Purification and Characterization of the Recombinant Chitinase ChiBlUV02 of Bacillus licheniformis UV01 with a Choleoptericidal Effect on Hive Beetle (Aethina tumida)

Abstract

The biotechnological applications of chitinases are diverse. They are used in industrial sectors such as pharmaceuticals, textiles, and agriculture, including the use of recombinant chitinases for pest control, since traditional treatments affect and contaminate hive products. Bacillus licheniformis UV01 bacterium is of interest, as it expresses genes for different enzymes, including chitinase. The Chibluv01 gene was cloned into the pHTP8 vector with a His/tag for purification using affinity chromatography. It showed a specific activity of 115 U/m. The optimal pH and temperature were 7.5 and 42 °C, respectively. The choleoptericidal activity (ability to kill beetles) of the enzyme was evaluated in the larvae and adult beetles of Aethina tumida treated with immersion in a purified enzyme extract at different concentrations, and the mortality was verified at 24, 48, and 72 h. Within 24 h of application, the mortality increased by 50% in the larval stage and 56.6% in adult beetles compared to the control groups. The LC₅₀ and LC₉₀ were obtained: 104.05 U/mL and 234.36 U/mL in larvae and 92.99 U/mL and 211.14 U/mL in adults, respectively. These results indicate the potential of the application of ChiBlUV02 chitinase in pest control.

1. Introduction

Chitinases (E.C. 3.2.2.14) are a group of enzymes that have a wide range of applications due to their ability to degrade chitin, obtaining biotechnologically important polymers such as chitosan. Chitinases catalyze the cleavage of β (1–4) links in native chitin [1], releasing N-acetyl-β-D-glucosamine units in the process. Likewise, chitinases play an important role in several organisms, such as the walls of fungi and insects, primarily in the remodeling of their exoskeletons and the acquisition of carbon sources, as well as the collection of energy for microorganisms that feed via the hydrolysis of this polymer [2].

The research, development, and optimization of these enzymes represent a significant advance in biotechnology, and parameters such as the pH and working temperature are fundamental data that, when obtained, can provide advantages for the application of chitinases. These include the management and control of some pests in agriculture, for example, some phytopathogenic fungi such as Botrytis cinerea or insect pests such as Helicoverpa armigera, that cause considerable financial losses yearly in this sector. In addition, chitinases can be used to manufacture biofuels using chitin as a raw material and to produce fermentable sugars through the conversion of waste from crustaceans [3,4,5,6].

Obtaining chitinases with industrial value has generated benefits in different sectors. For example, in the food industry, they are used for chitosan production. Chitosan is a polymer derived from chitin that is used as an antimicrobial agent and as a food preservative [3]. There are also reports of effects on insects of the Lepidoptera and Diptera orders [7].

Chitinases of bacterial origin can act as endochitinases and exochitinases depending on the site in the chitin chain where they act, and they also present a broad range of molecular weight, from 20 to 80 kDa. Among the bacterial genera that produce chitinases, Bacillus is one of the most interesting; species such as Bacillus thuringiensis possess a gene named bt15a3 that encodes for the protein called Chia1, which possesses chitinolytic activity that significantly inhibits the spore germination of four species of fungi [8].

Bacillus cereus strain NK91 produces an extracellular chitinase; the enzyme was tested against three phytopathogenic fungi, where it exhibited higher antifungal activity against Fusarium oxysporum (66.7%), Rhizoctonia solani (64.6%), and Colletotrichum gloeosporioides (63%) [9]. Native chitinase from Bacillus licheniformis NK-7 was tested as an antifungal agent against phytopathogenic fungi and for the isolation and formation of fungal protoplasts of Aspergillus niger, showing an inhibition in the radial growth of 69.44% in relation to the control [10].

Bacillus licheniformis is a Gram-positive facultative anaerobic bacterium that requires a simple medium for growth. It is used in industry to produce endogenous and exogenous enzymes due to its thermophilic bacterial characteristics and great variety of genes that express enzymes with different characteristics, such as proteases, amylases, lipases, and chitinases [11,12,13]. Slimene et al. reported that Bacillus licheniformis S213 produces a chitinase with a phytopathogenic effect on some fungi, such as Phoma medicaginis [14]. Kwon et al. indicated that Bacillus licheniformis PR2 exhibits antifungal activity against certain vegetables [15]. Bacillus licheniformis UV01, isolated in the thermal waters of San José Purua Michoacán, has a gene called Chibluv01 that codes for the chitinase ChiBlUV02. This enzyme showed a choleoptericidal effect on larvae of the species Aethina tumida.

