Construction and Evaluation of Engineered Yersinia entomophaga for Stable Inheritance of trans-Cry3Aa-T-HasA Against Monochamus alternatus

TL;DR

Scientists engineered a bacterium to stably produce insecticidal proteins, improving its ability to control a destructive forest pest.

Contribution

A genomically stable Yersinia strain was developed using conjugation transfer, preventing plasmid loss and enhancing insecticidal effectiveness.

Findings

The engineered strain retained its genes for 78 generations without plasmid loss.

The strain secreted the insecticidal Cry3Aa-T protein extracellularly.

The new strain showed significantly higher virulence and faster insecticidal action against Monochamus alternatus larvae.

Abstract

Monochamus alternatus larvae, as concealed trunk-boring pests, evade conventional insecticide contact due to their cryptic feeding niche. To overcome this limitation, previous studies have engineered strains of the naturally entomopathogenic bacterium Yersinia entomophaga. The lethality of these strains against M. alternatus was enhanced by incorporating extracellular secretion systems and enriching insecticidal proteins within the larval midgut. However, plasmid loss occurs during serial subculturing. Here, we established an engineered strain that expresses the red fluorescent protein gene mCherry to explore the applicability of bacterial conjugation transfer to Yersinia. We then constructed a chromosomally integrated strain (CSLH88-pCHSW) that incorporates extracellular secretion systems. The results of stability assays demonstrated 100% retention of the mCherry and Cry3Aa-T-HasA genes over 78 generations. SDS-PAGE and Western blot analyses confirmed the extracellular secretion of the Cry3Aa-T protein in the CSLH88-pCHSW strain. Bioassays revealed that the CSLH88-pCHSW strain was significantly more virulent against M. alternatus larvae than both the wild-type strain (CSLH88) and the plasmid-transformed strain (CSLH88-pCHKW), and exhibited markedly faster insecticidal kinetics. Our study reveals the application of bacterial conjugation transfer technology for constructing biocontrol strains. This genomically stabilized Yersinia strain eliminates the risks of failure associated with plasmid loss in the field, enabling the sustainable control of M. alternatus.

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1. Introduction

Pine wilt disease, caused by the pine wood nematode, is an immensely destructive forest disease that has spread to China, Japan, Korea, and Europe, resulting in massive annual economic losses in the forestry sector [1,2]. Pine sawyer beetles (Monochamus spp.) are key vectors of pine wilt disease, transmitting the pinewood nematode to healthy trees during feeding [3]. Monochamus alternatus Hope serves as a vector for this disease, which has caused the death of over 100 million pine trees, making it one of the country’s most devastating forest diseases [4,5]. Therefore, controlling M. alternatus is a critical strategy for preventing and managing pine wilt disease [6].

To effectively manage the population of M. alternatus, a variety of strategies are currently available, including efficient trapping, chemical control, and biological control [7,8,9]. Among these, biological control, particularly through the use of pathogenic microorganisms, has gained significant attention and application due to its numerous advantages, such as high safety, environmental friendliness, and target specificity [10,11]. Yersinia entomophaga Hurst, a Gram-negative bacterium in the family Enterobacteriaceae, exhibits potent insecticidal activity against diverse insect hosts, including M. alternatus [12]. This insecticidal activity is primarily mediated by a multi-subunit toxin complex (Yen-Tc) [13]. Upon ingestion by insects, Yen-Tc associated chitinase hydrolyzes the peritrophic matrix within the midgut, ultimately inducing insect mortality [12]. Yersinia entomophaga has a unique pathogenic mechanism that typically affects only insects, which gives it significant potential for application in biological control [14,15,16]. From the gut microbiota of M. alternatus, we successfully isolated an entomopathogenic strain of Y. entomophaga. To advance its utility in pest management, enhancing the efficacy of this strain is imperative. One promising strategy involves the genetic engineering of insect symbiotic bacteria for heterologous expression of specific insecticidal toxins, which could significantly improve targeted pest control [17,18].

