production using talc powder as a solid carrier,
the study recommends maintaining higher microbial concentra-
tions above 104 CFU/g to ensure adequate shelf life and sustained
efficacy.

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	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