Aethina tumida, or the small hive beetle, was discovered in 1867 [16]; it is a scavenging and parasitic insect native to Africa and has become a pest that causes catastrophic damage to Apis mellifera hives outside its natural range [17]. The beetles infest hives where they feed, develop, and oviposit. They feed primarily on bee brood (larvae and pupae), as well as honey, pollen, and dead bees [18]. Precise figures on monetary losses are not available due to a lack of recent and specific data. However, reports from previous years give an idea of the magnitude of economic losses in the international beekeeping sector.

The United States reported losses of USD 428 million in 2023 [19] and the European Union reported losses of EUR 1.4 billion due to pests and factors related to climate change [20].

This study focuses on the purification and characterization of chitinase ChiBlUV02 and the evaluation of its choleoptericidal effect against the Aethina tumida species, thus increasing its efficacy as a biological control agent.

2. Materials and Methods

2.1. Reactivation and Growth of Bacillus licheniformis UV01

Bacillus licheniformis strain UV01 was reactivated from a preserved culture at −80 °C in 30% glycerol, which was inoculated in Luria–Bertani medium (LB). The inoculate was grown at 50 °C under orbital shaking at 100 rpm for 24 h.

2.2. Chitinase Gene Amplification

The reaction was carried out using polymerase chain reaction (PCR) with the chromosomal DNA of Bacillus licheniformis UV01 and the previously designed oligonucleotides: FChiBLNd01 sequence (5′->3′): GGAATTCCATATATGAAGAAGCCGCTTCATC-; RChiBLBa02 sequence (5′->3′): -CGGGATCCAATTTCCTTTAAGCCTGTACTTT-. The reaction was carried out in a C1000™ Touch Thermal Cycler (Bio-Rad Laboratories, Inc.^® Hercules, CA, USA). The PCR conditions comprised a single cycle of 1 min at 94 °C, 30 cycles of 1 min at 94 °C, 1 min at 58 °C, 1 min at 72 °C, and a final single cycle of 5 min at 72 °C. The PCR products were stored at −20 °C until further use.

2.3. Purification and Quantification of the PCR Products

The purification of PCR products was accomplished through the utilization of the Wizard^® SV Gel™ kit and the PCR Clean-Up System™ (Promega^® Madison, Wisconsin, WI, USA) in accordance with the established protocols. The quantification of purity and concentration was achieved using the NanoDrop^® ND 2000 (Thermo Scientific™, Waltham, MA, USA), adhering to the established protocols. The concentration was determined based on the absorption ratio at 260 nanometers, while the purity was determined based on the ratio at 260/280 nanometers.

2.4. Cloning of the Chitinase Gene Chibluv01

Cloning of the chitinase gene was performed in the pHTP8 His-tag (NZYEasy vector Cloning & Expression Kit^®, Lisbon, Portugal) from the concentrations and purities estimated via Lehninger spectrometry [21]. With purity ratios for the gene from 1.8 to 2.0, we proceeded to conduct a single ligase-independent reaction mediated using NZYEasy enzyme mixture according to the user’s manual; the digested vector and insert were ligated in a 3:1 ratio and incubated at temperature cycles of 37 °C/60 min, 80 °C/10 min, and 30 °C/10 min. The resulting ligation product was used as a template for the transformation of competent E. coli BL 21 (DE3) cells and plated on LB agar plates containing kanamycin (50 µg/mL).

2.5. Recombinant Protein Expression

The expression of recombinant cells was performed in liquid culture media, LB-kanamycin (50 µg/mL). These previously activated cultures were incubated for 8 h at 37 °C under agitation at 150 rpm with 0.8 mM IPTG. At the end of the incubation, the cell culture was centrifuged at 14,000× g for 10 min at 4 °C, the cell package was collected and resuspended in phosphate buffer (pH 6.0, 100 mM), and it was subsequently sonicated in a sonicator (Yucheng Tech^® Zhejiang, Hangzhou, China) (20 kHz) for 20 cycles alternating between 15 s and 30 s. All of the steps of the protocol were carried out in the cold. The cell lysate was centrifuged under the same conditions, and the supernatant was recovered and stored at 4 °C.