Cry proteins have been extensively studied in biological pest control and genetically modified crops due to their remarkable insecticidal activity against diverse insect orders [19,20,21,22]. Among them, Cry3Aa exhibits specific toxicity against pests of the Coleoptera [23,24,25]. Guo et al. [25] developed a highly potent variant designated Cry3Aa-T protein against M. alternatus. However, the combined use of insect gut microbiota and Cry proteins for M. alternatus biocontrol faces significant challenges. A primary limitation is the absence of toxic protein secretion machinery in bacterial cells, leading to intracellular protein sequestration. This prevents protein release into the extracellular environment and functional activity within the insect gut [26]. To address this issue, a previous study constructed Y. entomophaga (CSLH88 strain) with the HasA heme transporter, demonstrating extracellular secretion of fluorescent reporters [27]. By integrating the Cry3Aa-T gene, Han et al. [28] generated recombinant CSLH88-pCHKW strains capable of secreting Cry3Aa-T toxin protein. These engineered strains achieve extracellular protein secretion, enhancing the targeted delivery of insecticidal toxins to the M. alternatus midgut. However, the frequency of the CSLH88-pCHKW strain carrying the insecticidal gene decreased after 50 generations.

Traditional genetic engineering strains often face issues such as plasmid loss and low genetic stability [29]. Compared to plasmid-based vectors, introducing transgenes using Tn5 transposons delivered via bacterial conjugation offers a significant advantage in diverse bacterial species [30]. These advantages include maintenance without antibiotic selection pressure, high long-term stability, the ability to create multiple insertions within a single cell, and the ability to accommodate relatively large transgene inserts [31,32]. Martinez-Garcia et al. [33] successfully constructed the entirely synthetic transposon vector pBAM1, which enables the insertion of functional genes into the genome of target strains. The synthesized transfer vector is a suicide plasmid, which cannot exist in other strains except for specific ones (such as Escherichia coli S17-1λpir). Wang et al. [34] modified the pBAM1 transposon plasmid to insert toxin genes into the genome of the mosquito symbiotic bacterium Serratia AS1, thereby inhibiting the development of Plasmodium falciparum Welch within mosquitoes. If the insecticidal protein gene could be integrated into the genome of Y. entomophaga via bacterial conjugation, it would facilitate the stable inheritance of the toxin protein.

In this study, we first constructed the suicide plasmid pMCSW and integrated the mCherry gene (expressing red fluorescent protein) in Y. entomophaga. We then assessed the genetic stability of the target gene through serial subculturing and employed fluorescence microscopy to detect red fluorescent protein expression. Subsequently, we replaced mCherry with the Cry3Aa-T-HasA gene to construct an engineered strain and evaluated its extracellular secretion capability and insecticidal activity. Our study successfully generated an engineered Yersinia strain (CSLH88-pCHSW) capable of stable inheritance and extracellular expression of the insecticidal protein gene. This strategy prevents the loss of insecticidal protein genes in future field applications, thereby enhancing the efficacy in controlling M. alternatus.

2. Results

2.1. Generation of Engineered Yersinia Strains CSLH88-pMCSW and CSLH88-pCHSW

The pMCSW plasmid was designed and contains a nptII promoter, a red fluorescent protein marker gene (mCherry), a spectinomycin resistance gene (Spec), a kanamycin resistance gene (Kan), a transposase gene (tnp), a conjugative transfer gene (OriT), a replication origin (R6KOriV), and recognition sites for transposase (ME-O/ME-I) (Figure S1A). Following heat-shock transformation into E. coli S17-1λpir, the plasmid was extracted. The expected size of the complete plasmid is 5459 bp. Double digestion of the extracted pMCSW plasmid with BamHI and SacI restriction enzymes yielded fragments measuring 3804 bp and 1655 bp, respectively. These sizes matched the bands observed on the electrophoresis gel (Figure S1B).