SDS-PAGE was performed (12%) to determine the molecular weight of the enzyme. The bands of protein were stained with Coomassie Brilliant blue G 250 (Sigma/Aldrich^®, Saint Louis, MO, USA) according to the protocol described in [22]. The banding pattern was observed using a white light transilluminator.

2.6. Preparation of Colloidal Chitin

Sigma/Aldrich brand colloidal chitin^® was prepared using a modification of the method of Hsu and Lockwood [23]. The paste obtained was sterilized for 15 min at 121 °C and 15 psi. It was stored at 4 °C until further use.

2.7. Quantification of the Protein Concentration

The protein concentration of the crude and purified extracts was measured using the spectrophotometric method described by Bradford [24]. A calibration curve with bovine serum albumin (BSA) and phosphate buffer pH 7 100 mM, with concentrations between 0.0 and 1.0 mg/mL, was used, and the absorbances were read in a Beckman Coulter TM spectrophotometer^® (Brea, CA, USA) at 595 nm.

2.8. Enzymatic Assay

For the determination of the enzymatic activity, 750 μL of 1% colloidal chitin was incubated with 750 µL of purified extract, and the reaction was incubated at 42 °C for 1 h. Then, the reaction mixture was centrifuged at 12,000× g for 10 min, and the release of N-acetyl-D-glucosamine was determined in 300 μL of supernatant using the DNS protocol (3,5-dinitrosalicylic acid) [25]. The absorbances were measured at 540 nm. A unit of chitinolytic activity was defined as the amount of enzyme that released 1 µM of sugar per 1 min at 42 °C (pH 7 100 mM).

2.9. Purification via Affinity Chromatography

The purification was performed using Ni-NTA Agarose (QIAGEN^® Hulsterweg Venlo, Nederland) Nickel-NTA. The column preparation was realized using the manufacturer’s recommended protocol. The activated Ni-NTA Agarose was packed in 1 × 5 cm column and equilibrated with 3 volumes of lysis buffer solution (100 mM/L NaH₂PO₄ 10 mM Tris-Cl and 10 mM/L imidazole, pH 8). The cell extract was mixed with lysis buffer and Ni-NTA agarose resin for 60 min at 200 rpm at 4 °C. The mixture was poured into the column and the filtrated mixture was recovered for SDS-PAGE analysis.

Column washing was performed using 4 mL of the wash solution (100 mM/L NaH₂PO₄, 10 mM Tris-Cl, and 20 mM/L imidazole), and 1 mL of the wash fractions was collected. The elution fractions were performed with elution buffer (100 mM/L NaH₂PO₄, 10 mM Tris-Cl, and 250 mM/L imidazole) up to 500 μL aliquots. This step was followed by polyacrylamide gel electrophoresis with 12% SDS, under dissociation conditions, according to the technique described by Gallagher [26].

2.10. Determination of the Optimal Temperature and pH for Chitinolytic Activity

The determination of the temperature for the maximum chitinolytic activity was evaluated between 20 and 80 °C. The reactions were performed as previously described. To determine the maximum pH, the assay was performed at the maximum activity temperature (42 °C) using acetate and phosphate buffer systems at a pH of 4.0–9.0, with 100 mM.

2.11. Adaptation of Aethina tumida

Adult Aethina tumida beetles were collected from apiaries in the Sotavento region of central Veracruz state. To maintain the biological cycle of A. tumida, they were conditioned in rearing chambers at 28 ± 2 °C and 80% relative humidity, following Haas et al. [27]. The beetles were deposited in 60 × 15 mm Petri dishes (Scientific Senna^®, Ocala, FL, USA) with a central perforation of 2 cm in diameter, covered with nylon mesh to allow for ventilation. The beetles were fed with 500 mg of a mixture of honey, pollen, soybean, and barley (1:1:1:1) every third day. After oviposition, the beetles were relocated into new conditioned Petri dishes, and the hatched larvae were feed with the same food mixture, humidity, and temperature conditions until the start of their normal biological cycle. For this last phase, plastic pupating chambers of 1 L were designed, and ¾ of the chambers were filled with a mix of soils of sand, clay, and silt. A Petri dish was adapted at the upper part of the chamber with a perforation at the base in order to allow for the larvae to descend and initiate the pupation stage. For the choleoptericidal activity, assays were used from the larvae and adult beetles of the third generation, 3–4 days after their hatching.

2.12. Choleoptericidal Activity on Aethina tumida

For the evaluation of the choleoptericidal activity of ChiBlUV02, five concentrations of extract were used (10, 30, 45, 70, and 115 U/mL), as well as a negative control. Purified recombinant chitinase was administered using the immersion method, which consists of using 10 beetles or 10 larvae, depending on the stage evaluated, for 30 s for the tests. The acute effect conditions were determined at 24, 48, and 72 h. All assays were carried out in triplicate. Any specimen that did not walk or did not respond to manipulation with mechanic manipulation was considered dead. In all assays, the beetles and larvae were kept at a temperature of 28 ± 2 °C in darkness. The response variable was the percentage of the mortality of the beetles and larvae at 24, 48, and 72 h after the assay conditions.

Damage to the exoskeletons of the beetles was observed from micrographs that were taken using an EBTOOL^® digital stereoscopic microscope.

2.13. Statistical Analysis

To determine the optimal pH and temperature conditions for ChiBlUV02 chitinase enzyme activity, a univariate analysis (ANOVA) was performed at a significance level of 0.05 via Tukey’s test using Minitab^® 19.0 software.

Mortality data were evaluated using the analysis of variance (ANOVA) at a significance level of 0.05 and comparison of means using Tukey’s test using SAS statistical software version 9.3. For the calculation of LC₅₀ and LC₉₀, a Probit analysis was used.

3. Results

3.1. Recovery of the Gene chiBLUV01

The PCR was carried out using chromosomal DNA from Bacillus licheniformis UV01 as a template. The amplicon of approximately 2000 bp was corroborated through 1% agarose gel (Figure 1). The sequence corresponded to a chitinase gene previously reported in National Center for Biotechnology Information (NCBI) (GeneBank access number CP000002.3)

3.2. Identification of the Transformed Cells

The construction of the pHTP-Chibluv01 vector was verified using the amplification of the pHTP-Chibluv01 via PCR of the Chibluv01 gene. Using plasmid DNA as a template, the transformed strains were analyzed on 1% agarose gel. Figure 2 shows the amplification of the Chibluv01 gene present in the transformed Escherichia coli BL21 cells (DE3).

3.3. Induction of Chibluv01 Gene Expression

Gene expression was induced according to the methodology previously described. The cells were harvested at different times after the induction and were lysed. A prominent band was observed on the polyacrylamide gel at approximately 80 kilodaltons (kDa), corresponding to the expected molecular weight of the protein of interest in the overexpressed sample. The highest expression was determined using polyacrylamide SDS-PAGE under dissociating conditions (Figure 3).

3.4. Determination of the Optimal pH and Temperature

The optimal enzyme temperature was 42 °C and the optimal pH was 7.5. The changes in the incubation temperature within the range studied (20–80 °C) showed relevance to the performance of the recombinant chitinase produced by B. licheniformis UV01. The maximum production was 115 U/mL between 42 and 45 °C (Figure 4).

The effect of different pHs on the chitinase ChiBlUV02 activity was measured from pH 4.0 to pH 9.0 (100 mM). The maximum value was found at pH 7.5 with an activity of 115 U\mL (defined as 100% relative activity), while the minimum activity was 3 U\mL at pH 4 (3.5% relative activity), as shown in Figure 5.