The Cry3Aa-T-HasA gene was amplified by PCR and cloned into pMCSW via the BmtI and XhoI enzyme cutting sites to replace the mCherry gene, generating pCHSW (Figure 1). Following transformation and extraction, the 7283 bp plasmid was confirmed by size. Double digestion of the extracted pMCSW plasmid with BamHI and SacI restriction enzymes yielded fragments measuring 5762 bp and 1521 bp, respectively. These sizes matched the bands observed on the electrophoresis gel (Figure S2B).

To confirm genomic integration of mCherry and Cry3Aa-T-HasA genes in the Y. entomophaga genome, genomic DNA from the engineered strains CSLH88-pMCSW and CSLH88-pCHSW was analyzed (Figure 2). PCR with mCherry primers amplified a 711 bp fragment in the CSLH88-pMCSW strain (Figure 3A), while nptII-Cry3Aa-T primers yielded a 2382 bp product exclusively in the CSLH88-pCHSW strain. These sizes matched the bands observed in the electrophoretogram (Figure 3B).

2.2. Fluorescent Characterization of CSLH88-pMCSW Strain

A fluorometric assessment revealed an absence of red fluorescence in the wild-type Y. entomophaga strain CSLH88, with a complete lack of signal in the microscopic fields examined (Figure 4A,B). In contrast, the recombinant strain CSLH88-pMCSW exhibited bright mCherry-derived red fluorescence upon laser excitation, confirming successful heterologous expression of the fluorescent protein in Y. entomophaga strain (Figure 4C,D).

2.3. Genetic Stability of the Engineered Strains

Successive subculturing was performed to determine the genetic stability of the engineered strains CSLH88-pMCSW and CSLH88-pCHKW. The retention frequency of the mCherry and Cry3Aa-T-HasA genes was then analyzed. The data revealed that, in the absence of antibiotic selection pressure, 100% of cells in both the CSLH88-pMCSW and CSLH88-pCHKW strains retained these genes over 78 generations (Table 1).

2.4. The Secretion Expression of the Cry3Aa-T-HasA Fusion Protein

SDS-PAGE results revealed abundant protein expression in both intracellular and extracellular fractions of the CSLH88-pCHSW strain. Western blot analysis confirmed the presence of Cry3Aa-T protein (~70 kDa) in both intracellular and extracellular fractions of the CSLH88-pCHSW strain (Figure 3C).

2.5. Insecticidal Activity of CSLH88-pCHSW Strain

Analysis of the results of the biological assay shows that the constructed entomopathogenic Yersinia strain CSLH88-pCHSW (LC_50_ = 1.278 × 10^6^ CFU mL^−1^) and the plasmid-based engineered strain CSLH88-pCHKW (LC_50_ = 5.016 × 10^6^ CFU mL^−1^) were both significantly more toxic than the wild-type strain CSLH88 (LC_50_ = 4.801 × 10^8^ CFU mL^−1^, p < 0.05) (Table 2). The probit model demonstrated a good fit to the data (Pearson chi-square test: χ^2^ = 3.929, df = 11, p = 0.972).

2.6. Proliferation of CSLH88-pCHSW Strain in the Gut of M. alternatus

Grinding and plating the intestines of fed M. alternatus larvae yielded results indicating rapid proliferation of CSLH88-pCHSW within their intestines. Post hoc Tukey’s test further showed that at the 1- and 2-day time points, the CFUs of CSLH88-pCHSW were significantly greater than those of the other times (p < 0.05). The number of CSLH88-pCHSW bacteria in the gut of M. alternatus larvae increased by more than 200-fold within 1 day post feeding (Figure 5A). Kaplan–Meier survival analysis showed that, compared with the wild-type strain (CSLH88) and the plasmid-based strain (CSLH88-pCHKW), the CSLH88-pCHSW strain reduced the survival of the test larvae to 0% within 15 days (Figure 5B). Statistical significance was determined using the log-rank (Mantel–Cox) test. The overall comparison among the three groups was highly significant (χ^2^ = 10.78, df = 2, p < 0.05) (Figure 5B).

3. Discussion

The larvae of M. alternatus exhibit concealed boring behavior within tree trunks, making it difficult for insecticidal agents to directly contact the insect body [3]. Engineered gut bacteria enable targeted oral delivery of insecticidal toxins. Upon ingestion, they colonize the gut and produce toxins locally, ensuring sustained accumulation and effective exposure without requiring direct spray contact [34,35]. Han et al. [28] previously constructed an engineered Yersinia strain, CSLH88-pCHKW, which secretes insecticidal toxins for controlling M. alternatus. However, this strain suffers from plasmid instability. Bacterial conjugation facilitates the unidirectional transfer of DNA via transposable elements [36]. This process requires coordinated interaction between donor strains, recipient strains, and transposon-carrying plasmids to achieve stable integration of foreign genes into the genome of the recipient [37]. In this study, we constructed two plasmids, pMCSW and pCHSW, harboring transposable elements along with the fluorescent protein gene mCherry and the extracellular secretion insecticidal protein gene Cry3Aa-T-HasA. The mCherry and Cry3Aa-T-HasA genes were inserted into the Yersinia genome via bacterial conjugation. First, successful expression of the mCherry gene was confirmed by red fluorescence detection, demonstrating the effective functioning of the transposition system in Yersinia. Second, protein detection verified the extracellular secretion of the insecticidal protein Cry3Aa-T by Yersinia. Western blot analysis detected Cry3Aa-T in the culture supernatant, indicating successful secretion in a soluble form. Consequently, the stably inherited engineered strains CSLH88-pMCSW and CSLH88-pCHSW were successfully constructed.

Genetic stability assessment of engineered strains constitutes a critical step in evaluating the adaptability of foreign genes within genetically modified bacteria [38]. The plasmid retention rate of the previously constructed strain CSLH88-pGHKW4 (which extracellularly expresses GFP) declined to 64% by the 64th generation [27]. The plasmid-based engineered strain CSLH88-pCHKW (extracellularly expressing the insecticidal protein Cry3Aa-T) exhibited a plasmid retention rate of 94% at the 50th generation [28]. In contrast, the engineered strains (CSLH88-pMCSW and CSLH88-pCHSW) developed in this study demonstrated superior genetic stability. When cultured to the 78th generation, both strains maintained 100% plasmid retention, achieving stable inheritance of the foreign genes. Our study represents the first successful construction of Yersinia engineered strains capable of stable inheritance and extracellular expression of insecticidal proteins, significantly reducing the risk of functional loss due to plasmid instability. The genomic stability of the engineered bacteria could be influenced by the copy number of the integrated gene [39]. While multi-copy integration might theoretically enhance transgene expression, it conversely risks imposing a significant metabolic burden, potentially compromising bacterial fitness and long-term stability [40]. Therefore, employing whole-genome sequencing in future work will be essential to conclusively determine the copy number and evaluate its impact on stability under field conditions.