3.5. ChiBlUV02 Chitinase Purification

The extracts obtained from the treatment with lysis buffer (100 mM/L NaH₂PO₄, 10 mM Tris-Cl, and 10 mM/L imidazole) were clarified via centrifugation, adjusted to pH 8.0, and loaded on to a Kimble Flex-Column^®. The matrix used was Ni-NTA agarose from Qiagen™. The column matrix was washed with the respective buffer (100 mM/L NaH₂PO₄, 10 mM/L Tris-Cl, and 20 mM/L imidazole), and the retained enzyme on the matrix was recovered using the elution buffer (100 mM/L NaH₂PO₄, 10 mM Tris-Cl, and 250 mM/L imidazole). The data from the purification show the fraction corresponding to ChiBlUV02 from the protein (Figure 6 has an increase of 90% with respect to the crude extract and 23% of yield).

3.6. Choleoptericidal Activity on the Larvae and Adults of Aethina tumida

Table 1. Choleoptericidal effect of the purified enzyme ChiBlUV02 at different time periods and concentrations of treatments on Aethina tumida larvae.

	24 h		48 h		72 h
Concentration (U/mL)	Live	Dead	Live	Dead	Live	Dead	Mortality
Control	30	0	30	0	30	0	0
10	30	0	30	0	30	0	0
30	26	4	26	4	26	3	13%
45	22	8	21	9	21	7	30%
70	19	11	18	12	18	12	43%
115	15	15	15	15	15	15	50%

shows the mortality of larvae treated with the purified enzyme via immersion for 30 seconds. The maximum mortality was observed at 24 h post treatment, which might suggest that the enzyme loses its choleoptericidal effect after that time.

Table 2. Choleoptericidal effect of the purified enzyme ChiBlUV02 at different time periods and concentrations of treatments on the adult stage of Aethina tumida.

	24 h		48 h		72 h
Concentration (U/mL)	Live	Dead	Live	Dead	Live	Dead	Mortality
Control	30	0	30	0	30	0	0
10	30	0	30	0	30	0	0
30	27	3	27	3	27	3	10%
45	23	7	23	7	23	7	23%
70	19	11	18	12	18	12	40%
115	13	17	13	17	13	17	56%

shows the results of the adult beetle mortality in the 30 s immersion assays. We observe that the enzyme, as with the larvae of Aethina tumida, manifested its maximum efficiency 24 h post treatment.

3.7. Determination of LC₅₀ and LC₉₀

The mortality data of larvae and adult beetles were used to perform a Probit analysis and calculate the chitinolytic activity units required to promote 50% (LC₅₀) (Figure 7) and 90% (LC₉₀) (Figure 8) mortality in the population of adult beetles, which were 97.58 U/mL and 222.94 U/mL for adult beetles and 104.05 U/mL and 234.36 U/mL for the larvae, respectively. The choleoptericidal effect exhibited by the purified chitinase ChiBluv02 from Bacillus licheniformis UV01 substantiates its potential utilization as an alternative method of Aethina tumida control.

The mortality rate increased as the enzymatic activity increased, obtaining the best result when using purified chitinase at 115 U/mL, which promoted 50% mortality of larvae and 56% of mortality in adult beetles of Aethina tumida at 24 h (Table 2). The units of activity evaluated in the present study did not allow us to obtain 100% mortality of the larvae and adult beetles, but the LC₅₀ and LC₉₀ could be calculated to determine the enzyme activity necessary to promote mortality in 50% and 90% of the population, respectively.

Probit analysis was performed on the data obtained, and the units were calculated for the chitinolytic activity required to promote 50 (LC₅₀) and 90 mortality % (LC ₉₀).

The choleoptericidal effect of the ChiBlUV02 enzyme was also observed in the architecture of the exoskeleton of the larvae and adults of Aethina tumida. A change in the structure of the exoskeleton was observed in the larvae (Figure 9).

Digital microscopic observation revealed changes at the structural level that could be attributable to the action of the chitinase contained in the purified extract. The cause of the wear of the exoskeleton in the adult beetle may be associated with the hydrolysis of chitin present in the beetle (Figure 9).

Oviposition of the treated adult beetles was affected, resulting in a 15-day prolongation of egg laying compared to the control group. This phenomenon is potentially attributable to the effect of the enzyme ChiBlUV02 (Figure 10). The oviposition of eggs from beetles surviving post treatment showed severe damage resulting in larval death. A change in eggshell coloration was observed, becoming darker. In addition, the egg walls were observed to be visibly thinner (Figure 10a,b), indicating an alteration in the structural integrity compared to the control group (Figure 10c,d).