To further evaluate the insecticidal efficacy of the engineered strains, bioassays were performed. The results showed that the constructed entomopathogenic Yersinia strain CSLH88-pCHSW exhibited an LC_50_ of 1.278 × 10^6^ CFU mL^−1^, significantly lower than that of the wild-type strain CSLH88 (LC_50_ = 4.801 × 10^8^ CFU mL^−1^). The potency of CSLH88-pCHSW was 3.9-fold lower than that of the CSLH88-pCHKW strain (LC_50_ = 5.016 × 10^6^ CFU mL^−1^). Previous studies have demonstrated the effectiveness of Y. entomophaga against Coleopteran insects [16,41]. The Cry toxin protein also exhibits specific toxicity against pests in the Coleoptera [42,43]. Our results confirm the synergistic insecticidal activity between Yersinia and Cry toxin protein, with the engineered strain showing significantly enhanced efficacy against M. alternatus. To elucidate the mechanism underlying this high virulence against M. alternatus larvae, gut proliferation dynamics of CSLH88-pCHSW were investigated. The engineered strain rapidly multiplied in the insect gut post-ingestion, concentrating insecticidal toxins within the digestive system and facilitating direct toxin-insect contact. This colonization mechanism likely contributes to optimized insecticidal efficacy. Compared with the plasmid-based strain CSLH88-pCHKW, the CSLH88-pCHSW strain exhibited markedly faster insecticidal kinetics due to its lower risk of plasmid loss. Crucially, the genomic integration strategy in CSLH88-pCHSW prevents toxin gene loss during field deployment, ensuring persistent insecticidal activity against M. alternatus. In order to translate our laboratory findings into practical applications, it is essential to evaluate the efficacy of biocontrol under greenhouse or field conditions. However, due to the extended time frame required for experiments involving wood-boring insects such as M. alternatus, data from these settings are currently unavailable. We therefore encourage future studies to conduct more experiments in greenhouses or fields with engineered strains to demonstrate the efficacy of biocontrol.

In practical field applications, the engineered strains can be fermented and formulated into a microbial agent that can be deployed efficiently via drones across large forests to manage M. alternatus. In the future, we plan to develop a viable powder formulation modeled on health-food probiotic powders to prolong the strain’s shelf life and facilitate field applications [44]. Nevertheless, the strain is still at the experimental stage and has not yet secured commercial product registration. Potential challenges in the future commercialization of the strain may include the lengthy and costly processes required for industrial-scale production, such as field efficacy trials, safety assessments for non-target animals, and environmental impact evaluations.

A key feature of our strains is the stable inheritance and expression of the foreign toxin gene. This stability ensures that the cells continuously produce and excrete the insecticidal toxin, resulting in a prolonged duration of toxin activity on treated surfaces. This prolonged activity is essential for increasing the likelihood of pests ingesting a lethal dose [45]. The summer and autumn seasons, when the new generation tunnels beneath the bark and creates fresh entry wounds, are the critical window [46,47]. At that time, a high-concentration suspension of our engineered strain can be sprayed directly onto the trunk and main branches, thoroughly coating the bark—especially around natural wounds, pruning scars, and existing beetle holes. As the larvae continue to bore, they ingest wood shavings laden with bacteria. Since our strain secretes its toxins extracellularly, this oral route is the primary mode of exposure. Larval movement then carries the bacteria from the treated surface into the moist, protected galleries, where they persist, proliferate, and ultimately infect the insect. While this strategy is promising, it is also crucial to acknowledge that the large-scale application of any engineered organism requires careful evaluation of potential biosafety concerns, such as horizontal gene transfer to other microbes or effects on non-target insects. Therefore, future research must include comprehensive assessments of these environmental risks to ensure the safe and sustainable use of this technology.

In this study, we initially constructed an engineered strain expressing the mCherry gene to evaluate bacterial conjugation applicability in Yersinia. Subsequently, we developed the entomopathogenic engineered strain CSLH88-pCHSW with chromosomally integrated genes for stable inheritance. SDS-PAGE and Western blot analyses confirmed extracellular secretion of the Cry3Aa-T insecticidal protein, with stability assays demonstrating sustained genetic integrity. Compared to the wild-type strain CSLH88 and plasmid-transformed control CSLH88-pCHKW, the engineered strain exhibited significantly enhanced virulence against M. alternatus larvae. This high-virulence strain provides a robust platform for field-deployable biocontrol of M. alternatus.

4. Materials and Methods

4.1. Experimental Materials

This experiment employed laboratory-reared second-instar larvae of the M. alternatus Hope FAFU strain. The Escherichia coli S17-1λpir used in the experiment was obtained from the China General Microbiological Culture Collection Centre. The Y. entomophaga CSLH88 and CSLH88-pCHKW strains were stored at −80 °C in 20% (v/v) glycerol. The characteristics and sources of the plasmids used in this experiment are shown in Table S1. All experiments in this study were conducted in accordance with standard microbiological practices.