4. Discussion

In recent decades, the use of biocontrol agents, such as enzymes or molecules of natural origin, to eliminate pests has gained interest due to the different advantages they offer compared to synthetic pesticides. They have specific activity against pests, a null or low level of contamination in the crops or animal products where they are applied, and a low residual activity. Within this group of biomolecules, microbial enzymes represent an important group as biocontrol agents; specifically, chitinases are a relevant group, given the nature of the pests that can affect agricultural and animal production. One of the microorganisms with an important biotechnological application is Bacillus licheniformis, which is a bacterium that produces an important variety of enzymes including chitinases, given its saprophytic nature [28]. In the present study, the recombinant chitinase named ChiBlUV02 showed a maximum activity of 115 U/mL with a temperature of 42 °C and pH 7.5. Similar studies have reported the expression and biochemistry characterization of native and recombinant chitinases from Bacillus licheniformis, such as that described by Jayanthi et al., where they performed the characterization of a native thermostable chitinase from Bacillus licheniformis B2, observing optimal activity at 50 °C and enzymatic stability up to 70 °C, with maximum activity at pH 7.0. As a result of these findings, the authors considered that the enzyme could be employed in the recycling of chitin residues [29]. Hassiba et al. cloned, purified, and characterized an extracellular chitinase of Bacillus licheniformis LHH100 isolated from wastewater samples in Algeria; the enzyme has optimal activity at pH 4.0 and 75 °C that enables it to be used in the bioconversion of chitin residues [30]. Menghiu et al. characterized the biochemical properties of a recombinant chitinase from Bacillus licheniformis DSM8785, expressed in Pichia pastoris KM71H. The optimal activity levels of the enzyme were reported to be 140 U/mL, at pH 4.0–5.0, and in a temperature range of 50–60 °C [31]. These characteristics of the optimum temperature and pH, as well as the activity, found in ChiBlUV02 are similar to those described in other works and make it appropriate for application as a biocontrol agent given the nature of the medium in which the enzyme could be applied.