The preparation of a 1 L batch of liquid LB medium involved the dissolution of 10 g of tryptone, 5 g of yeast extract, and 10 g of sodium chloride in distilled water. This mixture was then adjusted to a pH of 7.2 and transferred to conical flasks. After sealing the flasks with a membrane, the medium was sterilized at 121 °C for 20 min, making it ready for use.

The preparation of a solid LB medium (1 L) was carried out using the same proportions as the liquid LB medium previously mentioned. The mixture was poured into conical flasks, and 1.5% agar powder was added. The flasks were sealed with a membrane, and the medium was sterilized at 121 °C for 20 min to ensure it was ready for use.

The required antibiotics are dissolved in distilled water to create a mother solution with a concentration of 100 mg mL^−1^. This solution is then sterilized by filtering it through a disposable filter before being added to the culture medium in the correct proportions. The final antibiotic concentration in the medium is 100 μg mL^−1^. This process ensures the sterility of the antibiotics and their correct incorporation into the medium for experimental use.

4.2. Construction of Transposon Plasmid pMCSW and pCHSW Vector

In this experiment, we synthesized the pMCSW plasmid based on the pBAM1 plasmid (NCBI accession number: HQ908071) [33]. pBAM1 has a streamlined, restriction site-free and narrow-host-range replication frame that carries the R6KoriV, oriT sequences, and an ampicillin resistance marker. This plasmid is a suicide vector that can only grow in the E. coli S17-1λpir strain. The application of suicide plasmids eliminates the need for cumbersome verification steps. However, due to the ampicillin resistance of the experimental strain Y. entomophaga [27], the ampicillin gene (Amp) could not be used as the resistance gene. As the experimental strain is not resistant to spectinomycin, the Amp gene was replaced with the spectinomycin gene (Spec) to enable subsequent screening of the target strain. The nptII promoter and the mCherry gene were then inserted upstream of the kanamycin (Kan) gene to enable fluorescent labelling. These genes were assembled to construct the pMCSW plasmid (Figure S1; Table S1), which was synthesized by GenScript Biotech Corporation.

The pMCSW expression vector was digested using the BmtI and XhoI restriction enzymes, resulting in two fragments: the mCherry gene and the vector backbone. These fragments were gel-purified and set aside for subsequent use. The Cry3Aa-T-HasA gene was amplified via PCR using the pCHKW plasmid as a template and the following primers.

Forward primer: 5′-GCTAGCATGAATCCGAACAATCGAAGTG-3′,

Reverse primer: 5′-CCTTGACGACCGCCGGACTGAGCTC-3′

The resulting Cry3Aa-T-HasA gene was then ligated to the gel-purified vector backbone fragment using T4 DNA ligase (New England Biolabs, Ipswich, MA, USA). This ligation reaction generated the pCHSW plasmid (Figure 1).

The pMCSW and pCHSW plasmids obtained were introduced into the E. coli S17-1λpir strain via heat shock transformation. The transformed cells were spread onto LB agar plates containing a final concentration of 100 μg mL^−1^ spectinomycin and incubated at 37 °C for 12 h. The transformed colonies were then inoculated into a liquid LB medium containing spectinomycin and cultured at 150 rpm and 37 °C for 12 h [48]. Subsequently, plasmid extraction was performed, and the plasmids were digested using the BamHI and SacI restriction enzymes, followed by sequencing for verification. E. coli S17-1λpir strains carrying mCherry and Cry3Aa-T-HasA gene were obtained to provide donor strains for the subsequent conjugation transfer experiment.

4.3. Introduction of mCherry and Cry3Aa-T-HasA Genes into Yersinia by Bacterial Conjugation

The donor strain, E. coli S17-1λpir, which carries the target plasmids pMCSW or pCHSW, and the recipient strain, CSLH88, were cultured separately until they reached the logarithmic phase (Figure 2). The cultures were then washed with a 10 mM MgSO4 solution, resuspended to an OD600 of 0.1, mixed at a ratio of 1:1, and spread onto agar plates without antibiotics. The plates were incubated for 5 h at 37 °C. The bacterial lawn from these plates was scraped into an LB liquid medium containing 200 μg mL^−1^ of ampicillin and cultured for 12 h at 30 °C. Subsequently, streaking was performed on LB plates containing kanamycin (200 μg mL^−1^), and the plates were incubated for 12 h at 30 °C. Single colonies were then picked [33]. Genomic DNA was extracted from the strains, and sequencing identification was conducted first using bacterial 16S rRNA primers (8F: 5′-AGAGTTTGATCCTGGCTCAG-3′, 1492R: 5′-GGTTACCTTGTTACGACTT-3′). This revealed that the extracted DNA belonged to the Yersinia species.

Primers for mCherry (5′-CGGGATCCATGGTGAGCAAGGGCGAGG-3′, 5′-TACCTGCTCGACATGTTCATTGAGCTCCC-3′) and nptII-Cry3Aa-T (5′-CGAGCTCGTCAGGCTGTTACAGCTCAGAGAAA-3′,5′-CACGCGGCCACGGCATAGGCCTAGGCG-3′) were designed and used for PCR validation.

4.4. Fluorescent Detection of CSLH88-pMCSW Strain

The fluorescently labeled strain CSLH88-pMCHSW was directly observed under an inverted fluorescence microscope. The untreated CSLH88 strain served as a non-fluorescent control, with both strains examined for morphology under bright field and fluorescence field.

4.5. Genetic Stability Testing

In this study, the CSLH88-pMCSW and CSLH88-pCHSW strains were activated for 12 h and then inoculated (1% v/v) into antibiotic-free LB liquid medium, separately. The culture was incubated on a shaker (150 rpm, 30 °C) for 12 h, followed by repeated subculturing. After each odd-numbered subculture (e.g., 1st, 3rd, 5th), bacterial suspensions were serially diluted. For each subculturing time point, diluted samples were plated on antibiotic-free LB agar plates in triplicate. Plates were incubated at 30 °C for 24 h. A total of 100 distinct colonies from antibiotic-free plates were individually patched onto Spectinomycin-supplemented agar plates and antibiotic-free agar plates. After 12 h incubation, colonies on both plate types were counted [38]. The plasmid retention rate was calculated by dividing the colony count on Spectinomycin plates by that on antibiotic-free plates, assessing the genetic stability of the recombinant plasmid without selective pressure. The CSLH88 strain underwent identical treatment as a negative control.

4.6. Cry3Aa-T-HasA Fusion Protein Extraction and Immunoblot Analysis

Extracellular proteins were extracted by ultrafiltration. Bacterial cultures were centrifuged at 8000× g for 10 min at 4 °C, and supernatants were collected. The supernatant was concentrated using a pre-chilled ultrafiltration tube (Amicon® Ultra-15, Merck KGaA, Darmstadt, Germany) at 5000× g (4 °C, 20 min per cycle) with repeated concentration until 20-fold volume reduction. The concentrated protein fraction was transferred to cryovials and stored at −20 °C [34].

Intracellular proteins were extracted by ultrasonic lysis. Bacterial cell pellets were washed three times with PBS (140 mM sodium chloride (NaCl), 2.7 mM potassium chloride (KCl), 10 mM sodium hydrogen phosphate (Na_2_HPO_4_), 1.8 mM KH_2_PO_4_, pH 7.4), and centrifuged at 8000× g for 8 min at 4 °C. Resuspended cells in lysis buffer (50 mM Tris-HCl, pH 7.4, protease inhibitors) were sonicated on ice using a 3 mm probe at 50 Hz (10s pulse on/5s off, total duration 30 min). Lysates were centrifuged (8000× g, 8 min, 4 °C) and supernatants containing intracellular proteins were stored at −20 °C [48].