Chitinases with insecticidal capacity have been tested against a variety of fungal and insect pests; not only should the chitinase be considered as a control agent, but the route of administration and other biomolecules that help to present the fungicidal or insecticidal effect should also be considered. Brzezinska and Jankiewicz reported one chitinase produced by the Aspergillus niger LOCK 62 that inhibited the growth of the fungi phytopathogens: Fusarium culmorum, Fusarium solani, and Rhizoctonia; however, the crude extracts showed higher activity than the pure chitinase. This could be due to the presence of other antifungal enzymes such as glucanases or proteases [32]. Aoki et al. reported a chitinase produced by Trichoderma sp. that was tested against Botrytis cinerea inhibiting the growth of this fungus on cucumber leaves [33]. Essghaier et al. worked with the strain J24 from Bacillus licheniformis and expressed a gene coding for a chitinase. They found a fungicidal effect on Fusarium mangiferae with a 94% growth inhibition [34]. Moon et al. investigated the efficacy of the chitinase and the protease produced by Bacillus licheniformis PR2 on the termite Reticulitermes speratus in laboratory conditions, presenting a mortality of 88.9% [35]. Another chitinase that has been utilized for pest control is the one documented by Rajendran et al. In their study, the enzyme was expressed in Streptomyces mutabilis IMA8 and showed a larvicidal effect at different concentrations of chitinase against all stages of Anopheles stephensi and Aedes aegypti (I–IV) and pupae after 24 h of exposure [36]. Purified chitinase has been used as a biocontrol agent against the fungus Fusarium oxysporum and the Colorado potato beetle Leptinotarsa decemlineata. The enzyme was shown to be effective in reducing fungal growth and disrupting the chitin structure of the Colorado potato beetle [1]. The method of application is a critical factor to consider. The efficacy of the treatment may be influenced by the way in which it is administered. In an artificial diet, Spodoptera frugiperda larvae exhibited a decline in survival when administered chitinase AsChtII-C4B1 [37]. The purified enzyme ChiBlUV02 obtained a mortality rate of 50 and 56% in two phases (larvae and adults), respectively, of Aethina tumida. The purification process of the chitinase ChiBluv02 resulted in a significant increase in its enzymatic activity, which translated into a markedly improved efficacy in the treatment of Aethina tumida, an invasive beetle with relevant economic and ecological implications in global beekeeping. The enhanced activity can be attributed, in part, to the eradication of competing proteins, endogenous inhibitors, or structural contaminants present in the crude extracts, which have the potential to produce a grade of enzymatic inhibition (Aktas et al.) [38]. The purification process resulted in a more homogeneous and conformationally stable form of the enzyme. The effectiveness may be attributed to the sweeping of the cuticular relief of the exoskeleton and increased permeability, due to the hydrolyzed chitin, in addition to the acute effect caused by possible atrophy due to enzymatic action, which was manifested in the increase in the number of days for laying. The results obtained show that the recombinant chitinase ChiBlUV02 produces a choleoptericidal effect on Aethina tumida, as reflected by the mortalities obtained (50% in larvae and 56% in adults). In addition, evident morphological changes were observed in the exoskeleton of treated adult beetles, suggesting an enzymatic action of chitin hydrolysis. This alteration in the structure of the integument could have considerable repercussions on the physiology of the insect, particularly in the process of ecdysis. The research of Liu et al. provides a relevant point of comparison. In their study, by silencing the endogenous chitinase (CHI) gene in Spodoptera frugiperda species using iRNA, the authors reported a block in molting and alterations in carbohydrate metabolism such as trehalose and glucose. The reduction in enzyme activity inhibited the efficient degradation of the exoskeleton by modifying the expression of genes related to chitin biosynthesis, such as CHSB, GFAT, and UAP, causing stage interference. Although in the present study gene expression of the treated coleopteran was not evaluated and no silencing techniques such as RNAi were applied, the observed effects, particularly the structural damage to the exoskeleton and associated mortality in Aethina tumida, suggest that the chitinase ChiBlUV02 could be causing a comparable effect, interfering with normal chitin turnover during molting or affecting the integrity of internal structures (peritrophic membrane), as documented for CHI in S. frugiperda. This is consistent with the recognized role of chitinases in chitin degradation during the growth, metamorphosis, and immune defense processes in insects [39].

Biochemical parameters such as the temperature and pH of the recombinant chitinase ChiBlUV02 allowed for the establishment of initial conditions for the application of the enzyme in pest control as a bioinsecticide; therefore, this represents an important possibility for the use of these enzymatic technologies as an alternative to control infestations such as that represented by Aethina tumida in the beekeeping sector. In addition to not having the negative effect of traditional insecticides with chemicals, the chitinases ChiBlUV02 exhibit a residual activity of approximately 80% at the temperature conditions of hives (approximately 34 °C); therefore, it would require any special application conditions.

5. Conclusions

This study presents the development of an alternative treatment using recombinant chitinase ChiBlUV02 and its effects on Aethina tumida at two different life stages. In Mexico, there is currently no approved method for controlling this pest, which poses a significant threat to apiculture. The proposed approach offers a promising alternative to conventional chemical treatments, which can harm honeybees (Apis mellifera) and contaminate hive products, leading to economic losses for beekeepers.

This study found that ChiBlUV02 exhibited an enzymatic activity of 115 U/mL at 42 °C and pH 7.5, demonstrating a choleoptericidal effect on Aethina tumida, with a mortality rate of 50% in larvae and 56% in adults. Additionally, structural changes in the beetle’s exoskeleton were observed, suggesting enzymatic degradation and a possible effect on the metabolism via chitin during molt.

It is important to recognize some limiting factors that should be considered in order to take the use of the chitinase ChiBlUV02 to the next level, such as the fact that all trials were conducted under controlled laboratory conditions; hence, it is possible that the results in field trials will present variations due to the more complex environmental and biological variables, in addition to the possible biological response that the beetle could present as resistance mechanisms. Future research should prioritize the optimization of formulation and delivery mechanisms to improve the efficacy of this treatment under field conditions. Potential synergistic effects with other biological control agents will be crucial for technology transfer and eventual application in beekeeping. The development of this biotechnological approach has the potential to provide a sustainable and environmentally friendly solution for the management of Aethina tumida, thus benefiting the beekeeping industry.