Protein samples were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Electrophoresis was initiated at 80 V through the stacking gel and increased to 120 V for the separating gel. Gels were stained with 0.1% Coomassie Brilliant Blue R-250 in destaining solution (40% methanol, 10% acetic acid) for 1 h, then destained overnight in 10% acetic acid with gentle agitation. Protein bands were visualized using a gel documentation system (Bio-Rad ChemiDoc™, Hercules, CA, USA).

Western blot analysis was performed concurrently. Proteins separated by SDS-PAGE (10% gel) were transferred to nitrocellulose membranes at 100 V for 60 min in transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol). Membranes were blocked with 5% (w/v) non-fat milk in TBST for 1 h at room temperature, then incubated with primary antibody (Rabbit polyclonal anti-GFP-Tag, 1:4000 in TBST) overnight at 4 °C. After washing with TBST (3 × 10 min), membranes were incubated with HRP-conjugated Goat Anti-Rabbit IgG (1:5000 in TBST) for 1 h. Signals were detected using Syngene GBOX-CHEMI-XT4 (Syngene, Cambridge, UK) [25].

4.7. Bioassay of Insecticidal Activity of CSLH88-pCHSW Strain

Each strain (CSLH88-pCHSW, CSLH88-pCHKW, and CSLH88) was cultured separately in 100 mL of LB broth within 250 mL conical flasks and incubated at 30 °C with shaking at 150 rpm for 48 h, and then serially diluted. From each dilution, 100 μL was spread onto LB agar plates. Colonies were counted to calculate the initial bacterial density. Second-instar M. alternatus larvae were starved for 12 h prior to exposure. A total of 400 μL of the bacterial solution was applied uniformly to 1 g of sterile tissue paper. A 250 mg portion of this impregnated paper was then transferred to a sterile container containing one larva to provide a bacteria-treated substrate for feeding. The bacterial suspension was serially diluted to five concentrations: 10^5^, 10^6^, 10^7^, 10^8^, and 10^9^ CFU mL^−1^. For each concentration tested in the bioassay, 30 larvae were used in total. This comprised 3 independent replicate trials, with 10 larvae per replicate. All assays were conducted under standard laboratory conditions of 25 ± 1 °C and 70 ± 5% relative humidity, with a 16:8 h light:dark photoperiod. Larval status was monitored daily, and individuals were considered dead if unresponsive to gentle tactile stimulation. The larval mortality was calculated after 11 days.

Using the same procedure for preparing the bacterial suspensions as described above, suspensions of the three strains (CSLH88-pCHSW, CSLH88-pCHKW and CSLH88) were prepared. Second-instar larvae were treated with 100 μL of the bacterial suspension (10^6^ CFU mL^−1^) per larva. Larval survival was observed and recorded daily for 14 days, and Kaplan–Meier survival curves were plotted.

4.8. Detection of CSLH88-pCHSW Strain Proliferation in the Gut of M. alternatus

The CSLH88-pCHSW strain was cultured in 100 mL conical flasks for 24 h and diluted to 10^5^ CFU mL^−1^. This diluted bacterial solution was then mixed uniformly into the diet of M. alternatus larvae, which were subsequently fed. The intestines of the larvae were then dissected at different time intervals. The intestines were ground and spread onto kanamycin agar plates containing 100 mg mL^−1^. After incubating the plates in a culture chamber for 24 h, individual colonies were picked and subjected to PCR verification. The number of bacterial colonies containing the gene was counted to determine its proliferation in the gut of M. alternatus.

4.9. Statistical Analysis

The data from this experimental study were based on three independent biological replicates. Statistical analyses were performed using SPSS 22.0 [45]. Intergroup comparisons used one-way ANOVA with post hoc Tukey’s test, where p < 0.05 indicated statistical significance. LC_50_ values with 95% confidence intervals were calculated via probit regression modeling in SPSS. Figures were generated using GraphPad Prism 8.0.